Directrice Co

Lambert De Thèse

Claude

Etienne Invité

Keywords: protein microarray, autoantibodies, breast cancer diagnosis, predictive biomarkers, storage 4

Breast cancer becomes the most common cancer among women. In order to improve women's chances of survival and life quality, to be diagnosed at an early stage and to receive correct treatment are the most promising ways. In this context, we aim at developing an antigen microarray for screening serological biomarkers to diagnose breast cancer patients as early as possible. Among numerous potential biomarkers, recent researches showed that antibodies against heat shock proteins (HSPs) are associated with tumor genesis and would be good diagnostic and prognostic biomarkers for breast cancer. Therefore, we used customized antigen microarray to screen anti-HSP antibodies in 50 breast cancer patients and 26 healthy controls. Our results indicated clearly that combining multiplex detection of anti-HSPs antibodies could discriminate breast cancer patients from healthy controls with sensitivity 86% and specificity 100%. Then, we elaborated an antibody microarray to detect the concentration of urokinase type plasminogen activator (uPA) in 16 cytosolic extracts of breast tummor tissue. uPA is good prognostic and predictive biomarker for breast cancer, low levels of uPA (≤3 ng/mg of protein) is associated with low risk of recurrence and no benefit of chemotherapy for breast cancer patients, and vice versa. Our results showed that the results obtained from our antibody microarray were surface dependent compared with the results obtained from ELISA. Furthermore, the use of our antibody microarray requires 25 times less sample volume compared with ELISA kit, thus solving the main limitations of ELISA. Finally, we determined and optimized the parameters which affected the performances of protein microarray, e.g. microarray surface chemistry, experimental duration, the concentration of solutions, etc. Furthermore, we studied the storage conditions for both chemically functionalized microarray surface as well as printed protein microarray. Results showed that our protein microarrays retain efficient biological activity for at least 3 month of storage.

Results and discussion

General introduction

Breast cancer remains a major public health problem in the world. According to World Health Organization, in 2012 there were 1.7 million women who were diagnosed with breast cancer and the incidence has increased by more than 20% since 2008. Early diagnosis and monitoring disease development represent promising approaches to reduce the growing cancer burden. Conventional diagnostic methods include mammography, clinical breast examination, breast self-examination and magnetic resonance imaging, etc. However, the use of these procedures has limitations including false positive, high cost, unnecessary biopsy, over diagnosis and undue anxiety etc.

Recent researches showed that screening tumor biomarkers could aid the diagnosis of breast cancer, monitoring tumor progression and response to certain therapy. Protein microarrays have already demonstrated their great potential as screening tool. However, efficient protein microarray still remains a challenge due to protein variability. Various factors influence the performance of protein microarray including surface chemistry, spotting buffer, spotting concentrations, etc. So our purpose is to develop efficient protein microarray to screen biomarkers in breast cancer patients, thus providing diagnostic, prognostic and predictive value for each patient.

In chapter I, we introduced the worldwide situation of breast cancer. The most efficient way to reduce the heavy burden is to diagnose the patient at an early stage and provide correct therapy to each cancer patient. Several serum and tissue biomarkers have been already used in clinic for these two purposes. However, current serum biomarkers used lack sensitivity and specificity for diagnosis and new biomarkers are needed to be developed. Recent reviews described more 1200 proteins as potential new biomarkers, so the current challenge is not to find one more biomarker candidate but to evaluate and validate their clinical relevance in order to perform an efficient test. So, Chapter 1 deals with an overview of state of the art about serum and tissue biomarkers in view to select only those that are of high interest.

Recent studies showed that autoantibodies could be good diagnostic and prognostic biomarkers because they are stable and relatively easy to be detected in serum on one hand.

On the other hand, tissue biomarkers including urokinase type plasminogen activator (uPA) and its main inhibitor plasminogen activator inhibitor 1 (PAI-1) are advantageously used in clinic. In the two cases, tests are limited by the detection method-ELISA. In contrast, protein microarray is an efficient tool for screening both serum and tissue biomarkers by consuming small sample volume. The challenges of protein microarray will be also introduced in this chapter.

The purpose of the chapter II is using customized protein microarray to profil anti-heat shock proteins (HSPs) antibodies in breast cancer serum. As protein microarray is influenced by various factors, so we firstly optimized key parameters including surface chemistry, spotting concentration; then we detected the autoantibodies against hsps in breast cancer serum under the optimal conditions. In total, 50 breast cancer patients and 26 healthy controls were tested. Our results showed that combining multiplex detection of anti-HSPs antibodies could discriminate breast cancer patients from healthy controls with sensitivity of 86% and specificity of 100%.

Chapter III reports the fabrication and the use of antibody microarray to quantify the concentration of urokinase type plasminogen activator (uPA) and its main inhibitor plasminogen activator inhibitor 1 (PAI-1) in breast tumor tissue. We firstly optimized the various conditions for the immobilization of antibodies. Then the best conditions were chosen to detect the concentration of uPA in 16 cytosolic extracts of breast tumor tissue. Results

showed that the results obtained from our antibody microarray were surface dependent compared with the results obtained from ELISA.

Chapter IV aims to optimize the parameters that influence the performance of protein microarray. These factors concern experimental duration, the concentration of solutions, storage conditions, etc. For instance, the study of protein microarray storage conditions showed that printed proteins could retain their biological activity for at least 3 months.

Chapter 1

State of the Art

Breast cancer: Key data and detection techniques

Breast cancer remains a major public health problem in the world. According to World Health Organization, in 2012 there were 1.7 million women who were diagnosed with breast cancer and the incidence has increased by more than 20% since 2008 [1]. It is the most frequently diagnosed cancer among women in both more and less developed regions and now it represents 25% of all cancers in women, as shown in Figure 1. Compared to incidence rate, the mortality rate of breast cancer is much lesser, probably reflecting early diagnosis as well as improved treatment options [2]. The development of early diagnosis or/and disease monitoring represents promising ways to reduce the growing cancer burden [3]. Therefore, in the following, conventional diagnostic and disease monitoring methods for breast cancer will be discussed.

Figure 1 Estimated Incidence, Mortality rate of breast cancer worldwide in 2012 [2]

Early diagnosis is one of the most effective and affordable approaches to improve women's chances of survival, which could facilitate treatment of breast cancer patients in their preinvasive state prior to metastasis. It is reported that five-year survival of women with breast cancer is highly associated with tumor stage. For example, the 5-year survival of very early stage tumor (stages 0 and I) is approximately 98%, it decreases to 85% for stage II tumors, 60% to stage III tumors and only 20% for stage IV tumors [4]. Conventional diagnostic techniques of breast cancers include mammography, clinical breast examination, breast selfexamination and magnetic resonance imaging. However, the use of these procedures has potential harms including false positive, high cost, unnecessary biopsy, over diagnosis and undue anxiety [5,6].

Mammography is the best-studied breast cancer screening modality and the only recommended imaging tool for screening the general population of women. Guidelines for breast cancer screening vary within and among countries [7]. In the United States, the US Preventive Services Task Force recommends that women aged 50 to 74 years undergo a screening mammogram every 2 years. For women who are younger than 50 years old, examination should be based on the individual women's context, including her values regarding the benefits and risks [8]. In contrast, the American Cancer Society recommends that women should begin annual screening at age 40 [9]. Screening women 40 to 49 years of age is more controversial than older ages, with less evidence available to determine the riskbenefit balance.

A recent study published in 2014 has almost totally denied the benefit of the mammography. In this study, they divided women aged to 50 to 59 years into two groups, one group receiving both mammography and clinical breast examination while another receiving only clinical breast examination. Then they studied the 25-years cumulative mortality from these two groups. Results showed that the mortality was essentially equivalent between women who received mammography and clinical breast examination versus women who received only clinical breast examination [10]. Once the article has been published, it provoked heated debate in academic community. Some people defended that the clinical breast examinations were performed by well-trained clinicians, while community clinicians may not perform such high-quality clinical breast examinations, thus limiting the applicability of these results to general practice [5]. Others declaimed that the whole experiment design was questionable and the consequent results were null [11]. Evidently, controversy exists on the benefits of mammography as a routine screening test for women; therefore, it needs time to prove the real benefits of mammography for women.

MRI is a useful and sensitive tool for evaluating abnormalities and diagnosing breast cancer. While no studies have shown a mortality benefit for the general population from this screening, it is therefore not recommended as screening modalities for the general population.

However, it is recommended by both US Preventive Services Task Force and American Cancer Society to be used as a supplemental screening in special high-risk populations such as women with BRCA1 and BRCA2 mutations [12,13].

Other methods like Physical Examinations (regular breast self-examination and routine clinical breast examination) are not recommended. These Examinations could be harmful and they couldn't reduce breast cancer mortality, but double the number of biopsies. Methods like thermography and ultrasound are neither recommended because their benefits are unknown [5,6].

Currently, screening of tumor markers represents another approach for cancer diagnosis and receives considerable interest. Tumor markers are associated with tumor genesis; therefore, screening these biomarkers could aid early diagnosis as well as better management of breast cancer.

In parallel, the concept of personalized medicine is more and more accepted by academic as well as clinic research. According to US President's Council of Advisors on Science and Technology, personalized medicine refers to the tailoring of medical treatment to the specific characteristics of each patient. The goal of personalized medicine is to reduce the burden of disease by targeting prevention or treatment more effectively [14]. Ideally, personalized medicine delivers the right care to the right cancer patient at the right time and results in measurable improvements in outcomes and a reduction on health care costs [15]. In the case of cancer, considering that each solid tumor in each person is unique in cause, in rate of progression and in responsiveness to certain therapy, personalized medicine is particularly well adapted and necessary. The essence of personalized medicine lies in the use of biomarkers. Screening tissue or serum markers could provide prognostic value and predictive value. Beyond a help for diagnose, prognostic markers and predictive markers can estimate recurrence risk and predict therapy efficacy respectively. Classifying patients with low or high risk for recurrence and administering optimal therapies could avoid overtreatment for breast cancer patients. Currently, screening biomarkers are replacing the traditional "one size fits all" medicine. Personalized medicine based on biomarkers is already having a remarkable impact [16,17].

So in the following, the routine used tumor biomarkers in breast cancer will be introduced.

Tumor biomarkers

During tumor genesis, cancer or other cells of the body will produce substances in response to cancer conditions. These substances are called tumor markers and they are produced at much higher levels in cancerous conditions. Tumor markers can be found in blood, urine, tumor tissue, or other tissues or bodily fluids of cancer patients. Screening these markers could aid early detection, risk stratification, prediction and disease prognosis of breast cancer.

A number of researchers found that tumor markers could be detected several months prior to clinical detection of breast cancer, thus screening these biomarkers could let the patients to be diagnosed at a more earlier stage [4]. Prognostic markers were defined to be markers that have an association with some clinical outcomes, such as overall survival or recurrence-free survival. They enable distinguish the clinical outcomes of patients in the absence of therapy.

Moreover, prognostic markers give support to evaluate the efficiency of certain therapy. For example, increasing levels of several prognostic markers are associated with failure of certain therapy.

Predictive markers are generally used to make more specific choices between treatment options. They serve as indicators of the likely benefit to a specific patient of a specific treatment. A predictive marker might indicate that a patient expressing the marker will benefit more from a new treatment than from standard treatment, whereas a patient not expressing the marker will derive little or no benefit from the new treatment [18,19].

Among all kind of biomarkers, protein biomarkers represent one of the ultimate levels of cellular function and thus give a picture of cell health. Protein biomarkers could be antigens as well as antibodies produced by immune and hormonal responses. Today, we do not suffer from a lack of candidate protein biomarkers. More than 1200 protein biomarker candidates for cancer have been described in the scientific literature; however, only 9 have been approved as tumor associated antigens by US Food and Drug Administration (FDA) [20]. The rate of introduction of new protein biomarkers approved by FDA has remained flat over the past 15 years, with an average of 1.5 new proteins cleared per year (median of 1 per year) for all diseases [21].

Thus, we will focus on the serum and tissue biomarkers that have been approved by American Society of Clinical Oncology (ASCO) or European Group on Tumor Markers Recommendations (EGTM) for routine usage. But as performances of biomarkers are evaluated through various indicators such as true positive, true negative, false positive, false negative, sensitivity, specificity, positive predictive value and negative predictive value, receiver operating characteristic (ROC) curve, the area under the ROC curve (AUC) and p value, we will firstly define these indicators.

Indicators of biomarker value and data analysis

Methods for calculating the true positive, true negative, false positive, false negative, sensitivity, specificity, positive predictive value and negative predictive value were based on the methodology provided in Epidemiology [22].

As shown in Table 1, cell ′a′ represents the test which correctly diagnosed the disease, which are the true positives (TP). Cell ′b′ is those who have positive results for the test but do not have disease; the test has wrongly diagnosed the non-disease and it is false positives (FP).

Cell ′c′ represents those who have disease but have negative results with the test. The test has wrongly labeled a diseased person as ′normal′, which means false negatives. Cell ′d′ is those who have no disease as determined negative with the test and it represents true negatives (TN). Sensitivity is the ability of a test to correctly classify an individual as ′diseased′, which equals a/(a+c). The ability of a test to correctly classify an individual as disease-free is called the test's specificity (Specificity = d/(b+d)). Positive predictive value (PPV) is the percentage of patients with a positive test who actually have the disease (PPV = a/(a+b)). Likewise, negative predictive value (NPV) is the percentage of patients with a negative test who do not have the disease (NPV = d/(c+d)) [23].

Table 1 Calculation for sensitivity, specificity, positive predictive value and negative predictive value

ROC

The receiver operating characteristic (ROC) curve is commonly used in medical decision making. ROC graphs are two-dimensional graphs in which true positive (TP) rate is plotted on the Y axis and false positive (FP) rate is plotted on the X axis. Since TP rate is equivalent to sensitivity and FP rate is equal to 1 -specificity, the ROC graph is also called the sensitivity vs. (1 -specificity) plot. Figure 2 shows an ROC graph with five classifiers labeled A through D [24].

Figure 2 A basic ROC graph showing five discrete classifiers [24].

Several points in ROC space are important to note. The lower left point (0, 0) represents the strategy of never issuing a positive classification; such a classifier commits no false positive errors but also gains no true positives. The opposite strategy (1, 1) means unconditionally issuing positive classifications, which commits maximal true positives as well as maximal false positives. The point D (0, 1) represents perfect classification. The diagonal line y = x represents the strategy of randomly guessing a test. Point C is virtually random. Any classifier that appears in the lower right triangle performs worse than random guessing. In Figure 2, point B performs much worse than random. In contrast, the point in the top left corner of ROC space is better, e.g. point A, which means that TP rate is higher while FP rate is lower [24].

Generally, a diagnostic test contains several samples and each sample has a tested value. In order to construct a ROC curve, we rank all the tested value and produce a discrete classifier with a threshold, which means that if the tested value is higher than the threshold, the classifier produces a Yes, otherwise a No. Each threshold value produces a different point in ROC space. Figure 3 shows an example of ROC curve on a test set of 20 instances.

No The graph shows the corresponding ROC curve with each point labeled by the threshold that produces it [24].

The test contains 20 samples (10 positives and 10 negatives) and the samples were ranked by their tested values, from 0.9 to 0.1, as shown in Figure 3a. Threshold is the tested value of each sample and according to each threshold value, it produces a different point in ROC space (Figure 3b). For example, a threshold of +1 produces the point (0, 0). As we lower the threshold to 0.9, the first positive sample is classified positive, thus the sensitivity is 10%

(1/10); also no negative is classified positive, thus there is no false positive and the specificity is 100%, yielding (0, 0.1). As the threshold is further reduced to 0.7, two positive samples are classified positive, thus the sensitivity is 20%; also one negative is classified positive, thus the false positive rate is 10%, yield (0.2, 0.1). Similarly, as the threshold is further reduced, the curve climbs up and to the right, ending up at (1, 1) with a threshold of 0.1 [24].

One point in ROC space is better than another if it is close to the top left which means that TP rate is higher while FP rate is lower, e.g. Figure 2 point D. Likewise, if the ROC curve is more close to top left, it means that the classifier is good at identifying likely positives as well as likely negatives (high sensitivity and high specificity).

AUC

AUC is the area under ROC curve. The ideal test would have an AUC of 1, indicating a perfect situation with 100% sensitive and 100% specific; a random guess would have an AUC of 0.5, represented by the diagonal line from the lower left corner to the upper right, as shown in Figure 2. In general, ROC curves with an AUC≤0.75 are not clinically useful and an AUC≥0.97 has a very high clinical value [25].

P-value

Chi-square χ2 test was used to determine whether the frequency of biomarker in cancer serum was significantly higher than healthy controls. Two significant levels (P <0.05 and P<0.01) are commonly used [26].

Serum biomarkers used in clinic

In recent years the discovery of cancer biomarkers has become a major focus of cancer research. When we compared the clinical practice guidelines published by American Society of Clinical Oncology (ASCO) from 1999 to 2013, it is evident to find that more emphasis was given on cancer biomarkers for breast cancer management in last 20 years [27][28][29].

Serum tumor markers are soluble molecules in blood that can be detected by monoclonal antibodies. They are released into the blood by tumor cells or by other cells in response to tumor cells. Serum markers have appealing features. Based on the circulatory nature of blood through almost every part of the human body, the measurements of blood components could reflect the dynamic evolution of the disease. Furthermore, obtaining blood samples is poorlyinvasive, so their levels can be easily repeated when required [30,31]. Currently, serum biomarkers used in clinic for breast cancer patients include certain members of mucin glycoproteins family (MUC-1) and Carcino Embryonic Antigen (CEA), which are recommended by ASCO and European Group on Tumor Markers Recommendations (EGTM). So in the following part, we will focus on these biomarkers in breast cancer.

MUC-1 and CEA

MUC-1 is involved in tumor genesis through complex pathways, e.g. promoting receptor tyrosine kinase signaling, constitutive activation of growth and survival pathways, and down regulation of stress-induced death pathways. Soluble form of MUC-1 family include cancer antigen CA 15-3, CA 27-29, CA 549, among which CA 15-3 and CA 27-29 are widely used in breast cancer. Because of their similar diagnostic sensitivities and specificities, the use of one MUC-1 marker is enough [33].

CEA is an oncofetal glycoprotein and is also widely used in breast cancer. CEA levels are less commonly elevated than the levels of MUC-1. However, CEA measurement can provide additional complementary information with MUC-1. Therefore, the combination of one MUC-1 marker and CEA is a good serum marker panel for monitoring patients with breast cancer [34].

Recommendations by ASCO and EGTM

For screening and diagnosis

The panel of MUC-1 (CA 15-3 or CA 27-29) and CEA is not recommended for screening and early diagnosis of breast cancer due to their low sensitivity [34,35]. The soluble form of MUC-1 was identified as a more specific marker with respect to CEA. However, MUC-1 disclosed low sensitivity and specificity for the early diagnosis of breast cancer, since its sensitivity is 10-15%, 20-25% and 30-35% for stages I, II, and III, respectively [36].

Therefore, lack of sensitivity for early-stage disease combined with a lack of specificity precludes the use of these two serum markers for the early diagnosis of breast cancer.

For early detection of recurrence

Two well-designed studies have shown that after primary breast cancer therapy, elevated levels of MUC-1 and CEA in patients are associated with distant recurrence. It can predict recurrence with an average of 5 to 6 months before other symptoms or test [37,38]. However, both ASCO and EGTM are vigilant to recommend them for detecting recurrence for several reasons. Evidence was insufficient to demonstrate whether early detection of metastases leads to good outcomes like disease-free survival, overall survival, quality of life or toxicity.

Furthermore, intensive screening may induce extra expenses and anxiety [35,34]. Therefore, it is not recommended by ASCO [29], while EGTM [35] recommended for the follow-up of asymptomatic women, this panel should be determined every 2-4 months during the initial 5 years after diagnosis, then every 6 months during the next 3 years and at yearly intervals thereafter.

For therapy monitoring

According to ASCO, present data are insufficient to recommend the use of MUC-1 or CEA alone for monitoring response to treatment. However, in the absence of readily measurable disease, increasing levels of this panel may be used to indicate treatment failure. It should be noted that a spurious rising level of MUC-1 and CEA was observed during the first 4-6 weeks of a new therapy [34].

Conclusions

Numerous serum biomarkers candidates in breast cancer have been reported, while only few of them have been approved by FDA. Approved serum markers are useful for determining recurrence, predicting therapeutic response, maintaining surveillance after primary surgery, and monitoring therapy in patients with advanced disease. However, at present, none of them are available for an early diagnosis and screening of breast cancer because of their low sensitivity and specificity.

In addition to screening novel biomarkers, validating candidate markers is also important.

Furthermore, in order to reduce the gap between discovery and validation in the biomarker development pipeline, several points need to keep in mind. 1) Biological samples should be carefully chosen based on well-established guidelines for both patients and matched controls.

2) Objectives should be clear and methods of reporting results should be critical [39]. 3)

Large scale and effective methods were needed to measure biomarkers in a high throughput manner. Compared with widely used single immunoassays, multiple immunoassays show several advantages including increased efficiency at a reduced expense, greater output per sample volume ratios and higher throughput. Among multiple immunoassays, planar immunoassays e.g. protein microarrays, are relatively inexpensive and would be simple and efficient to conduct large-scale population screening [40]. Therefore, in order to validate more candidate biomarkers, large-scale validation and a transition of methods is necessary. Only these requirements were satisfied, biomarker research can become more efficient and have the chance to translate into clinical evaluation.

Tissue biomarkers used in clinic

In this part, we will focus on already tissue biomarkers used in clinic for breast cancers.

These biomarkers include estrogen receptor (ER), progesterone receptor (PR), human epidermal receptor 2 (HER-2), urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1). Table 2 summarizes the detailed information of these tissue biomarkers.

Table 2 An introduction of clinic used tissue biomarkers

Estrogen receptor (ER) and progesterone receptor (PR)

ER and PR are transcriptional factors which mediate the actions of estrogens and progesterone, respectively. Both receptors are now known to exist in two different isoforms.

For ER, these forms are known as ER-α and ER-ß; for PR the two forms are known as PRA and PRB. It appears that only ER-α is critical for mammary gland development and tumorigenesis. Currently, the determination of ER and PR is obligatory in all breast cancer patients by EGTM [35] as well as ASCO [34] guidelines. Existing assays for PR do not discriminate between the two forms [30].

Three well-established assays exist for measuring hormone receptors, namely ligand binding, ELISA and immunohistochemistry (IHC). Only IHC is recommended by EGTM to measure ER and PR levels. Compared with other two methods, IHC assays can be carried out on small tumors, including core needle biopsy material [35]. It is recommended by ASCO that for the IHC test of ER and PR in breast cancer, the levels as low as 1% positive carcinoma cells are associated with clinically significant responses to endocrine therapy. They also noticed that up to 20% of IHC determinations of ER/PR testing worldwide may be inaccurate (false positive or false negative) due to variations in pre-analytical variables, thresholds for positivity, use of relatively insensitive antibodies, and criteria for interpretation. Therefore, they proposed that specimens should be handled in a uniform manner. Furthermore, factors should be well-considered including cold ischemia time, handling of specimens obtained remotely, fixation time in neutral buffered formalin, and selection of an optimal sample for testing, etc. [41].

ER and PR could provide prognostic value. Generally, ER-positive patients have a better outcome than ER-negative patients. However, this impact only last 4-5 years, after this period, the favorable prognostic value is lost. A further limitation of ER as a prognostic factor is that it is of little value in lymph node negative patients. Patients with tumors expressing PR also tend to have a better prognosis than those lacking this receptor [42,30]. Since both ER and PR are relatively weak prognostic factors in breast cancer, these factors should not be used alone, they could be combined with established prognostic factors in determining outcome based on EGTM recommendation [35].

In addition of prognostic value, ER and PR status is also considered to be very strong predictors of response to hormonal therapy in breast cancer patients. Both early and advanced disease, hormone receptor-positive patients have a significantly greater probability of responding to hormone therapy than patients lacking receptors. Therefore, it is recommended by EGTM [35] that patients with hormone receptor-positive tumors should be treated with some form of endocrine therapy like Tamoxifen, while receptor-negative patients should receive an alternate form of therapy. While according to the guidelines of ASCO [34], the benefits endocrine therapy for hormone receptor-positive patients with ductal carcinoma in situ (DCIS) is not sufficient.

HER-2

Approximately 15% of breast cancers over-express oncoprotein human epidermal factor receptor (HER2) [43]. There are three methods for identifying HER-2 status, including immunohistochemistry (IHC), which measures the HER-2 full-length oncoprotein;

fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH); both methods measures the number of HER-2/neu gene copies by a fluorescent system and an enzyme-based system, respectively. Among these three methods, only IHC assay was recommended by ASCO guidelines [44,30,45].

Nowadays, HER2 test must be performed to all newly diagnosed breast cancer patients with metastatic tissue biopsy samples available. Tumors with HER-2 over-expression are associated with higher grade and worse prognosis. However, its prognostic value is weak, therefore, it is not recommended to use alone for determining prognosis [34]. However, notice that in presence of mutated BRCA1 gene, HER2 over-expression is negative.

All patients with positive HER-2 receptors have to be treated by immunotherapy with Herceptin® (trastuzumab). Herceptin® is a humanized monoclonal antibody that binds with high affinity to the extracellular domain of HER-2, thereby blocking its role in signal transduction. Herceptin® is now widely used for the treatment of HER-2-positive tumor patients with breast cancer [30].

uPA and PAI-1

Urokinase plasminogen activator (uPA) is an extracellular matrix-degrading protease involved in cancer invasion and metastasis. uPA interacts with its plasminogen activator inhibitor-1 (PAI-1). Both PAI-1 and uPA promote tumor progression and metastasis [46].

Consistent with the causative role of uPA and PAI-1 in cancer dissemination, several retrospective and prospective studies have shown that uPA and PAI-1 are good prognostic and predictive biomarkers.

Prognostic and predictive value

One large scale study involving 8377 breast cancer patients demonstrated that uPA and PAI-1 are strong and independent prognostic markers in primary breast cancer. In both lymph node-positive and lymph node-negative patients, high uPA and PAI-1 values were independently associated with poor relapse free survival and poor overall survival [47].

In addition to provide prognostic value, uPA and PAI-1 could also provide predictive value to chemotherapy. A large-scale study including 556 patients was conducted by Jänicke F et al.

Results showed that for patients with high levels of uPA and PAI-1 (uPA > 3 ng/mg of protein and PAI-1 > 14 ng/mg of protein), those receiving chemotherapy displayed 43.8% lower probability of disease recurrence at 3 years than observation group [48]. This predictive impact of uPA/PAI-1 regarding an enhanced benefit from adjuvant chemotherapy has also been demonstrated in a retrospective multicenter analysis [49].

Recently, the study of 10-year long-term Chemotherapy for node-negative (N0) breast cancer patients follow-up confirmed the prognostic and predictive impact of uPA/PAI-1 in node-negative breast cancer [50]. Patients with low uPA/PAI-1 levels (uPA ≤ 3 ng/mg of protein and PAI-1 ≤ 14 ng/mg of protein) displayed 10-year recurrence-free survival of 87.1% compared to 77% in high uPA/PAI-1 level patients (p = 0.011). 10-year overall survival (OS) was significantly better in low uPA/PAI-1 level patients (89.8% vs. 79.1%; p = 0.01).

Moreover, in a randomized comparison in high uPA/PAI-1 level patients, adjuvant chemotherapy significantly reduced risk of recurrence (p = 0.019).

In conclusion, low levels of uPA and PAI-1 are associated with a sufficiently low risk of recurrence and chemotherapy will only contribute minimal additional benefit. On the contrary, high levels of uPA and PAI-1 signify high risk of recurrence and adjuvant chemotherapy provides substantial benefit for patients. uPA and PAI-1 are defined with the highest level-of-evidence (LOE-1) [46]. Therefore, they are considered to be the only biomarkers appropriate for the routine assessment of prognosis in patients with newly diagnosed node-negative breast cancer according to the American Society of Clinical Oncology (ASCO) guidelines [34] as well as European Group on Tumor Markers (EGTM) [35].

Detection methods

The above clinical trials employed ELISA to measure uPA and PAI-1 levels (normalized to the total protein content of the extract) extracted from breast tumor tissue. The cut-off value defined for uPA and PAI-1 is 3 ng/mg and 14 ng/mg, respectively. Currently ELISA is the only method which is recommended by ASCO to titrate uPA and PAI-1 [34]. The commercially available ELISA test (Femtelle ®) was developed by Sekisui Diagnostics. This kit has good quality insurance and is widely used in clinic. However, ELISA requires 100-300 mg of fresh or frozen breast cancer tissue. This is problematic for two reasons. Firstly, as formalin-fixed, paraffin-embedded (FFPE) tissue is the main source of patient material worldwide, therefore, requirement of fresh or frozen tissue preclude its usage [51]. Secondly, ELISA requires large quantity of tissues. Thomassen et al. [52] used ELISA to compare the levels of uPA and PAI-1 in 10-30 mg core biopsy specimens and 90-300 mg tumor tissue taken from the same specimens. Results showed that using the smaller tissue specimen correctly classified risk in 95% of the patients surveyed; however, correlation between individual uPA and PAI-1 levels in the small biopsy specimens versus the larger tissue samples was only 0.789 and 0.907, respectively. Therefore, the feasibility of measuring the level of uPA and PAI-1 in core needle biopsy breast cancer specimens needs to be confirmed.

The need for large quantity of tissue requires a surgical biopsy or vacuum-assisted core biopsy with an 8-gauge needle [53] and precludes the use of 14-gauge needle-core biopsies that are more common in clinical practice [54]. Indeed, requirement of large volume of fresh tissue becomes the main limitation of ELISA assays.

Considering the challenges faced by ELISA, several other assay formats were also used but no one method has proven to be a reliable substitute for the ELISA assay. For example, immunohistochemistry (IHC) can detect uPA and PAI-1 in frozen or formalin-fixed, paraffinembedded (FFPE) tissues. However, there was not complete agreement in protein levels, immunohistochemical scoring, and patient outcome between IHC and ELISA. Correlation rates for uPA and PAI-1 between expression levels determined by ELISA and IHC are 0.78 and 077, respectively [55]. Furthermore, IHC analysis yields semi-quantitative information and it is impossible to estimate protein expression level above the level causing maximum staining due to saturation. All these factors indicate that the two techniques are not directly interchangeable.

Analyzing mRNA levels of uPA and PAI-1 also seems appealing, because it requires very small amounts of tumor tissue and delivers quantitative estimation of the mRNA expression.

Moreover, RNA-based analysis was feasible from paraffin-embedded tissue samples.

However, quantification of mRNA has also proven to be an unreliable substitute for ELISA in assessing the level of uPA and PAI-1 [56][57][58][59]. Lamy et al. compared ELISA protein levels and mRNA levels of uPA and PAI-1 in tumor tissues. Results showed that the concordance of uPA and PAI-1 is only 84% and 70%, respectively [56]. In another study [57], correlations between uPA/PAI-1 mRNA and protein were found to be distinctly weaker or not significant;

no correlation between PAI-1 mRNA and protein level was also reported in [59,58]. The discrepancy between protein and mRNA levels was possibly caused by post-transcriptional regulation, which also demonstrated that measuring mRNA levels cannot always reflect the real expression of proteins.

Protein microarrays have several advantages compared with traditional ELISA as they yield high sensitivity and consume tiny volume sample [60]. Antibody microarray was used to detect the level of uPA and PAI-1 in extracts from breast cancer tissues. Higher sensitivity was achieved compared with ELISA. However, antibody microarray could not normalize the total protein content [61]. Preliminary studies have shown reverse-phase protein arrays (RPPAs) for uPA and PAI-1 promising [62,63]. For RPPA arrays, protein lysates are spotted onto glass slides and total protein can be measured by Sypro-Ruby protein stain. Moreover, protein extracted from formalin-fixed, paraffin-embedded primary breast cancer tissues could be used in RPPAs. As protein microarrays require tiny sample volume, they show high appealing potential for detecting uPA and PAI-1 in breast tumor tissue.

Autoantibodies (AAbs) -Diagnostic and prognostic values

As mentioned before, no-serum biomarkers were sufficiently effective for an early diagnosis and screening of breast cancer because of their low sensitivity and specificity. In recent years, numerous studies have demonstrated that serum autoantibodies could have high value as diagnostic and prognostic biomarkers for breast cancer. In the 1960s, Robert W.

Baldwin found that the immune system is involved in tumor development, during which autoantibodies (AAbs) were produced against intracellular proteins that are mutated, modified, or aberrantly expressed in tumor cells. These proteins are called tumor-associated antigens (TAAs) [64]. Several pathways can explain this regulation, including opsonization, enhancement of dendritic cell-mediated antigen presentation to T cells, recruitment of natural killer cells to perform antibody-dependent cell-mediated toxicity, generation of tumor antigen-specific CD8 + T cells and complement-dependent cytotoxicity. However, these mechanisms are not sufficient to explain how exactly these natural autoantibodies originate [65].

Although little is known about the origin of this immune response, an increasing number of articles have demonstrated that autoantibodies could be used for early diagnosis and prognosis of cancer [66]. AAbs show highly appealing properties compared with current serum biomarkers. Firstly, tumor-specific immune responses seem likely to occur before clinically apparent carcinoma. For example, Lubin et al. detected P53-specific antibodies almost 1.5 years before clinically relevant lung cancer was diagnosed [67]. Thus, the identification of AAbs could potentially be used for screening and early diagnosis of cancer. Secondly, during the anti-tumoral response, the immune system performs a very efficient biological amplification, leading to high concentration of AAbs and allowing indirect detection of very small amounts of tumor antigen. Thirdly, antibodies are highly stable in serum samples and are not subject to proteolysis like other polypeptides, making sample handling much easier.

They show a long lifetime (T 1/2 between 7 and 30 days, depending on the subclass of immunoglobulin) in blood and may persist as long as the corresponding autoantigen elicited specific humoral response. Finally, antibodies are biochemically well known molecules, and many reagents and techniques are available for their detection, simplifying assay development [68][69][70][71][72][73].

Thus, in recent years, numerous studies have screened various AAbs against TAAs and evaluated their diagnostic and prognostic value in breast cancer. Among them, antibodies against heat shock proteins (HSPs) received great interest. Therefore, an overview of published studies on AAbs against HSPs and other TAAs in breast cancer will be summarized in the following.

Heat shock proteins (HSPs) and AAbs against HSPs

HSPs were first discovered as a cohort of proteins that are powerfully induced by heat shock and other chemical and physical stresses in a wide range of species [74]. HSP are a group of highly conserved proteins and are classified into six families according to their molecular weight (MW): HSP110, HSP90, HSP70, HSPD1, DNAJ and small HSPs (range between 13-42 kDa) including HSPB1 and HSP10. Glucose-regulated proteins (GRPs) are a related class of proteins which are localized in endoplasmic reticulum. For example, HSPA5 belongs to HSP70 family member and shares 60% amino acid identity with HSP70; HSP90B1 belongs to HSP90 family and shares 50% amino acid identity with HSP90 [75]. HSPs function predominantly as molecular chaperones. They also restore cellular homeostasis by ensuring proper formation of new proteins, preserving existing complexes, restoring function of denatured proteins, and solubilizing protein aggregates [76].

Heat shock proteins (HSPs) are overexpressed in a wide range of human cancers. Elevated HSPs expression in malignant cells plays a key role in protecting cells against the spontaneous apoptosis associated with malignancy. Several HSPs are associated with the prognosis of specific cancer. For example, the expression of HSPB1 is associated with poor prognosis in gastric, liver, and prostate carcinoma, and osteosarcomas; overexpression of HSP70 is correlated with poor prognosis in breast, endometrial, uterine cervical, and bladder carcinomas [77]. Increased HSP expression may also predict the response to some anticancer treatments. HSPB1 and HSP70 were shown to be involved in resistance to chemotherapy in breast cancer; HSPB1 predicted a poor response to chemotherapy in leukemia patients, whereas HSP70 expression predicted a better response to chemotherapy in osteosarcomas.

Furthermore, implication of HSP in tumor progression and response to therapy has led to its successful targeting in therapy [77]. Thus, the detection of HSPs as biomarkers of cancer could aid early diagnosis, determining prognosis, prospectively predicting response or resistance to specific therapies, surveillance after primary surgery, and monitoring therapy in patients with advanced disease [77,76].

Elevated HSPs expression in tumor can also stimulate the immune system to produce anti-HSP AAbs. Conroy SE et al. [78] found that AAbs against HSP90 were detectable in a significant proportion (37%) of patients with breast cancer but not in normal individuals or patients with benign breast tumors. AAbs against HSP are also related with prognosis. Same authors [79] demonstrated that mortality rate from breast carcinoma was greater in women tested positive for AAbs against HSP90 than those tested negative. Thus, AAbs against HSPs could have diagnostic and also prognostic values in cancer. In the following, we firstly give an overview of published studies on detection of single AAb against HSP for discriminating breast cancer patients from healthy controls; then multi-AAbs panels are presented.

Evaluation of single anti-HSP AAbs

We identified 6 reports describing the use of individual AAbs against HSP for discriminating breast cancer patients from healthy controls. The result is presented in Table 3.

Enzyme linked immunosorbent assay (ELISA) was the most commonly used techniques.

Among all 6 studies, 5 researchers utilized ELISA and only one study used western blot (WB)

for the detection of anti-HSP antibody. From these data, it was observed that the frequency of a single anti-HSP antibody in breast cancer patients ranged from 8%-48%, whereas the frequency in healthy controls ranged from 0 -5% (anti-HSP70 antibodies were not included). antibodies was found to be correlated with the development of metastasis even in patients without axillary nodal involvement [78]. Then, in another study, they analyzed the correlation between anti-HSP90 AAb and mortality rate. They found that mortality rate from breast carcinoma was greater in women tested positive for AAbs against HSP90 than those tested negative [79]. This research group also identified anti-HSPB1 and anti-HSP70 antibodies in breast cancer, still using ELISA. One of the largest sample cohorts was evaluated with 579 samples tested for anti-HSPB1 Abs and 369 samples tested for anti-HSP70 Abs. In comparison, the number of healthy controls (53 healthy female) were limited. Results showed that there was no significant difference in the frequency of anti-HSP70 antibodies in patients with breast cancer and healthy control subjects. In contrast, anti-HSPB1 antibodies were detectable in over one-third of breast cancer patients (37.8%) while only in one healthy individual (P < 0.001). Furthermore, the presence of anti-HSPB1 antibodies appeared to show a significant correlation with improved survival, particularly beyond the first 5 years [80]. As tumor is a heterogeneous disease, the use of single AAb as diagnostic biomarker remains limited due to their low sensitivity and frequency ranging from 8% to 48%. Over the past 10 years several researches demonstrated that multi-AAbs panels could greatly improve cancer sensitivity detection while preserving reasonable high level of specificity.

Evaluation of AAbs panels including anti-HSPs antibody

Thanks to novel emerging proteomic techniques, like phage display, serologic identification of antigens by recombinant expression (SEREX), serological proteome analysis (SERPA), various biomarkers which could discriminate cancers from healthy controls were discovered. In the following, we present two studies on multi-AAbs panels including one or more anti-HSPs (as shown in Table 4). One study utilized ELISA and the other utilized protein microarray. As could be seen on Table 4, two panels of 5 antigens, including only HSPD1 as common antigen, were evaluated [84,32]. Although the size (143 breast cancer serum for [84] and 29 breast cancer serum for [32]), composition of patient cohorts (60 early-stage primary breast cancer and 82 carcinoma in situ (CIS) for [84], no information for [32]) and the methods used Compared with the low frequency of single AAbs in breast cancer, multi-AAbs panels could greatly improve the sensitivity of cancer detection. For example, the maximal frequency of antibodies against a single HSPD1 is 47.5% [81], whereas a panel of 5 autoantibodies (anti-HSPD1 antibodies included) can increase the sensitivity up to 82.5% [32]. Therefore, screening a panel of biomarkers was indispensable in order to have high sensitivity.

(

Emerging trends of anti-HSPs AAbs detection in breast cancer

We compiled and compared data on anti-HSP AAbs frequency obtained from the various studies described above. Results are presented in Table 5. The most commonly studied anti-HSP AAb in breast cancer was anti-HSPD1, which was reported in 3 separate investigations.

Some studies on AAbs panels didn't provide the frequency for each AAbs so the frequency is unavailable. We can only compare the frequency of autoantibodies against HSPD1 and HSP90 reported in two different studies. The frequency of anti-HSPD1 antibodies was quite reproducible (range from 31.8%-47.5%), however, almost 5 times discrepancy was obtained concerning anti-HSP90 antibodies (range from 8%-36.8%). Sample sizes maybe explain this variation. One study involved 125 breast cancer patients [78] while another study contained only 13 patients [83]. Sample size is an important factor and large-scale investigations are obligatory for verifying the real frequency of autoantibodies in serum. Anti-HSPB1, anti-HSP70 and anti-HSP90 AAbs were also studied. HC: healthy controls; ELISA: enzyme linked immunosorbent assay; WB: western blot; PM: protein microarray.

In addition to diagnostic marker, AAbs against HSPs are also associated with tumor prognosis and could be prognostic markers. For example, antibodies against HSP90 were associated with decreased survival [79] while anti-HSPB1 antibodies were associated with improved survival [80]. AAbs against HSPD1 were also found to be correlated with breast tumor stage. They are significantly higher in higher-grade ductal carcinoma in situ [82]. So the detection of autoantibodies against HSPs can not only discriminate the breast cancer patients from healthy controls, they could also provide prognostic values.

Autoantibodies against other TAAs than HSPs

In addition to AAbs against HSPs, various AAbs against other tumor antigens were also reported. Tumor antigens can, in general, roughly be divided into nine subgroups: decoy proteins, stem cell antigens, viral antigens, oncogenic proteins, over-expressed proteins, frameshift antigens, nucleic acid-specific antigens, ganglioside-like antigens, and cancertestis antigens [85]. In the following, we firstly present TAAs and related AAbs that have been reported to discriminate breast cancer patients from healthy donors; then TAAs to tailormade panels of AAbs will be presented. Finally, we will summarize the frequency of each AAbs and the challenges that we are facing.

TAAs and related AAbs in breast cancer

Numerous tumor specific AAbs have been identified in the serum of breast cancer patients (Table 6), but only a few of these AAbs (anti-P53, anti-Her2/neu, anti-MUC1) have been examined in detail as potential diagnostic or prognostic markers, while other anti-TAA AAbs were only reported once. More than half of studies were based on ELISA technique for screening and evaluation of AAbs frequency. In the following, we will give an overview of these reported AAbs in breast cancer patients. breast cancer patients and healthy controls (P < 0.0001) for the presence of anti-P53 AAbs [START_REF] Desmetz | Identification of a new panel of serum autoantibodies associated with the presence of in situ carcinoma of the breast in younger women[END_REF].

Moreover, three large-scale and multi-institutional studies were conducted to identify the frequency of anti-P53 AAbs in various type of cancers [START_REF] Cretich | Protein and peptide arrays: recent trends and new directions[END_REF][START_REF] Lacombe | Identification and validation of new autoantibodies for the diagnosis of DCIS and node negative early-stage breast cancers[END_REF][START_REF] Dong | Combined measurement of CA 15-3 with novel autoantibodies improves diagnostic accuracy for breast cancer[END_REF]. Altogether, 1345 cancer patients and 1520 healthy controls were involved. The average of the frequency of anti-P53

AAbs in cancers ranges from 15% to 24%; while its frequency in healthy controls is very low (0%-1.02%), especially in the results reported by Muller. M, et al [START_REF] Dong | Combined measurement of CA 15-3 with novel autoantibodies improves diagnostic accuracy for breast cancer[END_REF]. They found that control group (436 healthy controls) was all negative for anti-P53 antibodies, which means that this antibody response occurred only in patients with malignant disease. Therefore, anti-P53 antibodies showed high specificity for malignancy. However, low sensitivity (range from 15% to 24%) prohibits the use of single anti-P53 antibody test to screen patients. The combination of anti-P53 antibody test and the measurement of established conventional biomarkers can increase sensitivity. As reported in [START_REF] Dong | Combined measurement of CA 15-3 with novel autoantibodies improves diagnostic accuracy for breast cancer[END_REF], the occurrence of anti-P53 antibodies is independent of the elevation of conventional tumor markers (CEA and CA15-3) with significant increase of sensitivity (6-11%) and without reduction of specificity in most cancers. However, in these three large-scale studies, only limited breast cancer patients were involved (the maximal sample is 71) [START_REF] Cretich | Protein and peptide arrays: recent trends and new directions[END_REF][START_REF] Lacombe | Identification and validation of new autoantibodies for the diagnosis of DCIS and node negative early-stage breast cancers[END_REF][START_REF] Dong | Combined measurement of CA 15-3 with novel autoantibodies improves diagnostic accuracy for breast cancer[END_REF]. In addition to large-scale studies summarized above, several studies identified antibodies against P53 only in breast cancers [START_REF] Jonkheijm | Chemical strategies for generating protein biochips[END_REF][START_REF] Zhong | Autoantibodies as potential biomarkers for breast cancer[END_REF][START_REF] Mange | Serum autoantibody signature of ductal carcinoma in situ progression to invasive breast cancer[END_REF][START_REF] Ye | Mini-array of multiple tumor-associated antigens (TAAs) in the immunodiagnosis of breast cancer[END_REF]. The sample size ranges from 61 [START_REF] Jonkheijm | Chemical strategies for generating protein biochips[END_REF] to 158 [START_REF] Mange | Serum autoantibody signature of ductal carcinoma in situ progression to invasive breast cancer[END_REF] and the frequency of anti-P53 antibodies in breast cancer ranges from 7.9% [START_REF] Zhong | Autoantibodies as potential biomarkers for breast cancer[END_REF] to 35% [START_REF] Jonkheijm | Chemical strategies for generating protein biochips[END_REF]. Sample size has a great influence on the frequency of autoantibodies. Generally, high sample size leads to low frequency.

The association of circulating anti-P53 antibodies with clinic pathological features was also studied and conflicting results were obtained. Dalifard et al. found that there is no association between anti-P53 antibodies and prognostic factors [START_REF] Zhong | Autoantibodies as potential biomarkers for breast cancer[END_REF]. In contrast, several other studies found that autoantibodies against P53 were associated with shorter survival and advanced tumor stage [START_REF] Jonkheijm | Chemical strategies for generating protein biochips[END_REF][START_REF] Mange | Serum autoantibody signature of ductal carcinoma in situ progression to invasive breast cancer[END_REF][START_REF] Ye | Mini-array of multiple tumor-associated antigens (TAAs) in the immunodiagnosis of breast cancer[END_REF][START_REF] Dong | Combined measurement of CA 15-3 with novel autoantibodies improves diagnostic accuracy for breast cancer[END_REF]112]. Conflicting results were also observed concerning to the association of circulating anti-P53 antibodies with other features like hormone receptors. A. . Results indicated that anti-HER2 antibodies were significantly present in early-stage breast carcinoma patients compared to healthy controls [START_REF] Seurynck-Servoss | Surface chemistries for antibody microarrays[END_REF][START_REF] Girish | Site-specific immobilization of proteins in a microarray using inteinmediated protein splicing[END_REF] and in higher level than in advanced-stage breast carcinoma patients [START_REF] Watzke | Site-selective protein immobilization by Staudinger ligation[END_REF]. These studies suggest that the humoral immune response to HER2 may have a role in limiting breast carcinoma progression.

Mucin (MUC) is a family of high molecular weight glycoproteins expressed on cell surface. MUC1 has been found to be expressed abundantly in breast cancer [114]. Circulating immune complex containing MUC1 has been detected in breast carcinoma but did not correlate with the stage of disease [START_REF] Kwon | Selective immobilization of proteins onto solid supports through split-intein-mediated protein trans-splicing[END_REF]. Anti-MUC1 antibodies were detected more often among women with benign disease than in women with breast cancer. Indeed, a negative correlation was observed between the presence of anti-MUC1 antibodies and the development of disease. These results suggested that a natural humoral immune response to MUC1 should be protective against disease progression, while lack of immune reaction could be associated with unfavorable outcome [98]. antibodies were able to discriminate between breast cancer patients and healthy controls (p< 0.001) and between breast cancer patients and those with benign breast disease (p< 0.001).

1
The prevalence of SOX2 antibodies was associated with higher tumor grade and positive nodal status [110].

Although numerous tumor specific AAbs have been detected in the serum of breast cancer patients, their frequency varies greatly (from 5% to 80%). The great variation maybe resulted by various factors including tumor heterogeneity, sample sizes, sample quality and origin, healthy individuals, method and proteins used. Furthermore, some of these AAbs were also found in other cancers. For example, elevated anti-P53 antibodies were detected in head and neck carcinoma (32%), esophageal carcinoma (30%), colorectal carcinoma (24%), and carcinoma of the uterus (23%) [70]; elevated anti-HER2 antibodies were detected in prostate cancer [73]. All these results underscore the questionable utility of a single autoantibody evaluation for breast cancer diagnosis and prognosis.

The use of anti-TAA AAbs panels in breast cancer diagnosis and prognosis

AAbs against TAAs display high level of specificity while sensitivity is low because of the heterogeneity of tumor. Recently, many researchers found that multi-AAbs panels can greatly improve sensitivity while preserving a reasonable high level of specificity. In this part, we give an overview of distinct panels of AAbs which were used to discriminate breast cancer patients from healthy controls. Analysis of the literature is presented in Table 7. Overall, 12

AAbs panels were evaluated for breast cancer detection with panel size ranging from 2 to 10. large with respect to the number of cases evaluated, the prediction will be out of range because of noise. Generally, when the ratio of cases to markers is less than 10, it is considered of potential bias [71]. The ratio of cases to markers in both two studies was less than 10 (about 9 for [123] and 5 for [130]), so the high level of discrimination may be caused by overfitting bias. Low multi-markers panel can avoid overfitting bias; however, small marker panel will lead to low sensitivity, as obtained by [104]. Therefore, it is better to keep a reasonable ratio between multi-markers panel and the number of cases.

The AUC of three panels reached more than 0.81 and yielded good performance for discriminating breast cancer patients from healthy controls [127,131,129]. No common AAbs were observed in these three panels, which also indicated that the origin of tumor is an extremely complex process and various molecules were involved in tumor genesis. In [131],

they identified autoantibodies against hnRNPF and FTH1 and results showed that this panel had a low specificity. However, when CA15-3 was added to the panel, it reached a sensitivity of 89.3% and a specificity of 93.8%. From this we can see that in order to keep the specificity at a high level, different kind of markers including autoantibodies and antigens can be combined.

Four studies gave detailed information about cancer patients and evaluated the association between AAbs panel and cancer stage [126,84,128,129]. Three of them found that a panel of AAbs could be used in support to mammography for the diagnosis of early primary breast cancer, especially in younger women with high risk of breast cancer where mammography is known to have reduced sensitivity and specificity [84,129,126]. A panel composed of seven autoantibodies was found in 64% of primary breast cancer patients and 45% of patients with (Ductal carcinoma in situ) DCIS, at a specificity of 85% [126]. In [84], five AAbs panel could also significantly discriminated primary breast cancer (AUC=0. Compared with low sensitivity of individual anti-TAA AAb, the combination of anti-TAA AAbs panel can greatly increase the sensitivity while preserved a reasonable high level of specificity. AAbs panels' signature could be particularly appropriate in complement to mammography for women with high breast density. Moreover, they could provide clinical implications for the management of breast cancer patients, thus avoiding the over-treatment of low risk patients and dictating more intensive treatment of aggressive tumors. Two points need to be noted. Firstly, methods used to identify AAbs panel need to be improved. Indeed, ELISA is mostly employed but this technique is not adapted for large screening multiplex detection. New technologies such as protein microarrays should be more efficient to screen AAbs panels. Secondly, over-fitting bias should be avoided. Over-fitting in biomarkers analyses occurs when a multi-marker panel is inappropriately large with respect to the number of cases being evaluated. In this case, the prediction will be out of range because of noise.

Thus in order to avoid overfitting bias, size of AAbs panel and sample should be welldesigned.

Conclusion

Among all anti-TAAs studied, 9 received much interest (Table 8). Anti-P53 AAb was the most reported, following by HER2, MUC1, c-myc, Survivin, cyclin B1. As shown in Table 8, a discrepancy across studies about the frequency of single anti-TAA AAb was observed. This discrepancy could be explained by various factors. 1) Study population varies greatly among studies. 2) Definition of the cutoff value is an important factor that decides the performance of the test; lower cutoff value result in higher sensitivity and lower specificity, and vice versa. Although little is known about the origin of the immune response against tumor, an increasing number of articles have demonstrated that autoantibodies directed against tumor antigens have great potential for early diagnosis and prognosis of cancer. In recent years, more and more anti-TAA autoantibodies have been reported. However, their real diagnostic value needs to be further confirmed by large scale investigation. Compared with individual anti-TAA autoantibody screening, anti-TAA autoantibodies panels can greatly improve the sensitivity because of heterogeneity of tumor. For example, the frequency of single anti-P53 antibody ranges from 7.9% to 35%, while the combined analysis of antibodies to a panel of TAAs can greatly increase the sensitivity. As reported in [123,124], the combined analysis of antibodies to P53, IMP1, p62, Koc, c-MYC, cyclin B1 and survivin increased both diagnostic specificity and sensitivity to 92% and 85% respectively.

Among all these autoantibodies reported, we chose to screen autoantibodies against heat shock proteins (HSPs) family in breast cancer serum. Among HSPs family, antibodies against several HSPs were repoted, e.g. antibodies against HSPB1, HSPD1 and HSP90 were overexpressed in breast cancers, while no reports for antibodies against other HSPs. Therefore, we want to profile antibodies against HSPs family in breast cancer serum and test the diagnostic and prognostic performance of this antibody panel. Moreover, we added anti-p53 antibody in the antibody panel because it is the most studied antibody in breast cancer.

The need of large scale and autoantibodies panels ask urgently the transition of techniques because traditional ELISA is time consuming and laborious for parallel multiple screening.

New screening methods like protein microarrays are more and more developed since last 30 years. These new techniques are capable of analyzing multiple samples in parallel and require tiny volume sample. So in the following, we will focus on protein microarray.

Protein microarrays

Microarray technology is a term that refers to the miniaturization of thousands of assays on one small plate. This concept was first introduced by Tse-Wen Chang in 1983 [132]. In the following decades, this concept was successfully transformed into the DNA microarray, a technology that determines mRNA expression levels of thousands of genes in parallel.

However, DNA microarray technology possesses some limitations because mRNA profiles do not always correlate with protein expression [133]. Moreover, proteins are the major driving force in almost all cellular processes. Therefore, protein microarrays were developed as a high-throughput tool to overcome the limitation of DNA microarrays and to provide a direct platform for protein function analysis.

A general scheme of typical protein array experiment is provided in Figure 4. Various proteins are printed on a solid support. After washing and blocking un-reacted surface sites, the array is incubated with a sample containing a variety of unrelated proteins. After incubation, the interaction can be detected by two methods: label-free methods (mass spectrometry, surface plasmon resonance, etc.) or label-dependent methods (fluorescence, chemiluminescence, etc.) [134]. Protein microarray provides a powerful platform for characterization of thousands of proteins in a highly parallel and high-throughput manner. It can be categorized into three major classes (Figure 5):

1) Analytical protein microarrays where antibodies or antigens are immobilized on the surface and used to detect proteins in sample by direct labeling or using secondary labeled antibody in sandwich assay format;

2) Functional protein microarrays where large number of purified proteins are immobilized on the surface for studying protein interactions (e.g. protein-protein, protein-lipid, proteinnucleic-acid and enzyme-substrate interactions);

3) Reverse-phase protein microarrays where cells, tissue or cell lysates are directly spotted on surface for the analysis of samples [135,136]. Furthermore, reduction of sample volume usage is another important factor that demonstrates the superiority of this technology compared to other techniques like traditional ELISA. This factor is extremely important for clinical research in which samples are specious and limited [60]. Despite the promising benefit, protein microarrays are still associated with numerous unsolved problems, mainly due to the complex nature of the proteins. So in the following, the challenges and limitations of proteins microarrays will be discussed.

Surface chemistry

Glass is typically preferred for optical sensors because of their transparency and low intrinsic fluorescence glass should be functionalized with different surface chemistries and this process is a crucial factor for the fabrication of protein microarray. An ideal surface should be compatible with diverse set of proteins while maintaining their integrity, native conformation, and biological function [141]. Unlike DNA, whose structure is uniform and exhibits a strong one-to-one interaction, the structure of proteins is much more complex and diverse, therefore, the requirements of surface for proteins' immobilization is more rigid. A good surface for proteins should satisfy the following requirements: (i) high binding capacity for target proteins and low binding capacity for un-target proteins; (ii) ability to retain activity of immobilized proteins;(iii) low variability between slides; (iv) high signal-to-noise ratios;

(v) long stability for printed proteins [142].

The immobilization strategy chosen to attach proteins to the surface can greatly determine the properties of protein microarrays. There are three main strategies: physical adsorption, covalent biding and affinity based binding. Physical adsorption of proteins occurs via van der Waals or ionic interactions and hydrogen bonds between proteins and the chemical functions on the surface [143][144][145]. This is the simplest immobilization strategy; however, it is not easily controlled and may result in high variability. Covalent binding requires the presence of reactive groups on the surface allowing reaction with functional groups of proteins [146][147][148].

Depending on the reactive groups targeting on both surface and proteins, this strategy can led to random or oriented immobilization of proteins. However, due to the attachment to the slide at multiple sites, covalent binding is more likely to lead to a loss of activity of printed proteins. Affinity based binding lead to uniformly oriented proteins on surfaces. However, it requires a pre-treatment of printed proteins [149][150][151]. Each strategy induces advantages and shortcomings, as reported in Table 9. In the following, we will focus on the performance of commercial surfaces.

Commercial surfaces for protein microarray

There are a number of slides with various surface chemistries commercially available. In the following, we will present several representative studies to compare the performance of these commercial slides. Results showed that poly-L-lysine and aldehyde surfaces have good signal-to-noise ratios and low inter-field coefficients of variation (less than 20%). For polyacrylamide-coated slides, they have lower detection limits and are more suitable for the detection of very low concentrations of antigen [153]. They also investigated the properties of surface in the context of antigen microarray. Human serum albumin (HSA) was printed on 8 surfaces and mean signal to spotted concentration ratio, LOD and coefficients of variation were evaluated.

Results showed that covalent binding on PEG-epoxy or dendrimer slides showed higher signal intensities compared with non-covalently binding surfaces like amine slides or poly-L-Lysine slides [154]. These researches compared various surfaces and some of them, e.g. epoxy-silane, poly-L-lysine and aldehyde surface showed good property, however, limited proteins were evaluated. Considering the complexity and diversity of structures, these surfaces may be not applicable for other proteins.

More proteins were involved in recent studies. For example, Shannon L. Seurynck-Servoss et al. evaluate the performance of 23 different antibodies on 16 commercially available slides (major slide surfaces include aminosilane-coated slides, epoxysilane-coated slides, Full Moon slides, aldehyde silane slides, Poly-L-Lysine). Each antibody was spotted with 8 replications in the same experiment and 3 replicate experiments were conducted on separate days. They compared the different slide types based on spot size and morphology, slide noise, spot background, lower limit of detection and reproducibility. Results showed that the properties of the slide surface affect the activity of immobilized antibodies and the quality of data produced. Three dimensional slide surfaces tend to have higher background than twodimensional surfaces, likely due to an inability to efficiently wash and/or block the surface.

Furthermore, non-covalent chemistries for antibody immobilization work nearly as well as covalent ones [155]. This approach provides a rigorous and quantitative system for comparing the performance of commercial slide types.

In addition to antibody microarray, study on antigen microarray was also conducted. In order to identify the subtle differences of autoantibodies in serum samples, Balboni et al.

optimized the surface conditions for their autoantigens microarray platform. Firstly, they spotted 10 autoantigens on 22 commercially available slide surfaces and evaluated overall background, uniformity, streaking, and smearing of features. Among all 22 slides, 10 slides were considered potentially suitable for printing autoantigen microarrays based on visual inspection. Secondly, these 10 surfaces were tested to confirm their suitability for autoantigen immobilization. For this round of screening, 6 antigens were spotted at different concentrations with eight replications. Considering the intra-slide and inter-slide CVs (less than 30%) and spotting smearing and streaking, FAST, poly-L-lysine and SuperEpoxy slides were chosen. However, the subsequent study found that many other antigens did not spot well on poly-L-lysine and SuperEpoxy slides, resulting to background streaking and smearing.

Therefore, these two surfaces were not included for the following serum detection. Thirdly, they chose FAST and two additional surfaces (SuperEpoxy2 and PATH®protein microarray slides). Approximately 50 antigens were printed with 8 replications on each slide. Results

showed that after optimizing the major variables in autoantigen microarray platform, the variance within and among microarrays are low enough to allow detection of subtle differences within a patient over time and among patients [156]. The results are different from Angenendt et al., which found that poly-L-lysine slides performed better than FAST slides.

The discrepancy maybe caused by difference in spotting type and spotted antigen, among which Angenendt printed HSA with solid pins while Balboni printed 50 antigens (HSA was not included) with quill pins. Considering that they didn't analyze same antigen-antibody interactions, it is difficult to directly compare the results obtained by these two laboratories.

Furthermore, it also indicated that the performance of surface slides was quite variable depending on antigen type.

Up to date, it is not possible to compare studies from different laboratories due to differences in experimental protocols and proteins used. However, one point is evident: no unique surface is suitable for all proteins' immobilization considering the complexity of protein structure. Each protein performs differently on each type of surface; therefore, it is needed to select optimal microarray coating based on experimental requirements.

Commercial protein microarray

Nowadays, a number of companies have developed commercial protein microarrays to detect and analyze proteins in human samples like serum, urine, tissue, etc. The commercialization of reagents and kits has undoubtedly contributed much to modern research, particularly in biological and clinical disciplines. It has enabled faster and higher-throughput experimental protocols, promoted higher uniformity and consistency between independent labs and helped to develop technologies and methodologies that would otherwise be inaccessible to individual labs. So in the following, several kinds of commercial protein microarrays that have been developed by big companies will be presented. Then their limitations and challenges will be discussed. Table 10 summarized representative commercial protein microarrays fabricated by several companies. . Results showed that all three kits differed from each other for different analytes and there was no clear pattern for any analyte studied. They concluded that results obtained from different systems cannot be combined and suggested that the dynamic range of the assay, sensitivity of the assay, cost of equipment, cost of consumables, ease of use and ease of data analysis need to be considered when choosing a system for use [157]. A more recent study was conducted by Gendie E. However, no publications were reported on this product.

Allergen testing microarray

Assessing allergen resources is of great importance for humans and currently lots of Technology Assessment. Therefore, the information provided by the manufacturer about the assay characteristics may not be adequate, and the analytical performance of the assay may not be fit for purpose [195]. Therefore, although commercially available kits might initially be viewed as a step forward by biomarker and proteomics researchers, the users of these kits are advised to proceed with great caution for several reasons.

companies
Firstly, commercial protein microarray generally lack of replications, as shown in Table 10.

The replication range from 2 to 4, which is not enough and reduce the feasibility of obtained results. Secondly, the maximal analysis of sample on each slide is 16; some slides can only analyze one sample, which reduces their functional utility and raises the actual cost per assay of interest. Thirdly, surface chemistry is also needed to be mentioned. As shown in Table 10, several commercial protein microarrays use nitrocellulose as surface. However, nitrocellulose induces some drawbacks such as considerable background auto-fluorescence in the visible spectrum using laser excitation [196]. Moreover, surface chemistry plays an important role in the performance of protein microarray, while for commercial protein microarrays, all proteins were printed on the same surface regardless of their high complexity and diversity. Therefore, future efforts should be made on using different surfaces for each targeted proteins and combining different surfaces on the same support.

All these limitations lead to the possibility of seeking custom array design or home-made printing proteins, which allows specific customization to research needs and solves the limitations. For example, we could choose printing proteins of interest instead of all proteins, thus reducing the cost; we could also increase replications of printed proteins for increasing the credibility of results obtained; furthermore, we could use different surfaces and increase number of sub-arrays on each slide.

Optimization of assay conditions

In addition to surface chemistry, a plethora of factors also influences microarray assay performances, such as the composition of the spotting buffer (additives and pH) [197][198][199]152], humidity during spotting [200], concentration of immobilized proteins, incubation time and drying of spotting solution [201], composition of blocking buffer and time of blocking [202,203], buffer used for sample dilution, sample incubation time and so on [204]. A major challenge to multiplex sandwich assay development on microarray is the optimization of the assays and identifying critical parameters and optimal level for each of them.

Storage conditions of protein microarray

The storage of protein microarray is an important and necessary process for their development in routine use. Two main types of slides have to be considered for the storage:

non-printed slides and printed protein microarrays. Various studies were reported on these two aspects.

For non-printed slides, the ability to store chemically functionalized slides prior to spotting is an important factor, since microarray technology allows mass production of slides and their subsequent continuous consumption in experimentation. Different surfaces behave differently under storage. Kusnezow compared the storage conditions of non-printed slides functionalized with (3-aminopropyl) trimethoxy silane (APTES) with cross-linker, and (3glycidoxypropyl) trimethoxy silane (GPTS). These two types of surfaces were stored in two conditions: with or without argon. Then their performances were evaluated after 2 weeks, 1 month, 2 months and 4 months by printing 2 antibodies. Results showed that cross-linkermodified APTES slides slightly improved their performance following 2 months of storage without argon while no such effect could been seen when the slides were stored in argon atmosphere. In contrast, GPTS slides showed strong increase in signal intensities after two months of storage under argon. Without argon, less increase in signal intensities was observed. Furthermore, the performance of non-printed slides was greatly dependent on analyzed antibody. Among 2 printed antibodies, one is more affected by storage conditions The conditions are more complex for the storage of printed protein microarray. enzyme-substrate pairs to optimize storage conditions. Results showed that unlike printed antibodies, enzymes were more sensitive to storage conditions. Printed β-Gal was completely inactive after 12 days of storage at 4°C and β-lac also lost significant amount of activity within this period. However storage of immobilized enzymes in 50% glycerol at -20°C retained their activity for up to 30 days.

In conclusion, storage efficiency of protein microarray depends greatly on surface chemistry as well as printed proteins. For printed protein microarray, the immobilization strategy between surface and proteins has great influence on the conditions of storage.

Generally, immobilized proteins through covalent binding will not diffuse into the solution as may be possible with proteins just adsorbed onto the surface. Therefore, protein immobilization through physical adsorption favors dry storage conditions, e g. nitrogen or argon; in contrast, covalent binding favors storage in aqueous solutions containing glycerol, trehalose, polyvinyl alcohol addition (PVA). However, due to the diversity of proteins, it is needed to test each protein on each surface to select the best storage conditions based on experimental requirements.

Aims of the thesis

The goal of this research work concerns the development of customized multiplex protein microarrays to screen breast cancer biomarker panels for diagnosis, early detection of recurrence or therapy monitoring.

For this, three objectives have been defined: folding and maturation [1,2]. HSPs are over-expressed in a wide range of human cancers leading to the production of autoantibodies against HSPs by the immune system [3,4].

Indeed, elevated levels of anti-HSP autoantibodies (e.g. autoantibodies against HSPB1, HSPD1, HSP70 and HSP90) were found in breast cancer serum. Moreover, some of them (e.g. autoantibodies against HSPB1 and HSP90) were shown to be associated with tumor metastasis [5][6][7][8][9][10][11]. Therefore, the screening of antibodies against HSPs could provide information about tumor stage, development of metastasis, treatment efficiency for breast cancer patients. However, Enzyme-Linked Immunosorbent Assay (ELISA), the most commonly used format reported in the literature to screen interactions between antibodies and antigens, is expensive for multiplex analysis and consumes large amounts of biological products. In contrast, protein microarray could provide high throughput data by consuming only minute sample amounts [12,13]. However efficient multiplex analysis still remains challenging due to biomarkers variability.

In this study, our purpose is to develop customized antigen microarray to detect anti-HSPs autoantibodies in breast cancer serum. Seven proteins belonging to HSP family (HSPB1, HSPD1, HSP70, HSP90, HSP110, HSPA5 and HSP90B1) and P53 were selected as antigens.

This choice was driven in collaboration with the Institute of Cancer Research of Montpellier (IRCM). One of the key parameters that influence the performance of protein microarray is surface chemistry [14,15]. In previous work, we have developed and characterized 6 surface chemistries for protein immobilization including carboxylic (COOH), N-hydroxy succinimide (NHS), chitosan, amine (APDMES), maleic anhydride (MAMVE) and carboxymethyl dextran (CMD) functionalized surfaces. We have shown that the extent of the interactions of immobilized antibodies with their antigens (and consequently the performance of protein microarray) was surface and protein-dependent [16]. Other parameters affect the performances of protein microarray such as pH of spotting buffer and concentration of spotted proteins [17]. Thus, firstly various immobilization conditions for each antigen were screened on the 6 different surface chemistries in order to determine optimal conditions allowing retaining biological activity. Then in these conditions, the presence of anti-HSP autoantibodies was evaluated in 50 serum from breast cancer patients and 26 serum from healthy donors.

Materials and methods

Materials

Borosilicate flat glass slides were purchased from Schott. All chemicals were of reagent grade or highest available commercial-grade quality and used as received unless otherwise stated [16][17].

Serum samples

Surface functionalization of glass slides

Microwells were generated on the surface of flat glass slides by photolithography and wet etching on the basis of previous work in our group [29]. There are 40 microwells on each slide. These microwells are homogenous with 3mm side length, 60 ± 1 µm depth, as well as 4.5 mm spacing between each well. The details of surface functionalization of glass slides are reported in [16][17]. Briefly, flat and microstructured glass slides were functionalized with 6 different chemistries: (Figure 1

Multiplex immunoassays on microstructured protein microarray

HSPB1, HSPD1, HSP70, HSP90, HSPA5, HSP90B1, HSP110 and P53 were spotted at their optimal concentration (sciFLEX-ARRAYER S3, Scienion, Germany) into microwells of COOH and chitosan functionalized glass slides, as indicated in Figure 3. On COOH surface, all proteins were spotted in acetate buffer (pH 4.5); on chitosan surface, carbonate buffer (pH 9.6) was used as spotting buffer; each protein was spotted in 5 replications. Buffer solution and streptavidin-F555 were spotted as negative and quality controls, respectively. After spotting, proteins were allowed to react with functionalized surfaces under saturated water vapors overnight at 4°C. Then slides were washed sequentially for 2 × 5 min with PBS, for 5 min with PBS-T (0.1%), and blocked with 10% BSA/PBS-T 0.1% solution 2h at R.T. to limit unspecific adsorption. Then slides were washed for 3 × 5 min with PBS-T 0.1% and dried by centrifugation 3min at 1300rpm. Micro-wells tested with purified biotin-labeled antibodies (biotin-labeled antibodies against HSPB1, HSPD1, HSP70 and P53) were incubated with streptavidin-F555 (0.01 mg/mL diluted in 1% BSA/PBS); microwells tested with purified unlabeled antibodies (antibodies against HSP90, HSPA5, HSP90B1 and HSP110) were incubated with Cy3-labeled goat antimouse IgG (0.01 µM in 1% BSA/PBS-T 0.1%); microwells tested with buffer were incubated with buffer; microwells tested with serum were incubated with Cy3-labeled goat anti-human IgG (0.01 µM in 1% BSA/PBS-T 0.1%). All incubations were left to react for 1h at R.T. in saturated water vapors, and then slides were washed for 3 × 5 min with PBS-T 0.1%, 10 seconds in DI water and dried.

Fluorescence scanning

After drying, slides were scanned with the Microarray scanner GenePix 4100A at wavelengths of 532 nm with the same photomultiplier tube (PMT) gain (PMT=600). Data mining was accomplished with GenePix 4100A software package (Axon Instruments). The fluorescence signal obtained for each antigen-antibody system was determined as the average of the median fluorescence signal of several replicates. The signal-to-noise ratio (SNR) was calculated as the ratio between the fluorescence signal of each antigen-antibody system and the fluorescence signal of buffer spots.

Data analysis

The sero-reactivities of breast cancer serum and healthy donor serum to immobilized antigens were compared using the Mann-Whitney test. Differences were considered statistically significant when P < 0.05. Individual and combined autoantibody performances were based on receiver operating characteristic (ROC) curves. The generalized ROC criterion finds the best linear combination (virtual marker) of tumor markers such as the area under the ROC curve (AUC) is maximized. Statistical analyses were performed using mROC [18].

Results

Optimization of tumor antigen microarray conditions

For the optimization of process, various concentrations of spotted tumor antigens HSPB1, HSPD1, HSP70, P53, HSP90, HSPA5, HSP90B1 and HSP110 were tested on the 6 different chemically functionalized glass slides. The biological activity of immobilized tumor antigens was evaluated by measuring the extent of their interaction with their corresponding antibodies.

Figure 4 shows fluorescent images corresponding to HSP110/anti-HSP110 antibody interaction on the 6 surface chemistries. For each image, the top line corresponds to buffer spots and the following 4 lines correspond to 4 concentrations of spotted HSP110 (0.005 mg/mL, 0.01 mg/mL, 0.05 mg/mL, and 0.1 mg/mL). At low concentrations (0.005mg/mL, 0.01mg/mL), biological recognition between immobilized HSP110 and anti-HSP110 antibody was merely (not) detected, indicating that the surface density of HSP110 was too low for the efficient detection of the anti-HSP110 antibody. Above 0.01 mg/mL, the fluorescence signal increased with increasing concentrations of spotted HSP110 on all surface chemistries.

Moreover, we can notice that fluorescent signal of buffer spots was not detectable (< 400 a.u.)

indicating low non-specific adsorption. Among the 6 surface chemistries, two were composed of amino groups as functional groups (APDMES and chitosan), two were activated with NHS groups (NHS and CMD), one was composed of amine-reactive group (MAMVE), and one presented carboxylic groups (COOH). For COOH, chitosan and APDMES surfaces, the binding between surface and protein was achieved through physical adsorption; while on NHS, NHS-activated CMD and MAMVE surfaces, protein immobilization was achieved by covalent linking. Physical adsorption is the easiest way for protein immobilization and can exhibit excellent binding capacities. However, owing to random interaction, non-covalent binding does not allow to control the amount and orientation of immobilized proteins. Thus efficiency, accuracy and reproducibility of the immobilization process may be variable. Furthermore, the background level is usually higher due to a higher non-specific protein adsorption [19,20]. Compared to physical adsorption, covalent binding represents a more robust approach. It requires the presence of reactive groups on the support which can react with probe molecules. Although covalent binding enhances the stability of immobilized proteins, it can lead to partial loss of biological activity of proteins [21,22]. Both binding strategies have advantages and shortcomings. Furthermore, the immobilization efficiency is closely dependent on probe proteins. There is no unique surface chemistry which is suitable for all proteins immobilization due to their highly diverse and complex structures. Our results showed that COOH and chitosan surfaces perform better for the detection of anti-HSP antibodies compared to other surfaces. This indicated that HSPs and P53 better retained their recognition activity towards anti-HSPs and anti-P53 antibodies when they were immobilized through physical adsorption. However, COOH and chitosan surfaces involved different kind of interactions as COOH is a silane monolayer with carboxylic groups, and chitosan is a polymer with amino groups. These opposite characteristics underlined the complexity of protein structure and interactions. Therefore, COOH and chitosan surfaces were selected for further evaluation of breast cancer serum (Table 2).

Next, we estimated the best immobilization concentration needed for each HSP and P53 on COOH and chitosan surface for efficient anti-HSP and anti-P53 antibodies detection. In order to reduce reagent consumption, and thus cost of the assay, we decided not to use the highest spotting concentration (0.1 mg/mL) except if fluorescence signal (SNR) is too low. From the variation of SNR with spotting concentration (Figure 5), the immobilization concentration for each HSP chosen is reported on Table 2. These concentrations allowed sensitive detection of anti-HSP and anti-P53 antibodies with high SNR values. Incubation with buffer solution allowed checking the quality of the surface chemistry, protein immobilization and non-specific adsorption (Figure 7b). Only the 2 spots of streptavidin-F555 were observed. Incubation with purified anti-HSP antibody allowed checking the biological activity of immobilized HSP and the cross-reactivity with the other antigen probes (Figure 7c). We can see that anti-HSP110 antibodies were well recognized by immobilized HSP110 on COOH surface and there was no cross-reactivity.

Incubation with breast cancer serum gaves the content in anti-HSP antibodies present in the tested sample (Figure 7d). In this example, breast cancer serum tested was only positive for the presence of anti-HSP110 antibody. Furthermore, the fluorescence signal is weaker compared to microwell incubated with purified antibody (Figure 7c). This was due to two reasons: on one hand, serum contained many proteins which could disturb the specific binding between antibody and its targeted antigens; on the other hand, the concentration of antibody in serum was probably lower than purified antibody. In (Figure 7c), the concentration of purified anti-HSP110 antibody was 0.1 µM, according to the results of optimization, lower concentration of Cy3-labeled goat anti-mouse IgG (0.01 µM) was enough to detect purified anti-HSP110 antibody (chapter 4). As the concentration of anti-HSP110 antibody in serum was lower than purified antibody, therefore, 0.01 µM Cy3-labeled goat anti-human IgG was enough to test anti-HSP110 antibody in serum.

Incubation with healthy donor serum (Figure 7e) gaves the level of anti-HSPs antibodies below which the discrimination between healthy serum and cancer serum couldn't be done.

Thus, fluorescence intensities (SNR) of each tested serum (50 breast cancers (BC) and 26 healthy controls (HC)) were analyzed for the positivity to each spotted antigen (HSPs and P53) on each surface chemistry (COOH and chitosan surfaces). Results are presented in Figure 8 where each point corresponds to one serum sample. For each antigen probe, BC and HC groups were compared using statistic test (Mann-Whitney test) in order to determine if the 2 groups are statistically different for the presence of target antibody, with a probability higher than 95% confidence (p value < 0.05). On COOH surface (Figure 8a), the presence of autoantibodies against HSPD1, HSP70, HSP90 and HSP90B1 was significantly different between breast cancer patients and healthy controls (p<0.05). On chitosan surface (Figure 8b), the presence of autoantibodies against HSPB1 and HSPA5 was significantly different between the two groups of serum. No significant difference was obtained for the presence of autoantibodies against P53 and HSP110 between these two groups on both COOH and chitosan surfaces.

Optimal surface chemistries for the detection of anti-HSPD1, anti-HSPB1 and anti-HSP90B1 autoantibodies in serum were in accordance with conclusions drawn in Table 2.

Indeed, COOH surface displayed the highest signal for detecting purified antibodies against HSPD1 and HSP90B1. Similarly, the detection of the anti-HSPD1 and anti-HSP90B1 autoantibodies in serum was significantly different between breast cancer patients and healthy controls on COOH surface. The same tendency was also observed for the detection of anti-HSPB1 antibody on chitosan surface. On the contrary, the detection of autoantibodies against HSP90 and HSPA5 was not consistent with the results obtained with purified antibodies. This inconsistence could be due to variable affinity between spotted antigen and purified antibody compared to antibodies present in serum. Moreover the complex nature of serum composition as opposed to purified model solution could induce changes in antigen/antibody interaction. Taking into account these results, we considered each best surface chemistry/antigen probe couple which could discriminate between breast cancer serum and healthy control serum for the presence of autoantibody, to construct the receiver operating characteristic (ROC) curve (Figure 9). The method of constructing ROC curve is shown in annex. For the detection of anti-HSP90 and anti-HSPA5 autoantibodies, we chose best surface chemistry as defined in Table 2. As individual markers, these autoantibodies showed relatively low sensitivity (<70%) and specificity (<70%) for discriminating breast cancer patients from healthy controls on both COOH and chitosan surfaces. Indeed, calculation of the area under ROC curve (AUC) for each tumor antigen/anti-tumor antigen antibody system (Table 3) indicated that it ranged from 0.581 to 0.732. Biomarkers displaying AUC≤0.75 are not clinically useful whereas biomarkers displaying AUC≥0.97 has very high clinical value [23]. However, the combination of autoantibodies panel (black line in Figure 9) displayed significantly improved performance for discriminating breast cancer patients from healthy controls. It achieved a sensitivity of 86% and a specificity of 100%. Expectedly, at higher sensitivity values, the overall specificity of the panel dropped to 96% (at 90% sensitivity) and to 70% (100% sensitivity). Moreover, AUC of 0.978 was obtained with the combination of autoantibodies panel which is nearly to best diagnosis test.

Figure 9 Receiver operating characteristic (ROC) curve analysis of individual auto-antibody and combination of 7 auto-antibodies to discriminate breast cancer patients from healthy controls. The detection of auto-antibodies against HSPD1, HSP70, HSP90 and HSP90B1 was obtained on COOH surface; the detection of antibody against HSPB1, P53 and HSPA5 was obtained on chitosan surface

Association of anti-HSPs autoantibodies profile with tumor stage

We also assessed the correlation between the presences of autoantibodies against HSPs and P53 with tumor stage (Figure 10).

On COOH surface (Figure 10a), only the presence of autoantibodies against HSP70 was significantly different between tumor stage I and stage III (p<0.05) indicating that these autoantibodies could be use as prognosis biomarker. As well, the presence of autoantibodies against P53 was significantly different on chitosan surface (Figure 10b) between Stage I and stage III, and thus could be defined as prognosis biomarker. For the other auto antibodies tested, their distribution was not significantly different between all stages of breast cancer.

Thus, the presence of anti-HSP antibodies seemed to be independent of tumor stage, which suggested that the detection of autoantibodies against HSPs could be useful for the diagnosis of early stage of breast cancer. However, further study with large scale population is needed to confirm this speculation.

Discussion and Conclusions

In this study, we have demonstrated that customized protein microarrays could be powerful tools for the rapid screening of cancer biomarkers. The performance of protein microarray is influenced by many parameters such as surface chemistry, spotting buffer and protein concentration. 6 different surface chemistries were evaluated for the immobilization of 7

proteins belonging to the heat shock protein family and one oncoprotein, P53 in various conditions. To retain biological activity of the immobilized antigen protein, the best conditions (surface chemistry and the concentration of immobilized antigens) were determined to implement miniaturized immunoassays. Two surface chemistries (COOH and chitosan) were selected and used to detect anti-HSP and anti-P53 autoantibodies in 50 breast cancer serum and 26 healthy donors' serum. The frequency of autoantibodies against HSPs reported in literatures was compared to our results (Table 4). As discussed above, these differences could be explained by the size and origin of the cohort: among researches, cohorts vary from 13 to 579 patients [6,10]. The more extent is the cohort, the more reliable should be the results. The definition of the cut-off value could also lead to variable results. Indeed, cut-off value is defined as a value greater than the mean of the healthy individuals plus two standard deviations (S.D.) [9], or mean plus three standard deviations [7]. Generally, lower cut-off value results in higher sensitivity and lower specificity, and vice versa. Another parameter which could affect frequency of autoantibodies is the tumor stage: some studies [6,8,9] provide detailed characteristics about breast cancer patients, like histologic stage, lymph node status, etc; while others didn't provide any information [10,24]. However, as the frequency of some anti-HSPs autoantibodies could vary with tumor stage, such as anti-HSP70, the choice of patients' serum and qualification are very important for data analysis and conclusion. At least, we could notice that the study methods could influence results obtained. As ELISA and western-blot analysis are gold standard methods for evaluation of immune response, they are mostly chosen by researches. However, with high throughput analysis, they became awful to use and expensive. Thus antigen microarrays could be an advantageous alternative, but process need to be standardized in order to be used in clinical trials.

Furthermore, due to the heterogeneity of tumor and individual immune response, the detection of one single autoantibody didn't allow to significantly discriminate breast cancer serum from healthy serum, whereas combining 7 autoantibodies (autoantibodies against HSPB1, HSPD1, HSP70, P53, HSP90, HSPA5 and HSP90B1) increases the sensitivity of the detection to 86% and specificity to 100%. Thus our antigen microarray achieved good sensitivity as well as the highest specificity, which is comparable with other studies obtained through ELISA [25][26][27][28]. Moreover, compared to ELISA, our customized antigen microarray is capable of providing high throughput data by consuming smaller sample amounts.

In the future, larger cohort of healthy donors and breast cancer patients are needed to validate performances of our antigen microarray. Furthermore, we will also try to detect other antibodies against common reported antigens like HER2, MUC1. Thus, progress in technology associated to standardization effort should lead to an emerging and powerful tool for cancer diagnosis and prognosis in clinical assays.

Introduction

As reported in Chapter 1, Urokinase type plasminogen activator (uPA) and its main inhibitor plasminogen activator inhibitor 1 (PAI-1) are involved in many human cancers, including those of breast, prostate, lung, brain, ovary. They participate in a wide variety of physiologic and pathologic processes, e.g. tumor growth, invasion and metastasis, through their effect on angiogenesis and cell migration [1,2]. Various retrospective and prospective studies have shown that uPA and PAI-1 are good prognostic and predictive biomarkers in breast cancer. Low levels of uPA (≤3 ng/mg of protein) and PAI-1 (≤14 ng/mg of protein) are associated with low risk of recurrence and no benefit of chemotherapy for breast cancer patients. On the contrary, high levels of uPA and PAI-1 is correlated with high risk of recurrence and adjuvant chemotherapy provides substantial benefit for breast cancer patients [3][4][5][6]. Recently, these two biomarkers have been demonstrated having the highest level-ofevidence (LOE-1) for providing the prognostic and predictive value for node-negative breast cancer patients [7].

Currently ELISA is the only method which is recommended by ASCO to detect the concentration of uPA and PAI-1 in protein extraction from fresh or frozen breast tumor tissue.

The commercially available ELISA test (Femtelle®) was developed by Sekisui Diagnostics.

This kit has a high sensitivity. The lower limit of detection (LOD) of the assay for uPA and PAI-1 are 25pg/ml and 125pg/ml of sample respectively. However, it requires a minimum of 100-300 mg of fresh or frozen breast cancer tissue [8]. The need for large quantity of tissue requires surgical biopsy or vacuum-assisted core biopsy with an 8-gauge needle [9] and precludes the use of 14-gauge needle-core biopsies that are more common in clinical practice [10]. Indeed, requirement of large volume of fresh tissue becomes the main limitation of ELISA assays. Considering the challenges faced by ELISA, several other assay formats were also used including immunohistochemistry (IHC) [11] and analyzing mRNA levels [12][13][14][15].

However, none of them has been proven to be a reliable substitute for ELISA assay.

Protein microarrays have several advantages compared to traditional ELISA including high sensitivity and tiny volume sample consumption [16,17]. The aim of this study was the elaboration of sensitive antibody microarray to quantify uPA and PAI-1 in protein extracts from breast tumor tissues. Various parameters such as surface chemistry, pH of spotting buffer and concentration of immobilized antibodies were evaluated in order to optimize antibody microarray performances [18][19][20]. Then using the best conditions, the quantification of uPA and PAI-1 in protein extracts from breast tumor tissues was achieved and compared to classical ELISA using the Femtelle assay. In Femtelle kit, captured antibodies against uPA and PAI-1 had high affinity with uPA and PAI-1 respectively. However, the captured antibodies were unknown. So we tested several antibodies and chose those with highest performances for further experiment to measure the concentration of uPA and PAI-1 in breast tumor tissue extractions. The Femtelle assay was conducted in collaboration with the Research Cancer Institute of Montpellier (IRCM).

Experiments

Materials

Anti-uPA antibodies (mouse monoclonal) were obtained from Santa Cruz

Biological samples

All human samples were prospectively collected at Research Cancer Institute of Montpellier, France at the time of surgery after obtaining written informed consent. 50µl of cytosolic extracts were prepared from frozen tissue samples and stored at -80°C. All samples were quantified for uPA and PAI-1 using the Femtelle test.

Surface functionalization of microstructured glass slides

Microwells were generated on the surface of flat glass slides by photolithography and wet etching on the basis of previous work in our group [21]. Then, microstructured glass slides were functionalized with 6 different chemistries as referred in part 2.2.3. These microwells allowed testing different experimental conditions; furthermore, they allowed screening several patient sera on the same slide (one patient per microwell) when the best conditions are selected.

Design and Optimization of antibody microarray

Concentrations of spotted and detection antibodies

Anti-uPA and anti-PAI-1 antibodies were spotted at three different concentrations (0.33 µM, 0.67 µM and 2.5 µM) on the 6 chemically functionalized microstructured glass slides according to Figure 1. Anti-uPA antibody was spotted in the first three lines of microwells, and anti-PAI-1 antibody in the following three lines. PBS 1X was used as spotting buffer.

Each antibody concentration was spotted with 6 replications in each microwell. PBS 1X and streptavidin-F555 were spotted as negative and quality controls, respectively.

After spotting, antibodies were allowed to react with surfaces under saturated water vapors overnight at 4°C. Then slides were washed sequentially for 2 × 5 min with PBS, for 5 min with PBS-T (0.1%), and blocked with 10% BSA/PBS-T solution for 2h at room temperature (R.T.) to limit unspecific adsorption, then washed for 3 × 5 min with PBS-T and dried.

Slides were then incubated with 6 different concentrations of uPA (0 ng/ml, 0.2 ng/ml, 0.5 ng/ml, 1 ng/ml, 1.5 ng/ml and 2 ng/ml) and PAI-1 (0 ng/ml, 2 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml and 20 ng/ml) prepared from Femtelle kit, as shown in Figure 1. Each concentration was incubated in two microwells. Slides were left to react for 1h at R.T. in saturated water vapors, then washed for 3 × 5 min with PBS-T and dried. Then slides were incubated with non-diluted and diluted (1:2) biotinylated antibodies prepared from Femtelle kit as shown in Figure 1. The incubations were left to react for 1h at R.T. in saturated water vapors, and then slides were washed for 3 × 5 min with PBS-T and dried by centrifugation. Then slides were incubated with strep-F555 (0.01mg/ml diluted in 1% BSA/PBS). All incubations were left to react for 1h at R.T. in saturated water vapors, then slides were washed for 3 × 5 min with PBS-T, 10 seconds in DI water and dried.

Spotting buffer for anti-PAI-1 scFv immobilization

Three buffer solutions were tested for the immobilization of anti-PAI-1 scFv antibodies on COOH, NHS and chitosan surfaces: PBS 1X (pH=7.4), acetate buffer (pH=4.6) and carbonate buffer (pH=9.6). Anti-PAI-1 scFv antibody was spotted at 5 µM in all three buffers. The design of antibody microarray is shown in Figure 2. All incubations and washing steps were the same as previously described in part 3.2.4.1. Eight concentrations of PAI-1 were tested (incubation 1): 0, 0.5 ng/ml, 1 ng/ml, 2 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml and 20 ng/ml. Biotinylated anti-PAI-1 antibody was prepared from Femtelle kit at dilution 1:2.

Evaluation of the biological activity of antibodies against uPA and PAI-1 with ELISA

Among five antibodies studied, no fluorescence signal was detected for the immobilization of anti-PAI-1 antibody (from both Santa Cruz Biotech and Abcam) on 6 surface chemistries.

In order to figure out the reasons, we evaluated the biological activity of all antibodies by the standard method-ELISA. Also we compared them with the results obtained from Femtelle kit.

The protocol of Femtelle kit was done according to their guidelines. The protocol of ELISA is as follows:

1. Dilute the capture antibody to the appropriate concentration (20µg/ml) allowing sufficient volume for 50 µl per well.

2. Add the diluted capture antibody to the plate, cover and incubate overnight at room temperature (R.T.).

3. Remove the solution and wash the plate with 200 µl per well wash buffer (PBS 1X 0.1% Triton X-100) for 3 x 5 minutes.

4. Add 300 µl blocking buffer (1% BSA in PBS-T 0.1%) per well, cover the plate and incubate for 2 hours at R.T.

5.

Remove the blocking buffer and wash the plate with 200 µl per well wash buffer. 12. Add 100 µl substrate solution (from Femtelle kit) to the plate, cover and incubate for 20 minutes at RT, a blue color will develop.

13. Stop the reaction by adding 50 µl stop solution (0.5N H 2 SO 4 ), the solution color will turn yellow.

14. Measure the absorbance at 450 nm within 30 minutes.

Quantification of uPA from breast tumor tissue extracts on antibody microarrays

Anti-uPA (from Thermo Scientific) was spotted using PBS 1X buffer (pH=7.4), on 3 chemically functionalized microstructured glass slides (COOH, NHS and chitosan surfaces), as shown in Figure 3. Two spotting concentrations were used: 3 µM and 6.6 µM with 14 replications each. PBS1X buffer and streptavidin-F555 were spotted as negative and quality controls, respectively. Then all incubations and washing steps were the same as previously described in part 3.2.4.1.

On each slide for the first incubation, 6 microwells were incubated with uPA prepared from Femtelle kit at six different concentrations (0 ng/ml, 0.2 ng/ml, 0.5 ng/ml, 1 ng/ml, 1.5 ng/ml and 2 ng/ml) to obtain a standard curve; 2 microwells were incubated with PBS 1X (pH=7.4) buffer for negative controls; 16 microwells were incubated with non diluted breast tumor tissue extracts; 16 microwells were incubated with diluted breast tumor tissue extracts; for dilution, samples with relatively low uPA concentration (according to Femtelle kit) were diluted 2 times; while samples with relatively high concentration were diluted 5 times (Figure 3: 1 st incubation). Then slides were sequentially incubated with biotinylated antibody diluted 1:2 prepared from Femtelle kit and with strep-F555 (0.01mg/ml diluted in 1% BSA/PBS).

Fluorescence scanning and data analysis

After drying, slides were scanned with the Microarray scanner GenePix 4100A at wavelengths of 532 nm with the same photomultiplier tube (PMT) gain (PMT=600). Data mining was accomplished with GenePix 4100A software package (Axon Instruments). SNR was calculated as described in part 2.2.6. The threshold value (cut-off) for the determination of LOD (Limit of Detection) was calculated as followed: Cut off =Mean of median buffer spots + 3 SD, where SD represents standard deviation.

The standard curve was obtained by plotting the mean SNR value calculated for each uPA standard concentration.

Results and discussion

Optimization of antibody microarray conditions

The implementation of efficient antibody microarrays requires optimizing important parameters such as surface chemistry, concentration of spotting and detection antibody, and spotting buffer. Thus, for the detection and quantification of uPA and PAI-1, firstly the 6 surface chemistries developed in our lab were evaluated for the efficient immobilization of anti-uPA and anti-PAI-1 antibodies. The biological activity of immobilized antibodies was determined by the level of recognition with uPA or PAI-1. Secondly, various spotting concentration of the antibodies were tested on the best surface chemistries. Thirdly, concentration of the biotinylated antibodies was also optimized to obtain good detection signal. At last, 3 different spotting buffers were tested for the immobilization of anti-PAI-1 scFv antibody, the concentration of the other antibodies did not allow changing buffer. The influence of these factors will be presented as follows.

Influence of surface chemistry

Surface chemistry affected greatly the performance of antibody microarray. Among 6 surface chemistries tested, three of them (COOH, NHS and chitosan surface) showed good performances for the immobilization of anti-uPA antibody and anti-PAI-1 antibody scFv.

Anti-uPA antibody

Anti-uPA antibodies (from Santa Cruz Biotech) were spotted on the 6 different surface chemistries and its biological activity was evaluated and compared (Figure 4). Moreover, on NHS surface, the signal reached a plateau value when uPA concentration reached 1.5ng/ml. In contrast, the dynamic range of anti-uPA antibody on COOH and chitosan surfaces was much larger compared to NHS surface. According to these results, COOH, NHS and chitosan surfaces were chosen for immobilizing anti-uPA antibody from other company (Thermo Scientific) for further experiment.

Anti-PAI-1 antibody

Two anti-PAI-1 antibodies (from Santa Cruz Biotech and Abcam) were immobilized on the various surface chemistries and tested for their biological activity to recognize PAI-1.

However, none of them exhibited biological activity after surface immobilization as shown in Several reasons may lead to the failures of these two anti-PAI-1 antibodies. Firstly, surface chemistries tested could modify the structure of antibodies during immobilization process, thus resulting in the loss of biological activity. Secondly, these antibodies were not biologically active before immobilization. In order to figure out the reasons, we further tested the biological activity of these 2 antibodies by ELISA. Results will be presented in later.

Anti-PAI-1 scFv antibody

As 2 entire antibodies against PAI-1 didn't exhibit biological activity, a single-chain variable fragment (scFv) against PAI-1was evaluated for the recognition towards PAI-1. scFv consists of variable regions of heavy (VH) and light (VL) chains, which are joined together by a flexible peptide linker. scFv is the smallest unit of immunoglobulin molecule with antigenbinding activity [22]. Figure 6 presented performance of anti-PAI-1 scFv on the 6 surface chemistries. Only COOH surface allowed efficient immobilization of anti-PAI-1 scFv to sensitively detect PAI-1.

Figure 6 Biological activity of anti-PAI-1 scFv on COOH, NHS, chitosan, APDMES, CMD

and MAMVE surfaces; anti-PAI-1 scFv was spotted at 5µM.

ELISA to test the biological activity of antibodies against uPA and PAI-1

We have tested the biological activity of five antibodies by ELISA and compared the results with those obtained from Femtelle kit. Antibodies tested include 2 anti-uPA antibodies (from Santa Cruz Biotech (Ab-1) and Thermo Scientific (Ab-2)), 2 anti-PAI-1 antibodies (from Santa Cruz Biotech (Ab-1) and Abcam (Ab-2)) and 1 anti-PAI-1 scFv (from Randox Life Science). Results of ELISA tests are presented in Figure 7. Results obtained with anti-PAI-1 scFv spotted at 2 concentrations (5 µM and 10 µM) on chitosan surface are presented in Figure 9. As previously described for anti-uPA, the sensitivity of the detection of PAI-1 was dependent on the capture antibody spotted concentration. Thus, at the highest spotted concentration of anti-PAI-1 scFv, LOD of PAI-1 reached was as low as 2ng/ml, with a dynamic range varying from 2 ng/ml to 20 ng/ml.

Figure 9 Detection of PAI-1 on chitosan surface, as a function of anti-PAI-1scFv spotted (5µM and 10µM); limit of detection (LOD) of PAI-1 for each concentration was indicated.

In conclusion, higher spotting concentration of antibodies results to better LOD and wide dynamic range of detection. Therefore, high spotted concentrations of anti-uPA were chosen to titrate uPA in breast tumor tissue extraction.

Influence of the concentration of biotin labeled detection antibody

The concentration of detection antibody also affected the performance of antibody microarray. In our study, biotinylated antibodies against uPA and PAI-1 were obtained from Femtelle kit. However, no information about their concentration was given by the supplier.

Thus, two conditions were tested: not diluted and diluted 1:2 in 4% BSA/PBS-T 0.1% solutions. Figure 10 showed the results obtained for anti-uPA antibody (from Santa Cruz Biotech) spotted on chitosan surface at 2.5 µM (Figure 10a) and results obtained for anti-PAI-1 scFv spotted on NHS surface at 5 µM (Figure 10b). For both antibody/antigen systems no significant difference was observed between the two conditions. Therefore, diluted

Influence of spotting buffer for anti-PAI-1 scFv immobilization

According to previous results, the best spotting buffer for antibody on COOH, NHS and chitosan surface is carbonate buffer (pH=9.6) [20]. However, anti-PAI-1 scFv is closer to antigen protein in terms of size and structure than to native antibody [22]. Therefore, 3 buffer solutions [PBS 1X (pH=7.4), acetate buffer (pH=4.6) and carbonate buffer (pH=9.6)] were tested for the immobilization of anti-PAI-1 scFv on COOH, NHS and chitosan surfaces. Anti-PAI-1 scFv antibody was spotted at 5 µM in all 3 buffers. Then biological activity of the immobilized anti-PAI-1 scFv was evaluated by mean of level of recognition of PAI-1.

Results are presented on Figure 11. On both COOH (Figure 11a) and NHS (Figure 11b) surfaces, only immobilization of anti-PAI-1 scFv with carbonate buffer allowed retaining the biological activity of the scFv. In contrast, on chitosan surface (Figure 11c) the best biological activity was obtained with PBS 1X. COOH and NHS surfaces are both silane monolayers, NHS surface was obtained by NHS activation of the COOH surface. Protein immobilization on COOH surface was achieved through physical adsorption, whereas on NHS surface it was through covalent binding. These 2 ways of immobilization on silane monolayer seemed to lead to complete denaturation of immobilized anti-PAI-1 scFv, resulting to complete loss of biological activity, or to very low amount of immobilized anti-PAI-1 scFv, resulting in not detectable signal, when acetate buffer or PBS 1X was used as spotting buffer. However, chitosan surface is a polymeric surface obtained from functionalization of NHS surface with chitosan polymer, a natural polysaccharide. The immobilization on chitosan surface was achieved through physical adsorption as well, but the surface area developed and hydrophilic character was higher than COOH surface [20]. Moreover, chitosan surface presents NH 2 groups instead of COOH groups on COOH surface. Thus, physical adsorption on chitosan surface should involve different mechanisms leading to better level and orientation of immobilized anti-PAI-1 scFv.

This immobilization process seems to be enhanced using PBS 1X as spotting buffer.

Conclusion

Performances of antibody microarrays are greatly influenced by the biological activity of immobilized antibodies and by the sensitivity of the detection system. Among parameters involved in biological activity of immobilized antibodies, surface chemistry as well as spotting conditions (concentration and buffer solution) are ones of the most critical. Thus, to elaborate efficient antibody microarrays for the quantification of uPA and PAI-1, these parameters were optimized and results are reported in Table 1. Among the 6 surface chemistries tested, 3 of them (COOH, NHS and chitosan surfaces) displayed good performances to retain biological activity of immobilized anti-uPA antibody and anti-PAI-1 antibody scFv. Moreover, higher spotting concentration of antibodies results in better LOD and wider dynamic range of detection under optimal spotting buffer. At last, same detection level was obtained with biotinylated antibody diluted 1:2 allowing to reduce consumption of biological reactive. The goal was to quantify the concentration of uPA in each cytosolic extracts by our customized antibody microarray and compare the value to those obtained with Femtelle kit.

For this, we elaborated three types of antibody microarrays, with COOH, NHS and chitosan surface chemistries considered as best surfaces according to previous results. Thus, the design of customized antibody microarrays was pictured in Figure 12. 10 lines of 4 microwells were etched on glass slides. Inside each microwell, the anti-uPA antibodies were spotted in PBS 1X buffer at two concentrations (3 µM and 6.6 µM) and buffer and strep F555 were added as negative and positive controls of microarray quality.

In order to elaborate nano-FLISA (Fluorescent immunoassay) for the quantification of uPA, titration curve should be included on the microarray. So, on each microstructured slide, the two first lines of microwells were dedicated to construct standard titration curve. 6 microwells were incubated with 6 different concentrations of uPA including 0 ng/ml, 0.2 ng/ml, 0.5 ng/ml, 1 ng/ml, 1.5 ng/ml and 2 ng/ml.

On COOH

On NHS

On Chitosan

Figure 13 Fluorescence image obtained on COOH surface; microwells were incubated with uPA including 0 ng/ml, 0.2 ng/ml, 0.5 ng/ml, 1 ng/ml, 1.5 ng/ml and 2 ng/ml and buffer (PBS 1X) (two microwells).

Whatever the surface chemistry, in each microwell, the 2 spots of streptavidin-F555 were easily detected which means that these surfaces had a good ability for protein immobilization.

Microwells incubated with buffer exhibited weak signal. Unfortunately, Figure 13 exhibits also a spurious fluorescence which affected the quantitative analysis of fluorescence. This high unspecific binding was observed when the microwell was incubated with uPA, which should be caused by inefficient blocking process or more probably by bad washing steps.

Remember that washing and rinsing step were performed without agitation. Fluorescent intensity signals were higher on COOH and NHS compared to chitosan surfaces. But whatever the surface chemistry, a similar behavior could be noticed. For microwells incubated with uPA, fluorescence signal obtained from immobilized anti-uPA antibody increased with the increasing concentration of incubated uPA.

Figure 14 gives the fluorescent signal (SNR) obtained for the detection of uPA on COOH, NHS and chitosan surfaces when the concentration of spotted anti-uPA antibody was at 3 µM and at 6.6 µM). SNR is defined as the ratio between the fluorescence signals of antibody spots over the fluorescent signals of buffer spots. As expected, SNR increased with the increasing of uPA concentrations. Among all three surfaces tested, the fluorescence signal obtained from COOH surface is the highest, followed by NHS and chitosan surface. Although SNR is different, the lowest limit of detection (LOD) of uPA on these three surfaces is the same, about 0.2 ng/ml. However, the dynamic range is strongly depending on microarray surface chemistry. On COOH surface, SNR reached a plateau when uPA is at 1.5 ng/ml, therefore, the dynamic range of uPA detection is between 0.2 ng/ml to 1.5 ng/ml. The same range was obtained for chitosan surface. In contrast, the dynamic range is wider on NHS surface, between 0.2 ng/ml to 2 ng/ml. Moreover, according to the trend of the curve, higher concentrations of uPA could be also detected on NHS surface but with weaker signals.

Comparison between the curves obtained from two different concentrations of spotted antibodies did not exhibit crucial discreapancy. The difference concerned mainly the high concentrations of uPA which exhibited stronger SNR for 6.6 µM of spotted anti-uPA antibodies.

Figure 14 Fluorescent signal analysis (SNR) of uPA detection on COOH, NHS and chitosan surface when the concentration of spotted anti-uPA antibody was at 3 µM (left) and at 6.6 µM (right).

For providing the quantification of uPA in cytosolic extracts, standard titration curve issued from our customized microarray should be drawn and also compared with uPA standard curve obtained from Femtelle kit. According to the protocol of Femtelle kit, the lowest limit of detection (LOD) for the assay for uPA is 0.025 ng/ml, which seems much lower than the results obtained by our antibody microarray (0.2 ng/ml). However, 100 µl uPA standard solutions were needed to add in order to obtain the standard titration curve in Femtelle kit. In contrast, only 1 µl uPA standard solution was used in our antibody microarray. Therefore, if we consider the total amount of uPA, LOD of our antibody microarray (0.2ng) is 10 times less than Femtelle kit (2.5ng).

For the dynamic range, we can see that it ranges between 0.01 ng/ml to 1 ng/ml for Femtelle kit (Figure 15 a). In comparison, the dynamic range of uPA detection by our antibody microarray is wider, for example, it ranges from 0.2 -2ng/ml on NHS surface extracts, among which line 1-4 were incubated with non-diluted cytosolic extracts, whereas According to the standard curve obtained from each surface, if the SNR of one sample is in SNR range (shown in Table 3), its concentration could be calculated by the formula obtained from the standard curve (shown in Figure 15), otherwise not. Therotically, as uPA concentration of all samples are in the dynamic range after dilution, therefore, their corresponding SNR are also in the SNR range. However, several samples whose SNR is out of range, therefore, their concentrations could not be calculated by the formula. We summarized the samples whose SNR are in the range, and then calculated the concentration of each sample and compared the results with those obtained from ELISA. About half samples tested on COOH surface, displayed results consistent with those from ELISA; while the results of the other samples were lower than ELISA (Figure 17a). Thus we compared SNR values of tested samples with the standard curve obtained at 3 µM anti-uPA antibody spotting concentration (Figure 17b). It appeared that SNR values of most samples were lower than SNR values of standard curve. This result could be explained by the high unspecific binding of standard samples on COOH surface (Figure 13). In contrast, on NHS surface, about half tested samples displayed higher concentration than with ELISA (Figure 17c). In this case, the SNR values of most samples were higher than the SNR values obtained with standard samples at 6.6 µM anti-uPA antibody spotting concentration (Figure 17d). These differences could be due to the partial lost of activity of standard uPA solutions with time; therefore, new standard uPA solutions should be used for further experiment.

At last, most samples tested on chitosan surface displayed results consistent with ELISA (Figure 17e). Moreover, SNR values of these samples were consistent with standard curve (Figure 17f). Although SNR values obtained on chitosan surface were the lowest (Table 3), the quantification of uPA of most samples were the best; therefore, SNR value is not the deciding factor for quantifying uPA. In contrast, the quality of standard curve is more critical. Among all 3 surfaces tested, unspecific binding was the lowest on chitosan surface (Figure 13).

Standard curve is critical for quantifying uPA and two points need to be improved in further experiments. Firstly, high unspecific binding was observed on COOH and NHS surface (Figure 13) and it greatly influences the quality of standard curve; therefore, it important to reduce unspecific binding, e.g increasing washing time and/or washing buffer composition. Secondly, on NHS and chitosan surfaces, higher uPA concentrations could be tested in order to increase their dynamic ranges (Figure 15 d, e andg).

In the literature, only one study reported on the use of antibody microarray to quantify uPA in breast tumor tissue extracts. They used commercial surface and tested 50 tissue samples.

Results showed that uPA values measured by antibody microarray were 40-50% lower than those obtained from ELISA. Furthermore, they analyzed the association between protein microarray and ELISA and found that the results obtained from protein microarray equal 0.06 + 0.56 ELISA [23]. In our study, we found that results obtained from antibody microarray were also different from ELISA and furthermore, surface dependent. For example, results obtained on NHS are higher than ELISA while those obtained on COOH surface are lower.

However, as our study is limited by sample size, we could not obtain the real association between our home-made surface with ELISA; therefore, future large scale study is needed.

Conclusions

In this study, we aimed at developing antibody microarray for the titration of uPA and PAI-1 in breast tumor tissue extracts. Using microstructured glass slides, we could rapidly screen various conditions (surface chemistries, antibody spotting concentrations and spotting buffers) to optimize performances of the antibody microarray. Among the six surface chemistries tested, three were selected (COOH, NHS and chitosan surfaces) for the titration of uPA in cytosolic extracts. The best antibody concentration was found to be 6.6 µM spotted in PBS 1X (pH=7.4). In these conditions, LOD was determined at 0.2 ng/mL and the dynamic range was 0.2-2 ng/mL. Then 16 breast tumor tissue extracts were titrated for uPA on our antibody microarray. The results indicated that the performances of our antibody microarray are surface dependent. However, as our study is limited by sample size, we could not obtain the representative association between antibody microarrays and ELISA; further large scale investigation is needed. Furthermore, high unspecific binding was observed on COOH surface; therefore, unspecific binding should be reduced in order to improve the quality of standard curve and quantification.

Concerning the quantification of PAI-1, among 3 anti-PAI-1 antibodies tested, only anti-PAI-1 scFv could retain its biological activity following immobilization on surfaces (chemically functionalized microarray and ELISA plates). Best performances were obtained with 10 µM anti-PAI-1 scFv spotted in PBS 1X (pH=7.4) leading to LOD of 2 ng/mL. In comparison, LOD for the Femtelle kit for PAI-1 is 0.125 ng/ml. As discussed before, if we consider the total amount of PAI-1, LOD of our antibody microarray is almost 6 times less than those from Femtelle kit. These results are very promising for the implementation of a nano-FLISA test. Indeed, our antibody microarray showed a higher sensitivity and a wider dynamic range compared to Femtelle ELISA kit. Furthermore, considering that one of the main limitations of Femtelle kit is that it requires 100-300mg of fresh or frozen samples, our antibody microarray shows high potential as it consumes 25 times less sample volume. Moreover, we planned to use formalinfixed paraffin-embedded (FFPE) tissue which is more easy to obtain and main source of patient material worldwide [24].

Introduction

Although protein microarrays show high potential for biomarkers screening, various factors influence microarray performances such as surface chemistry [1], humidity during spotting [2], composition of the spotting buffer (additives and pH) [3][4][5][6], concentration of immobilized proteins [7]. Considering protein microarray processing, critical factors are composition of blocking solution and duration [8,9], concentration of incubated solution and incubation time [10], buffer solution used for sample dilution, washing time, and so on.

A major challenge for miniaturized multiplex sandwich assay development is the optimization of these parameters in order to reduce processing time keeping high performances. In this chapter, we study the influence of several critical factors (blocking and incubation duration, concentration of incubated solutions) on the performances of protein microarray. We chose one-factor-at-a-time (OFAT) method for designing experiment. OFAT method is based on the variation of one single factor in each experiment while keeping the others constant and measuring the process output [11].

Furthermore, due to the physico-chemical nature of proteins, performances may also be affected by the delay between microarray fabrication and their use. Therefore on a practical point of view, storage condition of protein microarray slides is an important issue for preserving the integrity of microarray performances. It is a major concern for both microarray manufacturers and users. Generally, arrays are stored under aqueous conditions (glycerol, blocking solution, polyvinyl alcohol, etc) or dry condition (sealed under nitrogen or not) [12][13][14][15]. However, proteins have diverse structure and physico-chemical properties. Therefore, optimal storage condition is expected to be protein and surface dependent.

Herein, various storage conditions for antigen microarrays were studied. Our microarrays are designed for cancer diagnosis and are based on immobilized heat shock proteins (HSPs) microarray for the multiplex profiling of anti-HSPs antibodies in serum. These arrays will be usable for medical applications (not only research) providing that their performances should be maintained upon storage for a reasonable time span. F555 (0.01mg/ml) and acetate buffer (pH=4.6) were used as quality and negative controls, respectively. After spotting, proteins were allowed to react with functionalized surfaces under saturated water vapors overnight at 4°C. Then slides were washed sequentially for 2 × 5 min with PBS, for 1 × 5 min with PBS-T, and then blocked (blocking step) with 10% BSA/PBS-T solution at various time (30 min, 1 h, 2 h) and then washed for 3 × 5 min with PBS-T and dried.

For the biological recognition step, purified biotin-labeled antibodies or purified unlabeled antibodies were incubated at various times (30 min, 1 h). Different antibody concentration diluted in 1% BSA/PBS-T 0.1% were tested (0.1µM and 0.5µM). Then slides were washed for 3 × 5 min with PBS-T and dried.

For the detection step, streptavidin-F555 at various concentrations, diluted in 1% BSA/PBS (0.05µM, 0.19µM), or IgG-Cy3 at various concentrations (0.01µM, 0.05µM) was incubated for 30 min or 1 hr. Then slides were washed for 3 × 5 min with PBS-T, 10 seconds in DI water and then dried. The different experimental conditions used are reported in Table 1, In a second test, four antigen proteins (HSPD1, HSP70, HSPA5 and HSP110) were spotted at two concentrations (0.05 mg/ml and 0.1mg/ml) on flat glass slide functionalized with COOH and CMD surface chemistries, shown in Figure 3. Each concentration has 48 replications; streptavidin-F555 (Strep-cy3) at 0.01mg/ml and acetate buffer (pH=4.6) were spotted as quality and negative control, respectively. After blocking with 10% BSA/PBS-T solution, slides were stored at various times (1 month, 2 months, 3 months and 6 months) in 50% glycerol solution at 4°C. Reference biological activity of spotted antigens (T0) was obtained immediately after protein immobilization and blocking with 10% BSA/PBS-T (no storage). After storage, unblocked slides were blocked with 10% BSA/PBS-T solution 2hr at room temperature, then washed with PBS-T for 3 × 5 min and dried. "Strep-F555" spots were always incubated with buffer solution. HSPD1 and HSP70 spots were incubated with 0.5µM biotinylated-antibody, 1hr at room temperature (RT). After washing, they were incubated with streptavidin-cy3 (0.01mg/ml), 1hr at RT. HSPA5 and HSP110 spots were incubated with 0.5µM unlabeled antibody, 1hr at room RT and washed. Then they were incubated with goat-anti-mouse IgG-cy3 at 0.01µM, 1hr at RT. Then slides were washed for 3 × 5 min with PBS-T, 10 seconds in DI water and then dried.

Evaluation of protein microarray reproducibility

P53 (0.1mg/ml), biotin-labeled BSA (0.05mg/ml), anti-P53 antibody (0.15mg/ml) and streptavidin-F555 (0.01mg/ml) were spotted onto 3 chemically functionalized (COOH, NHS and chitosan surfaces) flat and micro-structured glass slides. On flat glass slides, 4 fields containing 72 replications of each spotted solution were defined according to Figure 4a. On micro-structured glass slides, each micro-well contains 18 replications of each spotted solution according to Figure 4b. As each protein was spotted in 4 micro-wells, so in total, each protein also had 72 replications. For chitosan surface, carbonate buffer (pH=9.6) was used as spotting buffer while for COOH and NHS surface, acetate buffer (pH=4.5) was used.

In order to compare inter-and intra-coefficient variation for each surface chemistry, 2 flat glass slides and 2 micro-structured glass slides were evaluated for each surface chemistry. The process of incubation for both flat glass slides and micro-structured slides is shown in Table 2. The detail of incubation and washing step is the same as in part 2.2.4.

Fluorescence scanning and data analysis

After drying, slides were scanned with the Microarray scanner GenePix 4100A at wavelengths of 532 nm with the same photomultiplier tube (PMT) gain (PMT=600). Data mining was accomplished with GenePix 4100A software package (Axon Instruments). SNR were calculated as referred in part 2.2.6.

Results and discussion

Optimization of elaboration and processing of protein microarray

Enzyme-linked immunosorbent assays (ELISA) performed in a 96-well plate is routinely used for detection of proteins. Figure 5 showed the general protocol of ELISA, during which antigen was captured on well, then the first antibody was added and then followed by a second labeled antibody. Differences on protocol exist between different commercial ELISA kit and home-made ELISA, e.g. blocking time, blocking solution, incubation time and the concentration of incubation solution.

ELISA is limited to screen large numbers of proteins in an efficient manner, particularly when sample volumes are limited. In contrast, protein microarray could simultaneous screen multiple proteins in small sample volumes. Furthermore, the theoretical detection limit for microarrays is significantly lower than that for 96-well plate assay [17]. However, microarrays have not matched their theoretical predictions. One reason is that their current experimental protocols originate from 96-well plate ELISA protocols and have not been fully optimized [18,19]. Various factors influence the performance of microarray and in this study, our interests focus on optimizing factors including time of incubation time and the concentration of incubated solution. We chose one-factor-at-a-time (OFAT) method for designing experiment. During the experiment design, we vary one single factor each time while keeping the others constant and then measure the process output. We determine the best condition for each factor one by one.

Influence of blocking, antibody and detection incubation times

In order to evaluate the duration of experiment, we spotted two antigens (HSPD1 and P53)

on NHS surface at four concentrations (0.01mg/ml, 0.025mg/ml, 0.05mg/ml and 0.1mg/ml) and then evaluated the blocking and incubation process at different time period, as described in Figure 6.

Figure 6

Protein microarray design for evaluation of blocking and incubation time. HSPD1 and P53 were spotted at 4 different concentrations on NHS surface; streptavidin-F555 (0.01mg/ml) and acetate buffer were used as quality and negative control, respectively. Three time periods (2hr, 1hr and 30min) were tested for blocking process; two time periods (1hr and 30min) were tested for the incubation of biotinylated antibody as well as streptavidin-F555.

Blocking process

The influence of blocking time on the efficiency of antigen/antibody recognition is presented in Figure 7. Firstly, for both antigens, biological recognition with their corresponding antibody is improved at 0.1 mg/mL of spotted concentration. Secondly, the influence of blocking incubation time on the level of antigen/antibody recognition is protein dependent. Indeed, P53/anti-P53 antibody system doesn't seem to be affected by blocking time (Figure 7a), whereas HSPD1/anti-HSPD1 antibody system is greatly influenced by blocking time (Figure 7b). 1hr of blocking leads to the best performance for HSPD1/anti-HSPD1 antibody system. Blocking process is critical because it could reduce unspecific binding and improve the performance of microarrays. As shown in Figure 8, the fluorescence signal of buffer decreased with the increasing of blocking time for both HSPD1 and p53. However, after 1 hr of incubation with blocking solution, no significant benefit is observed. Same results were obtained from [9]. They found that long time of blocking could reduce the background; however, it could also block the reactive sites of spotted proteins and lead to the decrease of signal. Therefore, 1hr of blocking is the best for our microarray performance, because it could reduce unspecific binding without affecting the efficiency of biological recognition. The incubation time is dependent on antigen-antibody micro-spot kinetics. It is influenced by two factors: (i) the transport of the analyte from the solution to the surface reaction area;

(ii) the subsequent binding process [10]. Binding process depends on the affinity between antigens and antibodies. P53/anti-P53 antibody recognition performs better under 1hr of incubation maybe because of the low affinity between both parts compared to HSPD1/anti-HSPD1 system.

Incubation process with streptavidin-F555

For the detection step, both antigen/antibody systems showed improved detection level after 30 min incubation with Strep-F555 instead of 1hr, and with 0.1 mg/mL spotted concentration. Figure 10 showed the influence of second incubation (strep-F555) on microarray performance. From the results, we can conclude that for both HSPD1 (Figure 10a) and P53 (Figure 10b), 30 min of incubation is better than 1hr. When we compared the fluorescent signal of buffer, we found that longer time of incubation leaded to higher signal of buffer (Figure 10c). Therefore, shorter time of incubation is better and it could reduce the unspecific binding. During our antigen microarray processing, the process of incubation with antibody is essential. We could not reduce the incubation time because of the antigen-antibody microspot kinetics. Unlike the recognition between antigen and antibody, the affinity between biotin and streptavidin is strong, thus short time of incubation is efficient; furthermore, it could reduce unspecific binding of streptavidin. For the blocking process, long time of blocking could not reduce the background. Therefore, taken all these experimental duration together, we could reduce experimental time from 4h to 2h30, as shown in Table 3. Antibody incubation time (hr) 1 1

Streptavidin-F555 incubation time (min) 60 30

Influence of detection antibodies and streptavidin-F555 concentrations

From previous results, it was shown that the efficiency of protein microarrays is very dependent on the biological system studied and on the surface chemistry. Thus, to optimize costs related to the elaboration and processing of protein microarray, we studied the influence of detection antibodies and streptavidin-F555 concentrations on the level of detection of interactions of all antigen/antibody systems studied. The 8 antigens were spotted at 2 concentrations on NHS, COOH and APDMES surfaces and processed according to Figure 11.

Figure 11

Scheme of protein microarray design for optimization of detection antibody and streptavidin-F555 concentrations. 8 antigens were spotted at 2 concentrations (0.05mg/ml and 0.1mg/ml) in acetate buffer (pH=4.6) on COOH, NHS and APDMES surfaces. In each microwell, acetate buffer and strepdavidin-F555 were used as negative control and quality control respectively; each solution was spotted in 4 replicates. Biotin-labeled or unlabeled antibodies were tested (Incubation 1) at two concentrations (0.1µM and 0.5µM); detection step (Incubation 2) was performed using strep-F555 (0.05µM and 0.19µM) or IgG-cy3 (0.01µM and 0.05µM).

As expected, the best detection signal for all antigens was obtained with 0.1 mg/mL of spotting concentration. Therefore, only results obtained with this concentration will be presented.

Figure 12 showed the influence of the concentration of incubated antibodies on the performance of antigen microarray. Figure 12a represents the fluorescence signal (SNR) obtained for HSP90B1/anti-HSP90B1 on COOH, NHS and APDMES surfaces at 2 different concentrations (0.1 µM and 0.5 µM) of first antibody. On COOH and NHS surfaces, no significant difference was observed between the both concentrations of first antibody. In contrast, the signal on APDMES surface was higher when the concentration of first antibody was lower (0.1 µM), which was caused by lower fluorescence signal of buffer, as shown in 4 summarized the optimized concentrations of spotting antigens, recognition antibodies and detection molecules. We can see that the ratio between spotted antigen concentration and first antibody concentration ranges between 10:1 and 18:1. For HSPB1, HSPD1, HSP70 and P53, the ratio between first antibody and streptavidin-F555 was 2:1; for HSP90, HSPA5, HSP90B1 and HSP110, the ratio between first antibody and second antibody (IgG-cy3) was 10:1. Among the results, the ratio between first antibody and streptavidin-F555 is the highest; maybe we could further reduce the concentration of streptavidin-F555. During the protocol, washing step will remove the majority of proteins in each step, therefore, less concentrated solutions are needed in the following step; otherwise, higher concentrated solution could lead to high unspecific binding. According to our results, a ratio about 10:1 on the concentration between the former and later solution was recommended. In addition to reduce cost, it could also improve the performance of antigen microarray.

Evaluation of the stability of protein microarray under storage

Storing protein microarray is a major concern for both microarray manufacturers and users.

The stability of protein microarray under storage can be studied at 2 levels: the stability of the surface chemistry, and the stability of spotted proteins.

Stability of the surface chemistries of protein microarray

The 6 surface chemistries used for the elaboration of protein microarray were developed in previous work [20]. They were chemically characterized by X-ray photoelectron spectroscopy (XPS), infrared spectroscopy (IR), mass spectrometry (Tof-SIMS). They were also characterized for their surface energy by contact angle measurement. Indeed, contact angle provides macroscopic information of the surface energy and it is related to the chemical characteristics of the surface. Measuring contact angle is a rapid characterization technique, and any change indicates modification of chemical functions on the surface. Thus, first the stability of the 6 surface chemistries was evaluated, under storage (in dry condition) at different times (from 0 to 4 months), from a macroscopic point of view by surface energy calculation. Second, the stability of surfaces was also studied from the molecular point of view by evaluating their capacity to immobilize proteins.

Total surface energy of all six surfaces was calculated from contact angle measurements according to Owens-Wendt model [16]. 3 different liquids (water, diiodomethane, ethylene glycol) were used in order to determine the dispersive and polar contributions to total surface energy. Results presented in Figure 15 showed that total surface energy remains constant until 4 months of storage in dry condition, for 3 surface chemistries (COOH, NHS and chitosan surfaces, but not for MAMVE, APDMES and CMD surfaces (Figure 15a). The drastic increase of total surface energy of MAMVE surface was mainly due to the increase of polar energy (Figure 15b). MAMVE polymer contains anhydride units which, in the presence of water, could be hydrolyzed leading to 2 carboxylic acid groups. The storage of chemically functionalized surfaces was performed under dry condition but not under sealed nitrogen atmosphere. It is likely that water molecules contained in ambient atmosphere could contribute to the hydrolysis of anhydride units. Then, the formation of carboxylic acid groups led to increase the amount of hydrogen bonds, so the polar energy of the surface. It is to note that the increase of polar energy of MAMVE surface is detected after 3 months of storage indicating that the hydrolysis of anhydride units should be very slow. The dispersive energy of MAMVE surface also increase but in a lower amount (Figure 15c). The hydrolysis of anhydride units leading to the opening of the cycle, more London interactions could be established between pendent chains.

Concerning APDMES surface, although total surface energy displayed very slight variation with time, its polar contribution clearly decreased (Figure 15(b)). Smith et al. [21] described the decrease of the aminosilane layer depth due to siloxane hydrolysis catalyzed by amino groups in the presence of water at 40°C during 1 or 2 days. In our case, the decrease of polar energy was observed after 3 months of storage. Thus, it is possible that the water contained in ambient atmosphere hydrolyzed slowly the siloxane link, leading to the partial loss of APDMES molecules, so to the decrease of polar energy.

At last, CMD surface displayed a slight decrease of its polar energy from month 3 suggesting a degradation of the surface. Indeed, we observed some blotches on the surface (Figure 16). Further characterizations will be needed in order to understand the phenomenon inducing these blotches and their composition. Table 5 showed the evolution of the quality of spots with time, for streptavidin-Cy3, IgG-cy3 and ODN-cy3 on chitosan and MAMVE surfaces. On chitosan surface (Table 5a), IgG and ODN spots showed good homogeneity and constant spot size. However, streptavidin spots, at both concentration tested, became less homogenous while spot size was constant. On MAMVE surface (Table 5b), the quality of all spots changed from month 3 in agreement with the modification of surface energy. Indeed, spots became larger indicating that the surface became more hydrophilic as its polar energy increased. Only streptavidin spots remained homogenous until 3 months of storage, IgG and ODN spots showing inhomogeneity. The same behavior was observed with CMD surface. From these observations we can conclude that modifications in the quality of spots are in agreement with variations in surface energy, but spots homogeneity is closely dependent on molecules and surface chemistry as already described in [22]. Thus, it will be very important to characterize at the molecular level the interactions between biomolecules and chemical groups grafted on solid support. In conclusion, all chemically functionalized glass slides used in this study for the elaboration of protein microarray were shown to be sensitive to storage in ambient atmosphere. Even though no significant difference in surface energy was observed between fresh and stored surfaces, their ability to immobilize molecules could change a lot indicating that properties at the molecular level could be affected with effect at macroscopic level.

Indeed, modifications of macroscopic physico-chemical properties were confirmed at the molecular level. Functionalized glass slides could retain efficient reactivity until 3 months of storage in ambient atmosphere, but the storage time could probably be improved in sealed nitrogen environment. Thus, further analysis on the stability of surface chemistries in the least condition would be interesting as well as on biological activity of immobilized molecules.

Stability of spotted protein microarray

In order to reduce the number of microarray slides used and experiments, we divided this study in 2 parts. In each part we chose 2 surface chemistries, one allowing covalent binding of proteins (NHS or CMD surfaces) and one allowing physical adsorption of proteins (COOH or chitosan surfaces). The first part was devoted to rapidly identify the best storage conditions (under solution or dry, with or without blocking) of spotted protein microarray, and the second part aimed at studying biological activity of spotted protein as a function of storage time in the best condition defined previously.

So for the first part, P53 and HSPD1 were immobilized onto NHS and chitosan surfaces and stored under various conditions for 1 month. Then their biological activity was evaluated by their capacity to recognize their specific antibody. Storage conditions were selected according to the literature [12][13][14][15]: in 50% glycerol solution, in 5% trehalose solution, under nitrogen atmosphere, without blocking step, after blocking step with PBS/BSA solution.

Results indicated that biological activity of immobilized antigens is better retained when protein microarray was stored after blocking with PBS/BSA solution than before, for both p53 (Figure 18 a) and HSPD1 (Figure 18 c) immobilized on NHS surface. This was caused by the lower unspecific binding of buffer after blocking process (Figure 18 b andd). The results of P53 and HSPD1 on chitosan surface led the same conclusion. The results were consistent with those obtained by Adarsh D. Radadia, they also found that spotted microarray had a higher signal after blocking process [23]. Then, if we compared biological activity of immobilized antigens before storage (T0) and after 1 month storage with blocking step, it appeared that the best storage condition depends on surface chemistry (Figure 19). Indeed, for both P53 and HSPD1 immobilized on NHS surface, the best storage conditions seemed to be in solution (Figure 19a) whereas on chitosan surface, nitrogen atmosphere gave better results (Figure 19b). The immobilization strategy is different on the two surfaces studied. On NHS surface, proteins are immobilized through covalent binding while on chitosan surface, immobilization of proteins is achieved through physical adsorption. Under wet condition, covalently linked proteins are more stable and could be less released from the surface compared to physical adsorption. Thus, storage of protein microarray in solution is better when covalent immobilization strategy is used, whereas storage under nitrogen atmosphere should be better using physical adsorption strategy. Same results were obtained from [14].

Moreover, we can notice that for both immobilized antigens on both surfaces (except for HSPD1 on chitosan surface), there is an increase of the fluorescent signal (SNR) after 1 month of storage under all conditions. This was due to the decrease of the fluorescent intensity of buffer spots (data not shown). The same kind of phenomenon was observed by other researchers studying storage conditions for protein microarray [14,15,23]. Increase of the biological activity of immobilized protein after storage could be attributed to the favorable rearrangement of immobilized antigens on the surface, thus resulting in better accessibility of biological recognition sites with antibodies. Considering that immobilized proteins would have higher possibility to change their conformation in solution than in gas atmosphere, we decided to store protein microarray in 50% glycerol solution for further experiment.

Then, in the second part, taking into account these results, we studied the stability of 5 proteins (streptavidin-F555, HSPD1, HSP70, HSP110 and HSPA5) immobilized on 2 different surface chemistries (COOH and CMD surfaces) after blocking and storage in 50% We can see that the fluorescence intensity of strep-F555 and buffer kept constant during storage on COOH surface. In contrast, the fluorescence intensity of strep-F555 decreased greatly on CMD surface after 1 month of storage, and then stayed constant; whereas the fluorescence intensity of buffer kept stable untill first 3 months and then increased greatly (Figure 20b). Considering that the fluorescence intensity of immobilized strep-F555 showed a high signal after 6 month of storage on both surfaces, we could conclude that the condition of 50% glycerol is efficient to store immobilized proteins.

The second parameter studied was the biological activity of spotted proteins. After immobilization of HSPD1, HSP70, HSP110 and HSPA5 onto COOH and CMD surfaces, their ability to be recognized by their specific antibodies was evaluated (Figure 21). During the first month of storage, the fluorescence signal decreased greatly for all spotted proteins on both surfaces, especially for HSPD1. In total, fluorescence intensity of spotted proteins decreased about 20% to 50% on both surfaces suggesting that the biological activity of immobilized antigens was lost in the same amount. Then the fluorescence intensity showed slight change between 1 month and 3 months of storage. Unexpected sharp increase of fluorescence intensity was observed for all spotted proteins on both surfaces after 3 months of storage. This increase was due to strong unspecific binding of surface. Indeed, after 3 months of storage, biological activity of spotted proteins was not detectable. However, our protein microarrays were enough stable for 3 months of storage under 50% glycerol solution and after blocking step, to retain biological activity to sensitively recognize their corresponding antibody. These results are in agreement with those reported in the literature [12][13][14][15]23].

Reproducibility of protein microarray

Reproducibility is a main challenge of protein microarray [24], in the view to replace ELISA. Thus, to evaluate the reproducibility of our protein microarray, the 8 antigens studied were spotted onto flat glass slides and microstructured glass slides functionalized with the different surface chemistries. Then after recognition with their antibody, inter-slides and intraslide coefficient variation (CV) were calculated. Intra-slide CV evaluates the repeatability of spotting and biological interactions on the same slide. Inter-slides CV evaluates the reproducibility between slides functionalized with the same surface chemistry, spotted with the same proteins and processed in the same way. Table 5 presents the results obtained for P53/anti-P53 system. We can see that the majority of intra-and inter-CV are lower than 25%, except on chitosan surface. Same tendency was obtained with the other antigen/antibody systems tested. Therefore, our protein microarrays displayed good repeatability and reproducibility to be use in clinical evaluation and routine experiments.

Conclusions

In this part, we have studied various experimental parameters involved in the performance of protein microarrays such as concentrations and incubation time of recognition and detection solutions, blocking time. Optimization of these parameters allowed reducing the time of processing protein microarray from 4 hours to 2 hours 30 minutes, and the cost by decreasing concentrations of biological solutions. Furthermore, we have shown that chemically functionalized glass slides could be store in ambient atmosphere up to 3 months, and may be more under sealed nitrogen atmosphere. For printed protein microarrays, they could be stored after blocking step in 50% glycerol, at 4°C, for 3 months. The biological activity of immobilized proteins decreased but remained sensitive enough for efficient antibody detection. At least, we evaluated the reproducibility and repeatability of our protein microarray and showed that they were in the same range as classical immunoassay such as ELISA.

Therefore, we didn't quantify PAI-1 in tumor tissue extraction. For antibody against uPA, three surface chemistries (COOH, NHS and chitosan) performed well and they were selected for further experiments. In total, we have tested 16 cytosolic extracts of tumor tissue. Results

showed that results obtained from our antibody microarray are surface dependent. For example, no difference was observed between results obtained from chitosan surface with ELISA. In contrast, results obtained on NHS are higher than ELISA and those obtained on COOH surface are lower than ELISA. These results are very promising. Firstly, our antibody microarray showed a higher sensitivity and a wider dynamic range compared to Femtelle ELISA kit. Secondly, considering that one of the main limitations of Femtelle kit is that this kit needs 100-300mg of fresh or frozen samples; our antibody microarray shows high potential as it consumes 25 times less sample volume if we take the dilution times into consideration.

For optimizing the parameters of proteins microarray, we evaluated various factors including experimental duration, the concentration of incubation solutions, etc. Considering that protein microarray is a miniaturized system, it requires less reaction time and less sample volume. Our results showed that we could improve the performance of our customized protein microarray as well as become more economical. We also analyzed the storage condition of surfaces chemistry of protein microarray as well as spotted protein microarray. Results

showed that our printed protein microarray could retain their biological activity for at least 3 month.

Consequently, our work demonstrated that our customized antigen and antibody microarray are efficient and powerful tools for rapid screening tumor biomarkers. Various factors influence the performance of protein microarray among which surface chemistry is critical.

As observed from our results, several proteins completely lost their biological activity after immobilized on several surfaces. This was maybe caused by the change of structure after immobilization. We proved the necessity to adapt surface chemistry to each protein in order to improve performances of protein microarrays. Furthermore, due to the complex structure of proteins, there is no unique surface which will be suitable for all proteins.

Several aspects need to be improved in the future. 1) As our study was limited by sample size, therefore, further large scale investigation is needed to validate the real diagnostic performance of our customized antigen microarray. 2) Antibodies against HSPs were also over-expressed in other cancers; therefore, we could also test the diagnostic performance of this panel in other cancers. Furthermore, considering the heterogeneity and complexity of tumor, we need to add other biomarkers in order to increase the sensitivity of the test, e.g. DNA, miRNA, etc. 3) In order to being a simple and powerful tool to be used in clinic like ELISA, the experimental processes of protein microarray need to be automated, like incubation process, data analysing, etc. Also, the process should be well controlled to obtain a good reproducibility. 4) Data analyzing methods should be standardized and complete.

Compared with defining cutoff value as a certain value, analyzing data in AUC is better and more comprehensive. Only when data analyzing methods was standardized, could we compare the results from different studies.

Annexe

The receiver operating characteristic (ROC) curve is commonly used in medical decision making. ROC graph is two-dimensional graph in which sensitivity is plotted on the Y axis and 1-specificity is plotted on the X axis. We take the results obtained from HSPA5 immobilized on chitosan surface for an example to explain how we construct ROC curve. As shown in If we take the value of the first sample as the cut off value, it means that only one cancer was diagnosed as positive, therefore, the sensitivity is 2%; as there is no healthy control diagnosed as positive, then the specificity is 100%, yielding (2%, 0). Then we continued from

No. 2 to No. 13, during which the sensitivity increased gradually while the specificity didn't change because no healthy control was diagnosed as positive. It corresponds to ROC curve as shown in Figure 1 a. Then we continue No. 14. As it is a healthy control, so if we take its value as cut off value, no more cancers were diagnosed as positive, therefore, the sensitivity didn't change; however, as one healthy control was diagnosed as positive, the specificity decreased, yielding (4%, 26%), shown in Figure 1 De nos jours, on parle de plus en plus de médecine personnalisée, qui consiste idéalement à donner le bon traitement au bon patient au bon moment. De ce concept, il est attendu une forte amélioration de la prise en charge des patients, une plus grande efficacité des traitements et une diminution globale des coûts de santé [10,11].

Une des clefs de la médecine personnalisée tient dans l'utilisation massive des biomarqueurs et en parallèle, le développement d'outils de classification haut débit. Qu'ils soient issus de tissus ou de sera, les biomarqueurs doivent fournir une valeur prédictive ou de pronostic fiable, ou permettre de pouvoir estimer le risque de récidive ou l'efficacité d'un traitement donné sur un patient donné. Ainsi, dans le cas des cancers du sein, l'objectif ultime serait par exemple de pouvoir classer les patients selon le risque plus ou moins élevé de taux de récidive, de leur administrer la thérapie la mieux adaptée et ainsi d'éviter des surtraitements et ainsi augmenter les succès de la thérapie [12,13].

Biomarqueurs sériques dans les tests cliniques

Les marqueurs tumoraux sériques sont des molécules solubles dans le sang qui peuvent être détectées à l'aide d'anticorps monoclonaux. Ces marqueurs sont produits par les cellules tumorales ou par les autres cellules en réponse à la présence de cellules tumorales [18,19]. A l'heure actuelle, les tests cliniques utilisant des biomarqueurs sériques pour le cancer du sein contiennent des glycoprotéines mucines (MUC-1) et des antigènes carci embryonnaires (CEA) [20], recommandé par l' ASCO et l' EGTM. Ces marqueurs peuvent être utilisés soit pour le dépistage et le diagnostic de la maladie, soit pour la détection précoce de la récidive, soit pour le suivi thérapeutique. Selon l'objectif du test, leur mesure peut être plus ou moins pertinente.

La famille des MUC-1 est impliquée dans les différentes voies du processus complexe de la génèse tumorale (signalisation des recepteurs de la tyrosine kinase, prolifération et mort des cellules..) et inclus des antigènes de cancer tels que CA 15-3, CA 27-29, CA 549, largement utilisés pour le diagnostic de cancer du sein. [21].

CEA est une glycoprotéine oncofétale également utilisée pour les cancers du sein. Leurs taux sont généralement moins élevés que ceux de MUC-1. Mais les mesures de CEA peuvent donner des informations complémentaires, c'est pourquoi, la combinaison de ces 2 types de biomarqueurs semble pertinent pour suivre les patientes atteintes d'un cancer du sein [22].

Cependant, cette combinaison n'est pas recommandée pour un dépistage systématique ou un diagnostic précoce du fait de leur faible sensibilité et spécificité dans les premiers stades de la tumeur [22,23]. Par contre, la sensibilité de MUC-1 augmente fortement avec l'avancée de la tumeur passant 10-15% à 20-25% puis 30-35% pour les stades I, II, et III, respectivement [24].

Dans le cas de la détection d'une récidive, deux études bien construites ont montré qu'après un traitement thérapeutique, des niveaux élevés de MUC-1 et CEA sont corrélés avec une récidive. Ils permettent de prédire une récidive en moyenne 6 mois plus tôt que d'autres symptômes ou tests [25,26]. Cependant, l'ASCO et l' EGTM sont trés prudents dans leur recommandations d'utilisation car il n'est pas encore suffisamment démontré qu'une détection précoce de métastases a une incidence importante sur le taux de survie et la qualité de vie du patient… [22,23]. Ainsi, si le suivi n'est pas recommandé par l'ASCO, l'EGTM préconise tout de même de suivre les femmes asymptomatiques avec ce panel tous les 2-4 mois Durant les 5 premières années après le diagnostic , puis chaque 6 moiss dans les trois années suivantes puis ensuite une fois par an.

En ce qui concerne le suivi thérapeutique, selon l'ASCO, les données actuelles sont insuffisantes pour préconiser un contrôle avec cet unique panel de biomarqueurs.

L'exemple illustré par MUC-1 et CEA montre que le repérage de nouveaux biomarqueurs est loin d'être suffisant pour qu'ils soient validés et approuvés. Afin de diminuer le gap entre la découverte d'un biomarqueur potentiel et sa validation en tant que tel, il est nécessaire de poursuivre de vastes études en gardant en tête plusieurs points :

1) les échantillons biologiques doivent être soigneusement choisis avec des procédures bien établis de la banque de patients et des contrôles.

2) les objectifs des études doivent être clairement définis et les résultats doivent être rapportés de manière claire [27]. Ainsi, les immunoassays multiplexes, les immunoassays planaires (tels que les microarrays à proteines) peuvent s'avérer comme des outils efficace et simple pour mener des études à grande échelle permettant de transférer plus rapidement la découverte de nouveaux biomarqueurs dans les évaluations cliniques et ceci à moindre coût [28].

Biomarqueurs tissulaires dans les tests cliniques

Dans cette partie, on se focalisera sur les biomarqueurs des tissus déjà utilisés en tests cliniques pour les cancers du sein. Ces biomarqueurs sont ER (récepteurs estrogène), PR (les récepteurs progestérone), HER-2 (récepteurs 2 du facteur de croissance épidermique humain), uPA (activateur du plasminogène de type urokinase) et son inhibiteur principal (PAI-1).

ER et PR sont des facteurs de transcription qui régulent les actions des estrogènes et de la progesterone respectivement. [18]. Actuellement la détermination des ER et PR est rendue obligatoire pour toutes les patientes atteintes d'un cancer du sein selon les recommandations de l'EGTM [23] et l'ASCO [22]. En général, les patientes positives en ER ont un meilleur pronostic que celles négatives. Cette différence s'atténue au bout de 4-5 ans. Une limitation en tant que facteur pronostique des ER est qu'il est de faible valeur pour des cas non ganglionnaires. Les patientes avec des tumeurs exprimant PR ont aussi tendance à avoir un meilleur pronostique que celles qui manquent de ce récepteur [18,29]. En plus de leur valeur pronostique, ER et PR sont considérés comme d'importants indicateurs pour l'analyse de la réponse aux thérapies hormonales. Ainsi, pour les malades à un stade précoce comme à un stade avancé, la présence de récepteurs hormonaux donne une probabilité de réponse à la thérapie hormonale beaucoup plus importante que pour les patientes manquant de ces récepteurs.

Notons qu'il existe 3 types de tests bien établis pour mesurer ces récepteurs hormonaux : par liaison de ligand, ELISA ou immunohistochimie (IHC). Seul ce dernier test est recommandé par l'EGTM [23]. Il faut toutefois souligner qu'environ 20% de détermination de taux de ER/PR par IHC serait inopérante (faux positif ou faux négatif) au niveau mondial du fait de variations des variables pré-analytiques, des seuils de positivité, de l'utilisation d'anticorps relativement inefficaces, et des critères d'interprétation. [30].

L'oncoprotéine HER2 est surexprimée dans environ 15% des cancers du sein [31]. Toutes les patientes positives aux récepteurs HER2 doivent être traitées par immunothérapie avec l'Herceptin® (trastuzumab). [18]. 3 méthodes permettent de déterminer le taux d'HER2: l'immunohistochimie (IHC), l'hybridation in situ par fluorescence (FISH), et l'hybridation in situ chromogénique (CISH). Mais, seule l' IHC est recommandée l' ASCO [18,32,33]. L'activateur du plasminogène de type urokinase uPA est une protéase dégradant la matrice extracellulaire et impliquée dans l'invasion du cancer et des métastases. uPA interagit aussi avec son inhibiteur (PAI-1). Ainsi, les deux (PAI-1 et uPA) favorisent la progression de la tumeur et des métastases. [34]. La présence de uPA et PAI-1 est un fort indicateur de dissémination du cancer pour des patientes avec ou sans envahissement ganglionnaire. Des taux élevés sont associés à un faible taux de survie sans rechute et même à un faible taux de survie globale. Par contre, des études montrent que des niveaux bas de uPA and PAI-1 sont associés à des risques de récidive suffisamment faibles qui font qu'une chimiothérapie n'ajoute pas un bénéfice substantiel [35]. Ces deux marqueurs sont donc considérés comme particulièrement pertinents par l'American Society of Clinical Oncology (ASCO) [22] et l'European Group on Tumor Markers (EGTM) [23].

Actuellement, le test ELISA est la seule méthode préconisée par l'ASCO. Il existe un test disponible commercialement : le ELISA test (Femtelle ®) développé par Sekisui Diagnostics.

Ce Kit assure une bonne qualité et est largement utilisé en clinique. Cependant, le test ELISA nécessite un minimum de 300 mg de tissu frais ou congelé de cancer du sein, ce qui peut être problématique notamment dans le cas de tumeurs de très petites tailles [36]. Cependant, en recherche, comparés aux tests ELISA, les microarrays à protéines présentent certains avantages tels qu'une bonne sensibilité avec de très faibles volumes d'échantillons nécessaires [37].

En 1960, Robert W. Baldwin démontra que le système immunitaire était impliqué dans le développement tumoral. En effet, au cours du développement tumoral, des protéines intracellulaires mutées, modifiées ou exprimées de manière aberrante dans les cellules tumorales, appelées TAA (tumor-associated antigens), peuvent être la cible du système immunitaire conduisant alors à la production d'auto-anticorps (AAb) contre ces TAAs. [38].

Ces auto-anticorps peuvent être utilisés pour des marqueurs de diagnostics précoces de cancer

Conclusion

De ce travail, il ressort que les microarrays microstructurés sont des outils puissants permettant de cribler rapidement un très grand nombre de biomarqueurs et qui ne nécessitent

Résumé en Français

Le cancer du sein demeure un problème de santé publique majeure dans le monde. Afin d'améliorer les chances de survie et la qualité de vie des femmes, il est nécessaire d'effectuer le diagnostic à un stade précoce et d'appliquer le traitement. Dans ce contexte, un des objectifs de cette thèse est de développer des puces à protéines pour le diagnostic et le pronostic du cancer du sein. Parmi les nombreux marqueurs biologiques potentiels, des recherches récentes ont montré que des anticorps anti-heat shock proteins (anti-HSPs) sont associés à la genèse tumorale. Ces anticorps seraient donc de bons biomarqueurs diagnostiques et pronostiques pour le cancer du sein. Par conséquent, nous avons élaboré une puce à antigènes afin de détecter les anticorps anti-HSP dans le sérum de 50 patients atteints de cancer du sein et de 26 témoins sains. Nos résultats indiquent clairement que la la détection multiplex d'une combinaison d'anticorps anti-HSP permet de discriminer les patients atteints de cancer du sein des témoins sains avec une sensibilité de 86% et une spécificité de 100%. Ensuite, nous avons élaboré une puce à anticorps pour doser la concentration de l'activateur du plasminogène de type urokinase (uPA) et de son inhibiteur principal (PAI-1) dans 16 extraits cytosoliques de tissus tumoraux. uPA et PAI-1 sont décrits comme étant de bons biomarqueurs pronostiques et prédictifs du cancer du sein. De faibles taux de uPA (≤3 ng / mg de protéine) et PAI-1 (≤14 ng / mg de protéine) sont associés à un faible risque de récidive et pas de bénéfice d'une chimiothérapie pour les patients atteints de cancer du sein. Les résultats obtenus à partir de puces à anticorps étaient surface dépendante par rapport aux résultats obtenus sous forme ELISA. En outre, l'utilisation de nos puces à anticorps nécessite 25 fois moins de volume d'échantillon par rapport à un dosage ELISA, résolvant ainsi les principales limites de la méthode ELISA. Enfin, nous avons déterminé et optimisé les paramètres influençant les performances des puces à protéines, comme par exemple la chimie de surface, la durée expérimentale, la concentration des solutions, etc. Nous avons également étudié les conditions de stockage à la fois pour des surfaces chimiquement fonctionnalisées et pour les puces à protéines. Les résultats ont montré que les puces à protéines conservent leur activité biologique jusqu'à trois mois de stockage.

Mots clés: puces à protéines, anticorps, le diagnostic de cancer du sein, biomarqueurs prédictifs, le stockage

Figure 3

 3 Figure 3 ROC ''curve'' created by threshold a test set. (a) The Table shows the values of 20 samples obtained from one test and ranked from 0.9 to 0.1; "P" means positive, "N" means negative. (b) The graph shows the corresponding ROC curve with each point labeled by the

  : Not available; IHC: immunohistochemistry; FFPE: formalin-fixed, paraffin-embedded.

  73) and CIS (AUC=0.80) from healthy individuals. Moreover, this combination can discriminate CIS from healthy controls in women under the age of 50 years (AUC=0.85). The results are very important for young women who are at high risk of developing invasive and aggressive tumors. However, it is to note that the number of patients under the age of 50 years was limited (n = 14), so more investigations are needed to confirmed conclusion. Alain Mangé et al. identified another five AAbs panel and results showed that this panel can significantly discriminate early stage cancer from healthy individuals (AUC=0.81). Moreover, this value was high in both nodenegative early-stage primary breast cancer (AUC = 0.81) and DCIS (AUC = 0.85) populations. Therefore, the authors concluded that this autoantibody panel could be useful in screening strategy of early-stage invasive breast cancer and pre-invasive breast cancer[129].The same group also identified a panel of five AAbs in ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC) patients. Results showed that this signature significantly discriminated DCIS from IBC (AUC = 0.794), with an overall sensitivity of 86.1% and an overall specificity of 75.0%. Furthermore, this panel could highly distinguish low-grade DCIS from high-grade DCIS exhibiting an AUC of 0.749. Moreover, the authors compared local recurrence and absence of recurrence in a population of DCIS patients followed for 5 years.Results showed that the autoantibody signature could divide the DCIS patients into a poorprognosis group (local recurrence) and a good-prognosis group (recurrence free, P = 0.011).Therefore, they concluded that this autoantibody panel signature could have clinical implications for the management of DCIS, which may help to avoid the over-treatment of low risk patients and dictate more intensive treatment of aggressive DCIS [128]. If we compared these two studies conducted by the same group [128, 129], no common AAbs were observed in AAbs panels. This was maybe caused by the difference of cancer patients studied and it also reflects the fact that patients in different tumor stage have their own AAbs signature. Furthermore, it maybe caused by the different methods used for choosing AAbs. In [128], they used protein microarray for choosing AAbs panel while in [129], they used twodimensional gel electrophoresis (2D-GE) and mass spectrometry (MS) to choose the potential AAbs panel.

Figure 4

 4 Figure 4 General scheme of typical protein microarray experiment. A set of capture ligands (proteins, antibodies, peptides) is arrayed onto an appropriate solid support. After blocking unreacted sites of the surface, the array is probed by incubation with a sample containing the target molecules. If a molecular recognition event occurs, a signal is revealed either by direct detection or by a labelled probe. MS: mass spectrometry, SPR: surface plasmon resonance, AFM: atomic force microscopy, QCM: quartz crystal microbalance [134]

Figure 5

 5 Figure 5 Three categories of protein microarrays. (a) Analytical protein; (b) Functional protein microarrays; (c) Reverse-phase protein microarrays [135].

  Eric W. Olle et al. compared 4 commercial slides (nitrocellulose FAST, hydrogel, SuperAldehyde and epoxy-silane ES) by printing one antibody on these solid slides. Based on spot fluorescence signal and background intensity, they concluded that the optimal slide substrate for antibody was epoxy-silane ES microarray slides [152]. Angenendt et al. have screened 11 different array surfaces by immobilizing five different antibodies onto each type of microarray support. Then they evaluated detection limit, inter-and intra-chip variation.

[

  205]. Similarly, slides coated with poly-L-lysine produced better results if they were left untouched for about 1 month before being used for DNA array preparation[205]. In contrast, slides functionalized with NHS-ester are sensitive to water and performs worsen after storage unless stored under dry, cool conditions[142]. In a word, storage conditions of non-printed slides depend on surface chemistry and influences properties of immobilized proteins.

  Chitosan was kindly provided by Dr. T. Delair (Laboratoire des Matériaux Polymères et Biomatériaux, Université de Lyon). Dextran (Mw=40000 g/mol) was obtained from Pharmacosmos and maleic anhydride-alt-methyl vinyl ether (MAMVE, Mw=216000 g/mol) from Sigma-Aldrich. HSPB1, HSP70, HSP90, mouse-anti human anti-HSPB1 antibody-biotin, mouse-anti human anti-HSP70 antibody-biotin and mouse-anti human anti-HSP90 antibody were purchased from Enzo life science (Switzerland); HSPD1, HSPA5, HSP90B1, HSP110, mouse-anti human anti-HSPD1 antibody-biotin and mouse-anti human anti-HSP90B1 antibody were obtained from Abcam (UK); P53 and mouse-anti human anti-P53 antibody-biotin were obtained from Sigma and Thermo Scientific (USA), respectively; mouse-anti human anti-HSPA5 antibody and mouse-anti human anti-HSP110 antibody were obtained from R&D Systems (USA); F555-labeled streptavidin was purchased from Invitrogen; Cy3-labeled goat anti-human antibody immunoglobulins G (IgG) and Cy3-labeled goat anti-mouse antibody IgG were purchased from Jackson Immuno Research (USA). All proteins were stored as aliquot at -20°C or -80°C following manufacturer specifications.Bovine serum albumin (BSA) lyophilized powder was obtained from Sigma. 0.01 M PBS or PBS 1X (pH 7.4) was prepared by dissolving the content of one pouch of dried powder in 1 L of ultrapure water. 0.02 M sodium carbonate buffers at pH 10.7 were prepared from 0.1 M NaHCO3 and 0.1 M Na2CO3 solutions in ultrapure water. 0.01 M 2-(Nmorpholino) ethanesulfonic acid (MES) (pH=6.2) was prepared by dissolving the content of one pouch into 1 L ultrapure water and adjust pH up to 6.2. Washing buffer contained PBS 1X and 0.1% Tween 20 (PBS-T) at pH 7.4. Blocking solution was prepared by dissolving 10 g of BSA in 100 ml of PBS-T 0.1%.

  ): Carboxylic surface (COOH) was obtained after hydrolysis of the tert-butyl esters from tert-butyl-11-(dimethylamino) silylundecanoate silanized surface (TDSUM surface); NHS surface was obtained from N-hydroxy succinimide activation of COOH surface; Chitosan surface was obtained by functionalization of the NHS surface with 1mg/ml chitosan solution; APDMES surface was obtained by silanization with (3aminopropyl) dimethylethoxysilane; MAMVE and CMD surfaces were obtained by functionalization of APDMES surface with maleic anhydride-methyl vinyl ether copolymer Fields tested with purified biotin-labeled antibodies (biotin-labeled antibodies against HSPB1, HSPD1, HSP70 and P53) were then incubated with streptavidin-F555 (0.01 mg/mL diluted in 1% BSA/PBS); fields tested with purified unlabeled antibodies (antibodies against HSP90, HSPA5, HSP90B1 and HSP110) were incubated with Cy3-labeled goat anti-mouse IgG (0.01 µM in 1% BSA/PBS-T 0.1%). All incubations were left to react for 1h at R.T. in saturated water vapors and then slides were washed for 3 × 5 min with PBS-T 0.1%, 10 seconds in DI water and then dried.

Figure 2

 2 Figure 2 Design of protein microarray. 4 proteins were spotted per slide. Each field includes buffer (negative control), streptavidin-F555 (quality control), one protein at 4 different concentrations; each solution has 8 replications. HSPB1, HSPD1, HSP70 and P53 were spotted on the same slide; HSP90, HSPA5, HSP90B1 and HSP110 were spotted on another slide. For the incubation, HSPB1, HSPD1, HSP70 and P53 were firstly incubated with biotinlabeled antibodies then with streptavidin-F555; HSP90, HSPA5, HSP90B1 and HSP110 were firstly incubated with non-labeled antibodies and then with Cy3-labeled secondary antibody.

Figure 3

 3 Figure 3 Design of microstructured protein microarray. Each microwell contains streptavidin-F555, buffer solution, HSPB1, HSPD1, HSP70, HSP90, HSPA5, HSP90B1, HSP110 and P53 spotted in 5 replicates. Line 1 and 2 were incubated with purified antibody solution for positive control; line 3 was incubated with buffer solution for negative control; lines 4 to 10 were incubated with serum from breast cancer patients and healthy donors.

Figure 4

 4 Figure 4 Fluorescent scanning images of anti-HSP110 antibody detection on the 6 surface chemistries; line 1: buffer; line 2: 0.005 mg/ml, line3: 0.01 mg/ml, line 4: 0.05 mg/ml, line 5: 0.1 mg/ml (HSP110 spotting concentration).

Figure 5

 5 Figure 5 represents the fluorescent signal (SNR) obtained for the detection of anti-HSPD1antibody (Figure5a) and anti-HSP70 antibody (Figure5b) on the 6 surface chemistries, respectively. As expected, SNR increased with increasing of immobilized tumor antigen concentrations. Moreover, the dynamic range of SNR depended on microarray surface chemistry. The detection of anti-HSPD1 antibody and anti-HSP70 antibody on NHS, APDMES and MAMVE surface was not efficient whatever the spotting concentration of the tumor antigens. In contrast, the detection of anti-HSP antibodies on COOH and chitosan

Figure 5

 5 Figure 5 Fluorescent signal analysis (SNR) of anti-HSPD1 antibody (a) and anti-HSP70 antibody (b) detection on 6 surface chemistries.

Figure 6

 6 Figure 6 gathered the results obtained for all studied antigens immobilized on the 6 different chemically functionalized glass slides, at 0.1mg/ml. For almost all antigens, NHS, APDMES and MAMVE surfaces displayed very low SNR even when the spotted antigen concentration is the highest, which suggest that these 3 surfaces didn't lead to efficient immobilization of HSPs and P53 allowing the sensitive detection of anti-HSPs and anti-P53 antibodies. In contrast, antigens immobilized on COOH and chitosan surfaces displayed enhanced detection of their corresponding antibodies. Thus surface chemistry greatly influences the performance of antigen microarray.

Figure 6

 6 Figure 6 Signal analysis of the recognition between 8 immobilized antigens and their antibodies on the 6 different chemically functionalized glass slides. Antigens were immobilized at 0.1 mg/ml.

Figure 7

 7 Figure 7 Fluorescence images of characteristic microwells of antigen microarray on COOH surface. (a) spotting map of each microwell, (b) microwell incubated with PBS 1X buffer solution (blank), (c) microwell incubated with purified anti-HSP110 antibody, (d) microwell incubated with breast cancer serum, (e) microwell incubated with healthy donor serum.

Figure 8

 8 Figure 8 Scatter plots of SNR values of autoantibodies against 7 HSPs (HSPB1, HSPD1, HSP70, HSP90, HSPA5, HSP90B1 and HSP110) and P53 in breast cancer patients (n=50) and healthy controls (n=26); (a) and (b) represent the results on COOH and chitosan surfaces, respectively. HC: healthy controls; BC: breast cancer; *: p < 0.05 (p value was calculated by Mann-Whitney test).

Figure 10

 10 Figure 10 Correlation between the presence of autoantibodies against HSPs and P53 with tumor stage obtained on COOH(a) and chitosan (b) surfaces; *: p < 0.05 (p value was calculated by Mann-Whitney test).

  Biotech and Thermo Scientific (Clone number: U-16); anti-PAI-1 antibodies (mouse monoclonal) were purchased from Santa Cruz Biotech and Abcam (clone number: 3A120); anti-PAI-1 scFv antibody (sheep monoclonal, clone number: 1040.1518.5H8) was obtained from Randox Life Science; Femtelle test for uPA and PAI-1 was purchased from American Diagnostica Inc; F555-labeled streptavidin was purchased from Invitrogen. All proteins were stored as aliquot at -20°C or -80°C following manufacturer specifications. Bovine serum albumin (BSA) lyophilized powder was obtained from Sigma. 0.01 M PBS or PBS 1X (pH 7.4) was prepared by dissolving the content of one pouch of dried powder in 1 L of ultrapure water. 0.02 M sodium carbonate buffer at pH 10.7 was prepared from 0.1 M NaHCO 3 and 0.1 M Na 2 CO 3 solutions in ultrapure water. Washing solution contained PBS 1X and 0.1% Tween 20 (PBS-T) at pH 7.4. Blocking solution was prepared by dissolving 10 g of BSA in 100 ml of PBS-T.

Figure 1

 1 Figure 1 Scheme of antibody microarray design for optimizing the concentration of spotted antibodies and biotin-labeled detection antibodies. Spotting: lines 1 to 3 were anti-uPA antibody; lines 4 to 6 were anti-PAI-1 antibody; each antibody was spotted in PBS buffer at 3 concentrations (0.33 µM, 0.67 µM and 2.5 µM) with 6 replications for each concentration. 1 st incubation: lines 1 to 3, uPA at 6 different concentrations (each concentration incubated in two microwells); lines 4 to 6, PAI-1at 6 different concentrations (each concentration incubated in two microwells). 2 nd incubation was performed with not diluted and diluted (1:2) biotinylated antibodies.

Figure 2

 2 Figure 2 Scheme of antibody microarray design for optimizing the spotting buffer of anti-PAI-1 scFv antibody. Spotting: anti-PAI-1 scFv antibody was spotted at 5 µM in all three buffers; lines 1, 2: acetate buffer (pH=4.6); lines 3, 4: PBS 1X (pH=7.4); lines 5, 6: carbonate buffer (pH=9.6). 1 st incubation: 8 concentrations of PAI-1 were tested; 2 nd incubation: biotinylated anti-PAI-1 antibodies were incubated at dilution 1:2.

Figure 3

 3 Figure 3 Design of antibody microarray for the quantification of uPA in breast tumor tissue extracts. In each micro-well, anti-uPA antibody was spotted at two concentrations: 3 µM and 6.6 µM with 14 replications each, PBS 1X (pH=7.4) was used as spotting buffer; PBS 1X (pH=7.4) and streptavidin-F555 were spotted as negative and quality controls, respectively. First incubation was performed with uPA at six different concentrations (6 microwells)); with PBS 1X buffer (2 microwells); breast tumor tissue extracts not diluted (16 microwells); breast tumor tissue extracts diluted 1:2 (for samples with relatively low concentration) or 1:5 (for samples with relatively high concentration) (16 microwells).

Figure 4

 4 Figure 4 Fluorescent signal analysis of uPA detection on COOH, NHS, chitosan, APDMES, CMD and MAMVE surfaces. Spotted concentration of anti-uPA antibody was 2.5µM.

Figure 5 .Figure 5

 55 Figure 5.

Figure 7

 7 Figure 7 ELISA test from Femtelle kit and antibodies against uPA (a) and PAI-1 (b). In (a), anti-uPA antibody 1 and 2 are from Santa Cruz Biotech and Thermo Scientific respectively; in (b), anti-PAI-1 antibody 1 and 2 are from Santa Cruz Biotech and Abcam respectively.

Figure 7a indicated that

  Figure7aindicated that both anti-uPA antibodies tested displayed good biological activity for detecting uPA in ELISA, which is consistent with results obtained from antibody microarray. However, their biological recognition towards uPA appeared less sensitive compared to commercial Femtelle kit. Indeed in commercial ELISA kit such as Femtelle kit, antibody/antigen affinity is very high as the process is optimized to get best sensitivity and specificity of the assay.

Figure 8

 8 Figure8 Detection of uPA on chitosan surface as a function of anti-uPA antibody spotted concentration of (3µM and 6.6 µM); limit of detection (LOD) of uPA for each spotted concentration was indicated.

Figure 10

 10 Figure 10 Comparison of diluted and non-diluted biotinylated antibody against uPA and PAI-1 on the detection of uPA with capture anti-uPA (from Santa Cruz Biotech) immobilized on chitosan surface at 2.5 µM (a) and on the detection of PAI-1 with capture anti-PAI-1 scFv immobilized on NHS surface at 5 µM (b).

Figure 11

 11 Figure 11 Evaluation of the biological activity of immobilized anti-PAI-1 antibody scFv (5 µM) on COOH (a), NHS (b) and chitosan (c) surfaces using three different spotting buffers: acetate buffer (pH=4.6), PBS 1X (pH=7.4), carbonate buffer (pH=9.6).

  Moreover, in order to match the dynamic range of the standard titration curve, and taking account of the expected value given by Femtelle tests, cytosolic extracts were tested on antibody microarray under two conditions: non-diluted (microwell lines 3 to 6) and diluted 1:2 (No.1-11) or 1:5 (No.12-16) (microwell lines 7 to 10).

Figure 12 3 . 3 . 2 . 1

 123321 Figure 12 Scheme of antibody microarray design for uPA concentration measurement

Figure 15 Figure 15

 1515 Figure 15 (a) represented uPA standard curve obtained from Femtelle kit; (b) and (c) represented uPA standard curve obtained from our customized antibody microarray when anti-uPA antibody was immobilized on COOH surface at 3 µM and 6.6 µM respectively. (d) and (e) corresponds to NHS surface and (f) and (g) to chitosan surface.

line 5 -Figure 16

 516 Figure 16 (a) represents the scheme of antibody microarray design; (b) represents the fluorescence image obtained on COOH surface for detecting the concentration of uPA in tumor cytosolic extracts: line 1-4 were incubated with non-diluted cytosolic extracts; line 5-8 were incubated with corresponding diluted cytosolic extracts.

Figure 17 (

 17 a) (c) and (e) compared the results of ELISA with those obtained from protein microarray with COOH, NHS and chitosan surface, respectively. Figure 17 (a) (c) and (e) compared the results of ELISA with those obtained from protein microarray with COOH, NHS and chitosan surface, respectively; (b) (d) and (f) compared the SNR of samples with standard curve obtained from COOH, NHS and chitosan surface, respectively.

2

 2 Surface functionalization of flat and microstructured glass slides 4.2.3 Elaboration and processing of protein microarray 4.2.4 Stability of chemically functionalized glass slides 4.2.4.1 Contact angle measurement 4.2.4.2 Immobilization of probe molecules 4.2.5 Storage of spotted slides 4.2.6 Evaluation of protein microarray reproducibility 4.2.7 Fluorescence scanning and data analysis 4.3 Results and discussion 4.3.1 Optimization of elaboration and processing of protein microarray 4.3.1.1 Influence of blocking, antibody and detection incubation time 4.3.1.2 Influence of detection antibodies and streptavidin-F555 concentrations 4.3.2 Evaluation of the stability of protein microarray under storage 4.3.2.1 Stability of the surface chemistries of protein microarray 4.3.2.2 Stability of spotted protein microarray 4.3.3 Reproducibility of protein microarray 4.4 Conclusion References

  (a) and (b) represent the design for the optimization of experimental time and concentration of solution respectively.

Table 1

 1 Experiment design of optimization. (a) and (b) represent the optimization of experimental time and concentration of solution respectively.

Figure 3

 3 Figure 3 Scheme of protein microarray design. 4 antigen proteins (HSPD1, HSP70, HSPA5 and HSP110) and streptavidin-cy3 (Strep-cy3) were spotted onto COOH and CMD functionalized flat glass slides. Each field contains buffer spots and one protein at two concentrations (0.05 mg/mL and 0.1 mg/mL), each concentration has 48 replications."Strep-cy3" field was incubated with buffer solution; HSPD1 and HSP70 fields were firstly incubated with biotinylated antibody and then with streptavidin-F555; HSPA5 and HSP110 fields were firstly incubated with unlabeled mouse antibody and then with goat-anti-mouse IgG-cy3.

Figure 4

 4 Figure 4 Design of protein spotting on flat glass slide (a) and micro-structured glass slide (b). On both slides, 4 proteins (P53, BSA-biotin, anti-P53 antibody and streptavidin-F555) were spotted and each of them has 72 replications.

Figure 5

 5 Figure 5 Scheme of ELISA; C: concentration; Vol: volume; R.T. room temperature.

Figure 7

 7 Figure 7 Influence of blocking time (2hr, 1hr and 30min) on the efficiency of antigen/antibody recognition on NHS surface, as a function of spotted antigen concentration. (a) HSPD1/anti-HSPD1 antibody recognition; (b) P53/anti-P53 antibody recognition.

Figure 8 Figure 9 Figure 9

 899 Figure 8 Influence of blocking time (30min, 1hr and 2hr) on the fluorescence signal of buffer of HSPD1 and p53.

Figure 10

 10 Figure 10 Influence of detection time (30min and 1hr) on the efficiency of antigen/antibody recognition on NHS surface. (a) HSPD1/anti-HSPD1recognition, (b) P53/anti-P53 recognition, (c) Fluorescence intensity (FI) of buffer

Figure 12b .Figure 12

 12b12 Figure 12b. Higher concentration of first antibody could increase unspecific binding;therefore, in order to improve the performance as well as to be more economic, 0.1 µM of first antibody will be used in further experiment.

Figure 14 Table 4

 144 Figure14 Influence of the concentration of IgG-cy3 on the performance of HSP90/anti-HSP90 detection; 2 concentrations (0.01µM and 0.05µM) were tested.

Figure 15

 15 Figure 15 Variation of total surface energy (a), polar energy (b) and dispersive energy (c) of the 6 surface chemistries developed for protein microarray with storage time under dry condition.

Figure 16

 16 Figure 16 Blotch observed on CMD surface after storage through optical microscope.

Table 5 Figure 17

 517 Figure 17 Fluorescent intensity of streptavidin-Cy3, IgG-cy3 and ODN-cy3 spotted on chitosan surface (a) and MAMVE surface (b) as a function of storage time.

Figure 18

 18 Figure 18 Biological recognition activity of P53 (a), fluorescence intensity of buffer spots (b), biological recognition activity of HSPD1 (c), fluorescence intensity of buffer spots (d) immobilized on NHS surface after 1 month of storage in various conditions (50% glycerol solution, 5% trehalose solution, N2 atmosphere).

Figure 19

 19 Figure 19 Biological recognition activity of P53 and HSPD1 immobilized on NHS (a) and chitosan (b) surfaces after 1 month of storage after blocking in various conditions (50% glycerol solution, 5% trehalose solution, N2 atmosphere).

Figure 20

 20 Figure 20 Fluorescence signal of immobilized streptavidin-F555 (a) and spotted buffer solution (b) on COOH and CMD surfaces after different time of storage in 50% glycerol solution.

Figure 21

 21 Figure 21 Evaluation of the biological activity of immobilized antigens (HSPD1, HSP110 and HSPA5) on COOH (a) and CMD (b) surfaces after storage in 50% glycerol solution after blocking, at different time; FI: fluorescence intensity.

Figure 1 français 1 . 1

 111 Figure 1 c. Then we continued and we could obtain the complete ROC curve for HSPA5 immobilized on chitosan surface.

Figure 1 .

 1 Figure 1. Incidence estimée et Taux de mortalité des cancers au niveau mondial en 2012 [2]

Chapitre 1 :

 1 Dans ce contexte, grâce à leur capacité d'analyse haut débit et le faible volume d'échantillon nécessaire, les puces à protéine (protein microarray) présentent de nombreux avantages pour le criblage des biomarqueurs et la mise au point d'outils de diagnostic et de pronostic à visée de médecine personnalisée. De nombreux biomarqueurs du cancer du sein, qu'ils soient sériques ou tissulaires, sont décrits dans la littérature avec des valeurs diagnostiques et pronostiques faibles lorsqu'ils sont pris indépendamment. Un des challenges est donc d'identifier les combinaisons de biomarqueurs permettant d'atteindre des valeurs diagnostiques et pronostiques élevées. Ceci constitue un des objectifs de cette thèse. Pour ce faire, nous avons élaboré des puces à antigènes à façon, afin d'optimiser leurs performances. La chimie de surface, les conditions d'immobilisation des antigènes sondes ainsi que les conditions de reconnaissance avec leurs anticorps ont été optimisés et validés par l'étude d'une cohorte de 50 patientes. Un autre objectif de cette thèse était de développer une puce à anticorps pour le dosage des marqueurs tissulaires du cancer du sein, uPA et PAI-1, permettant une décision thérapeutique pour les malades. Les performances de notre test miniaturisé ont été comparées à celles du test commercial ELISA sur 16 échantillons biologiques. Le manuscrit est donc présenté sous forme de 4 chapitres. Le chapitre 1 dresse un état de l'art des biomarqueurs sérologiques et tissulaires décrits dans le cancer du sein ainsi que les récents développements d'outils de criblage comme les puces à protéines. Le chapitre 2 est consacré à l'élaboration des puces à antigènes pour l'analyse des profils d'expression d'anticorps anti-heat shock protein (anti-hsps) chez des patientes atteintes de cancer du sein. Le chapitre 3 concerne l'élaboration d'un immunoassay miniaturisé pour le dosage des protéines uPA et PAI-1 à partir d'extraits cytosoliques de tissus tumoraux de cancer du sein. Le chapitre 4 traite de l'optimisation des différentes étapes d'élaboration et d'utilisation des puces à protéines. Etat de l'artAu cours de la génèse tumorale, les cellules cancéreuses ou d'autres cellules du corps vont produire des substances en réponse aux nouvelles conditions induites par la progression du cancer. Ces substances appelées marqueurs tumoraux peuvent avoir des niveaux d'expression différents et se trouvent dans le sang, les urines, les tissus tumoraux ou autres fluides des patients. Dépister ces marqueurs peut aider non seulement à la détection précoce de cancer mais aussi à choisir l'option la plus pertinente parmi les différents traitements disponibles. Le suivi de certains marqueurs surexprimés doit permettre de vérifier et valider le bénéfice du traitement choisi[14, 15]. La problématique actuelle n'est pas le manque de candidats biomarqueurs mais plutôt la validation de leur pertinence. Ainsi, plus de 1200 protéines candidates ont été décrites dans la littérature comme biomarqueurs potentiels mais seulement 9 antigènes tumoraux ont été approuvés par la FDA (US Food and Drug Administration). Le taux d'introduction de nouvelles protéines approuvées par la FDA stagne à environ une par an sur les 15 dernières années pour l'ensemble des maladies.Dans la suite, nous nous focaliserons sur les marqueurs sériques et ceux des tissus recommandés par l'ASCO (American Society of Clinical Oncology) et l'EGTM (European Group on Tumor Markers Recommandations) pour une utilisation en routine.Les performances des biomarqueurs en qualité de tests diagnostiques sont évaluées à l'aide de plusieurs indicateurs tels que les positifs, les négatifs, les faux positifs, les faux négatifs, la sensibilité, la spécificité, la valeur prédictive positive et la valeur prédictive négative. Les méthodes pour calculer ces paramètres sont issues de la méthodologie biostatistique utilisées en Epidemiologie[16] avec notamment la détermination de la courbe de caractéristique de performance d'un test (courbe ROC -Receiver Operating Characteristic), l'aire sous cette courbe notée AUC (area under the ROC curve) et la valeur p qui représente la valeur de significativité de l'hypothèse. En épidémiologie, deux niveaux de significativité sont généralement utilisés (P <0.05 and P<0.01)[17].Les courbes ROC sont particulièrement utilisées en statistiques lorsque le seuil de discrimination varie. Cela est le cas en biologie moléculaire compte tenu d'une part de la complexité des fluides et d'autre part de la grande variabilité des niveaux de seuil d'un individu à l'autre.

3 )

 3 les études doivent être menées à grande échelle, ce qui nécessite le développement d'outils haut débit. Comparés aux analyses actuelles par simple immunoassays, les systèmes multiplexes présentent d'indéniables avantages tels que l'augmentation de l'efficacité, la réduction des coûts, un plus grand nombre de paramètres mesurés pour un même volume d'échantillons et le traitement en parallèle d'un très grand nombre d'échantillons.

[ 39 ]

 39 . Par exemple, Lubin et al. ont détecté des anticorps p53 spécifiques environ 18 mois avant qu'un diagnostic de cancer du poumon soit établi cliniquement [40]. Le système immunitaire permet une amplification biologique efficace conduisant à une concentration élevée d'auto-anticorps permettant une détection indirecte de très faibles quantités d'antigènes tumoraux. De plus , ces auto-anticorps sont très stables dans les sérums et ont une durée de vie relativement longue (T1/2 entre 7 and 30 jours, selon la sous classe d'immunoglobuline) [41-46].Ces dernières années, plusieurs études ont été menées sur différents AAbs contre desTAAs en évaluant leurs valeurs diagnostiques et pronostiques notamment pour les cancers du sein. En particulier, un vif intérêt a été porté sur des anticorps dirigés contre des protéines hsp (heat shock proteins). Les Hsp sont des protéines de forte conservation classées en 6 familles selon leur poids moléculaire (MW): hsp110, hsp90, hsp70, hsp60, hsp40 et un ensemble de petites protéines hsp (dans la gamme de 13-42kD) incluant hsp27 and hsp10.[47]. Les protéines hsp sont surexprimées dans une grande partie des cancers humains. Cette surexpression élevée des hsp dans les cellules malignes joue un rôle important de protection des cellules contre l'apoptose spontanée induite par la malignité[48]. Dans notre étude bibliographique, nous avons trouvé 6 rapports décrivant l'utilisation de tests unitaires d'hsps dirigés contre les auto-anticorps (AAbs) afin de discriminer les patientes atteintes d'un cancer du sein des contrôles sains. En utilisant des tests ELISA, Conroy et al. ont mené en 1995 la première étude pour identifier la présence d'auto-anticorps anti-hsp90 sur des patientes ayant un cancer du sein diagnostiqué. Ils ont trouvé que les anticorps ciblés étaient détectables dans 46 cas sur 125 (36.8%) patientes atteintes d'un cancer mais pas dans le cas d'individus sains ou ayant une tumeur bénigne. De plus, la présence de ces anticorps était corrélée avec le développement de métastases même sur les personnes non atteintes aux ganglions lymphatiques axillaires [49].Le tableau suivant regroupe les résultats publiés.

  stratégies d'immobilisation de protéines sur un support Jusqu'à présent; il est impossible de comparer les études issues de différents laboratoires compte tenu des différences dans les protocoles expérimentaux et les protéines utilisées.Cependant, de la revue bibliographique, un point parait évident : il n'y a pas une surface unique parfaitement adaptée à l'immobilisation de toutes les protéines compte tenu de la complexité de leur structure. Chaque type de protéine a un comportement différent sur chacune des surfaces. Aussi, il est nécessaire de sélectionner finement les microarrays avec les surfaces les mieux appropriées.En plus de la chimie de surface, de nombreux facteurs influencent les performances des microarrays tels que (a) la composition de la solution de spotting , (b) le taux d'humidité Durant le spotting (c) la concentration des cAbs, (d) le temps d'incubation et le séchage de la solution de spotting (e) la composition du tampon de blocage (f) le temps de blocage, (g) le tampon utilisé pour la dilution des échantillons, (h) le temps d'incubation des échantillons (i) la température, (j) le niveau d' agitation et de mélange durant l' incubation, (k) la composition du tampon de rinçage (l) l'agitation durant le rinçage, (m) la composition du tampon de détection, (n) la concentration des dAbs, (o) le temps d'incubation des dAbs, (p) l'affinité et la stabilité des dAbs, (q) la concentration des marqueurs de détection (e.g., Ab secondaire ou la streptavidine marquée, (r) la composition du tampon buffer, (s) le temps de l'incubation (t) la nature du marqueur de détection (fluorescent ou enzyme), etc…[98]. Un enjeu important pour le développement futur des microarrays multiplexes est d'identifier les facteurs critiques et de les optimiser.Aujourd'hui, plusieurs sociétés ont commercialisé des microarrays à protéines pour la détection et l'analyse de protéines dans des échantillons humains tels que le serum, l'urine, le tissu, etc…Le tableau suivant recense ces différents dispositifs et listent leurs caractéristiques.

Chapitre 2 :

 2 Criblage d'autoanticorps Anti HSP dans les sera de cancer du sein à l'aide de microarrays à protéines faits à façon Le chapitre 2 est consacré à l'élaboration des puces à antigènes pour l'analyse des profils d'expression d'anticorps anti-heat shock protein (anti-hsps) chez des patientes atteintes de cancer du sein. En effet, de nombreuses études ont mis en évidence des taux élevés d'anticorps anti-hsps dans le sérum de patientes atteintes de cancer du sein, certains d'entreeux étant associés à la progression de la maladie. Toutefois ces études ont porté sur l'analyse d'un ou deux anticorps anti-hsps simultanément, ce qui n'a pas permis de dégager un réel intérêt diagnostique ou pronostique de tels marqueurs. Ainsi, en collaboration avec le CHU de Montpellier, nous avons sélectionné 7 protéines appartenant à la famille des « heat shock proteins » (hsp27, hsp60, hsp70, hsp90, hsp110, grp78, grp94) comme antigènes sondes pour l'élaboration des puces à antigène. Nous avons également inclus dans ce panel, la protéine p53 largement décrite pour induire la production d'anticorps anti-p53 associés à un mauvais pronostic du cancer du sein.Dans un premier temps, nous avons étudié l'influence de la chimie de surface et des conditions d'immobilisation des antigènes sondes sur les performances de la reconnaissance antigène-anticorps afin de définir les conditions optimales pour le criblage des sérums de patientes. En effet, lors d'une étude précédente, 6 chimies de surfaces différentes ont été développées dans l'équipe pour l'immobilisation covalente ou non de protéines sur support de verre. Il s'agit de surfaces fonctionnalisées avec un silane carboxylé (surface COOH), avec le silane carboxylé activé (surface NHS), avec du chitosan (surface chitosan), avec un silane aminé (surface APDMES), avec un carboxyméthyl dextran (surface CMD), ou encore avec un polymère d'anhydride maléique (surface MAMVE).Quatre concentrations de dépôt d'antigène sonde (de 0.005 mg/mL à 0.1 mg/mL) ont été testées pour leur capacité à reconnaitre de façon spécifique et sensible les anticorps correspondants. La figure suivante montre les rapports signal sur bruit (SNR) obtenus après la reconnaissance entre antigènes sondes immobilisées à 0.1 mg/ml et les anticorps purifiés incubés sur les 6 chimies de surface étudiées. Parmi les 6 chimies de surface évaluées, 3 surfaces (COOH, chitosan et CMD) permettent une détection efficace des 8 anticorps testés et ceci même pour une concentration de 0,05 mg/mL en antigène sonde déposée. En tenant compte de ces conditions, 50 sérums de patientes atteintes de cancer du sein et 26 sérums de donneurs sains ont été évalués pour la présence des anti-hsps et de l'anti-p53. Des lames de verres micro-structurées et fonctionnalisées avec les chimies de surface COOH, chitosan et CMD ont été utilisées. La figure suivante montre la conception d'une lame sur laquelle sont gravées 40 micropuits. Dans chaque micropuits, sont spottées 8 protéines différentes (7 hsp et P53 ) en 5 réplicas dans leur concentrations optimales et avec le tampon adéquat qui dépend de la chimie de surface faite préalablement sur la surface ; ainsi sur des surfaces COOH, on utilise un tampon acetate (pH= 4.5) tandis que sur des surfaces de chitosan, on utilise un tampon carbonate de pH 9.6. Enfin, des plots contenant uniquement de la solution tampon ou de la streptavidine-F555 sont réalisés pour les contrôles négatifs et de qualité du microarray. Après le spotting, les lames sont laissées une nuit à 4°C sous hygrométrie contrôlée pour laisser la réaction d'immobilisation se faire. Les lames sont ensuite rincées soigneusement dans du PBS puis une étape de blocage avec une solution de 10% BSA/PBS-T 0.1% à température ambiante est réalisée pendant 2 heures afin de limiter ultérieurement les phénomènes d'adsorption non spécifique. Enfin les lames sont rincées 3X5 minutes dans du PBS-T 0.1% et séchées 3 minutes par centrifugation à 1300 tours par minute (rpm). Incubation avec des Anticorps purifiés Incubation avec Tampon Incubation avec des sera de cancer du sein Incubation avec des sera de patients sains Dans chaque micropuits En présence de sérum, les lames fonctionnalisées avec le CMD présentaient un bruit de fond très fort, empêchant leur exploitation. En revanche, la surface COOH permet de discriminer de façon significative (p<0,05) les sérums cancéreux des sérums sains vis-à-vis de la présence des anticorps anti-hsp60, anti-hsp70, anti-hsp90 et anti-grp94. Les taux d'anticorps anti-hsp27 et anti-grp78 sont significativement différents entre les sérums cancéreux et les sérums sains (p< 0,05) lorsqu'ils sont évalués sur la surface fonctionnalisée avec le chitosan. Par contre, aucune des surfaces testées ne permet de discriminer les 2 populations de sérums par rapport aux anticorps anti-hsp110 et anti-p53. A partir de ces résultats, nous avons construit les courbes ROC (Receiver Operating Characteristic) et calculer l'aire sous la courbe correspondante (AUC) pour chaque anticorps dans ses meilleures conditions de détection et de discrimination. Pour qu'un biomarqueur soit utilisable en clinique pour du diagnostic ou du pronostic, il faut que l'AUC soit supérieur à 0,75. Ainsi, si on considère chaque anticorps anti-hsp indépendamment, les valeurs des AUC varient de 0,576 à 0,731, indiquant que la détection unique d'un anti-hsp n'a pas de valeur clinique. Toutefois, si on considère la détection simultanée des 7 anticorps (anti-hsp27, anti-hsp60, anti-hsp70, anti-hsp90, anti-grp78, anti-grp94 et anti-p53) dans les conditions optimales, la valeur AUC obtenue est de 0,912. Ceci confirme que la détection d'un panel de biomarqueurs dans des conditions optimales permet d'augmenter significativement la valeur diagnostique et/ou pronostique du test. Aucune corrélation n'a pu être mise en évidence entre la présence des anticorps anti-hsp et anti-p53 et le stade de la maladie. Courbes ROC des autoanticorps pris individuellement et en noir pour une combinaison de 7 autoanticorps et table rassemblant les valeurs de AUC extraites des courbes afin de discriminer les patientes ayant un cancer du sein des contrôles sains Anti-HSP antibody AUC (95% CI) Anti-HSPB1 antibody* 0.631 (0.468-0.739) Anti-HSPD1 antibody** 0.683 (0.592-0.781) autoanticorps anti-HSPs étudiés dans notre travail ont été évalués dans d'autres travaux. Le tableau suivant donne un premier aperçu de nos résultats comparés à ces travaux. Cependant, la taille de notre échantillonnage (50 patientes atteintes de cancer et 26 donneurs sains) est trop limitée pour pouvoir conclure définitivement. Il sera donc nécessaire d'évaluer ces paramètres sur un échantillonnage incluant plusieurs centaines de personnes.

Chapitre 3 :Chapitre 4 :

 34 Elaboration d'un microarray à anticorps pour le dosage des biomarqueurs tissulaires uPA et PAI-1 dans les tumeurs du sein Le chapitre 3 concerne l'élaboration d'un immunoassay miniaturisé pour le dosage des protéines uPA et PAI-1 à partir d'extraits cytosoliques de tissus tumoraux de cancer du sein. En effet, uPA (urokinase Plasminogen Activator) et PAI-1 (Plasminogen Activator Inhibitor-1) sont 2 biomarqueurs tissulaires reconnus du développement du cancer du sein et de sa dissémination métastatique. Leur niveau d'expression dans les tumeurs du sein permet de classifier les patientes et de leur apporter un traitement adapté. De faibles taux d'uPA (< 3 ng/mg de protéines totales) et de PAI-1 (< 14 ng/mg de protéines totales) sont de bon pronostique et permettent d'éviter un traitement lourd sans réel bénéfice pour les patientes. Alors que de forts taux d'uPA et de PAI-1 sont corrélés à un haut risque de récidive, un traitement de chimiothérapie adjuvante est nécessaire afin de réduire ce risque. A l'heure actuelle, un seul test (Femtelle®, Sekisui Diagnostics) basé sur une méthode immunologique (ELISA) permet de doser ces 2 biomarqueurs dans des extraits cytosoliques obtenus à partir de tissus tumoraux frais ou congelés. Cependant, cela nécessite de disposer au minimum de 300 mg de tissu tumoral, ce qui est une limitation importante notamment pour des tumeurs à un stade très précoce. Une solution proposée est donc de développer un test immunologique miniaturisé basé sur la technologie des biopuces à protéine, et permettant de doser les 2 biomarqueurs considérés à partir de quelques milligrammes de tissu tumoral. Ce travail a été réalisé en collaboration avec l'Institut de Cancer de Montpellier. Les études précédentes menées dans l'équipe ont permis de développer des chimies de surface adaptées à l'immobilisation des protéines. Elles ont également mis en évidence l'importance d'adapter la chimie de surface et les conditions d'immobilisation (concentration, solution tampon de dépôt) à la protéine à immobiliser afin de conserver au maximum son activité biologique. Ainsi, dans un premier temps, nous avons optimisé les conditions d'immobilisation des anticorps anti-uPA et anti-PAI-1 de façon à avoir une détection sensible des marqueurs uPA et PAI-1. Puis nous avons réalisé, dans les conditions optimales définies, le dosage de uPA dans des extraits cytosoliques de tissus tumoraux de cancer du sein, fournis par le Centre de Ressources Biologiques de Montpellier. Nous avons alors comparés nos résultats avec ceux obtenus par le kit Femtelle. des quantités 10 fois inférieures en uPA, et 6 fois inférieures en PAI-1 peuvent être détectées et dosées par notre système, et ceci en consommant 100 fois moins d'échantillon biologique. Il reste cependant à évaluer les performances de notre microarray pour le dosage de PAI-1 en échantillons cytosoliques, puis à évaluer le dosage des 2 biomarqueurs dans des échantillons issus de tissus parafinés. Optimisation de l'élaboration des microarrays à protéines et de leurs conditions d'utilisation Le chapitre 4 traite de l'optimisation des différentes étapes d'élaboration et d'utilisation des puces à protéines. En effet, dans un but d'utilisation en clinique, il est essentiel de contrôler, d'optimiser et de stabiliser chaque étape de fabrication des puces depuis la fonctionnalisation chimique de la surface jusqu'à l'étape de détection de l'évènement de reconnaissance biologique.La stabilité des 6 chimies de surface utilisées a donc été évaluée au cours du temps (tous les mois pendant 6 mois) après stockage dans une enceinte contenant un desséchant. Cette évaluation a été réalisée par mesure de l'angle de contact et calcul de l'énergie de surface, ainsi que leur capacité à immobiliser des protéines marquées par un fluorophore. En effet, l'énergie de surface est directement corrélée à l'état de la surface et à sa composition chimique. Toute variation de l'énergie de surface implique donc une modification de la composition chimique de surface. Les résultats obtenus mettent en évidence la grande stabilité des chimies de surface COOH, NHS, chitosan, APDMES et CMD dans ces conditions. Seule la chimie de surface MAMVE présente une forte augmentation de son énergie de surface au cours du temps, notamment de la contribution polaire. Ceci peut s'expliquer par l'hydrolyse des fonctions anhydride maléique suite à l'exposition à l'humidité ambiante, conduisant à la formation de groupements carboxyliques à caractère plus polaire. Par ailleurs, l'immobilisation de différentes molécules marquées (streptavidine-Cy3, anticorps-Cy3, ADN-Cy3) sur les 6 chimies de surface, diminue de façon plus ou moins importante selon les molécules et les surfaces après 3 mois de stockage. Ces résultats combinés aux précédents suggèrent que les chimies de surface développées au laboratoire pour l'élaboration de puces à protéines sont stables jusqu'à 3 mois de stockage dans une enceinte en présence de desséchant.Nous avons également évalué la stabilité des protéines sondes (hsp60, hsp70, hsp110, grp78 et p53) immobilisées sur les puces (surfaces COOH, NHS, chitosan et CMD) après stockage dans différentes conditions (sous azote, dans une solution à 50% de glycérol, dans une solution à 5% de tréhalose, avant « capping » de la surface ou après). Cette étude a été réalisée par mesure de l'activité biologique des protéines immobilisées, c'est-à-dire leur capacité à reconnaitre leur anticorps spécifique. Les résultats indiquent que la stabilité des protéines sondes immobilisées est meilleure lorsqu'elles sont stockées après « capping » dans une solution à 50% de glycérol. Cependant leur activité biologique décroit avec le temps de stockage, mais permet une détection sensible des anticorps jusqu'à 3 mois de stockage. Ceci est en accord avec les résultats précédents concernant la stabilité des chimies de surface.Nous avons ensuite optimisé les temps d'incubation relatifs aux différentes étapes de traitement des puces à protéines. Ainsi, après le dépôt des protéines sondes, un blocage de la surface avec une solution 10% BSA/PBS 1X pendant 1 heure est suffisant pour saturer tous les sites non spécifiques. Puis l'anticorps de détection biotinylé doit être incubé 1 heure à une concentration de 0,1 µM au lieu de 0,5 µM pour avoir une détection sensible de la reconnaissance antigène-anticorps. Enfin, il est possible de réduire de moitié le temps d'incubation avec la streptavidine-Cy3 (soit 30 minutes au lieu d'1 heure), et la concentration d'un facteur 4 (soit 0,05 µM au lieu de 0,2 µM). Ainsi, l'utilisation de systèmes miniaturisés tels que les puces à protéines pour étudier les interactions antigène-anticorps permet de réduire d'une part le temps d'analyse et d'autre part le coût par rapport à des systèmes classiques tels l'ELISA.

Table of contents

 of

	Acknowledge
	Abstract
	Abbreviations
	Table of contents

General introduction Chapter 1 State of the art 13 1.1 Breast cancer: Key data and detection techniques 1.2 Tumor biomarkers

1.2.1 Indicators of biomarker value and data analysis 1.2.2 Serum biomarkers used in clinic 1.2.3 Tissue biomarkers used in clinic 1.3 Autoantibodies (AAbs)-Diagnostic and prognostic value 1.3.1 Heat shock proteins (HSPs) and AAbs against HSPs 1.3.2 Autoantibodies against other TAAs than HSPs 1.4 Protein microarray 1.4.1 Surface chemistry 1.4.2 Commercial protein microarray 1.4.3 Optimization of assay conditions 1.4.4 Storage conditions of protein microarray 1.

5 Aims of this thesis References Chapter 2 Anti-heat shock proteins autoantibodies profiling in breast cancer serum using customized protein microarrays 85

	2.2.6 Fluorescence scanning
	2.2.7 Data analysis
	2.3 Results
	2.3.1 Optimization of tumor antigen microarray conditions
	2.3.2 Detection of autoantibodies against HSPs and P53 in breast cancer
	sera by multiplex immunoassays on antigen microarray
	2.4
	2.1 Introduction
	2.2 Materials and methods
	2.2.1 Materials
	2.2.2 Serum samples
	2.2.3 Surface functionalization of glass slides
	2.2.4 Design of protein immobilization on flat glass slides
	2.2.5 Multiplex immunoassays on micro-structured protein microarray

Discussion and Conclusions References Chapter 3 Antibody microarray for the quantification of uPA and PAI-1 in breast tumor tissue 3.1 Introduction 3.2 Experiments

3.2.1 Materials 3.2.2 Biological samples 3.2.3 Surface functionalization of microstructured glass slides 3.2.4 Design and Optimization of antibody microarray 3.2.5 Evaluation of the biological activity of antibodies against uPA and PAI-1 with ELISA 3.2.6 Quantification of uPA from breast tumor tissue extracts on antibody microarrays 3.2.7 Fluorescence scanning and data analysis

  . Class Threshold Value No. Class Threshold Value

	1	P	0.9	11	P	0.4
	2	P	0.8	12	N	0.39
	3	N	0.7	13	P	0.38
	4	P	0.6	14	N	0.37
	5	P	0.55	15	N	0.36
	6	P	0.54	16	N	0.35
	7	N	0.53	17	P	0.34
	8	N	0.52	18	N	0.33
	9	P	0.51	19	P	0.30
	10	N	0.505	20	N	0.1

Table 3

 3 Frequency of single autoantibodies in breast cancer patients, benign subjects and

	healthy controls
			sample size (N)	AAb frequency %
	HSP	method	cases	HC	benign	cases	HC	benign	P value reference
	HSPB1 ELISA	579	53	-	37.8%	1.9%	-	p<0.001	[80]
	HSP70 ELISA	369	53	-	40.9%	35.9%	-	-	[80]
	HSP90 ELISA	125	-	-	36.8%	-	-	-	[78]
	HSPD1	WB	40	42	-	47.5%	4.7%	-	p < 0.01	[81]
	HSPD1 ELISA	107	93	-	31.8%	4.3%	-	p<0.0001	[82]
	HSP90 ELISA	13	22	10	8%	0	0	-	[83]
	HC: healthy controls; WB: western blot; ELISA: enzyme linked immunosorbent assay.
	Using ELISA, in 1995, Conroy et al. conducted the first study to identify anti-HSP90
	autoantibody in patients diagnosed with breast cancer. They found that antibodies targeting
	purified HSP90 were detectable in 46/125 (36.8%) breast carcinoma patients but not in
	healthy individuals, or patients with benign breast tumors. Furthermore, the presence of these

Table 4

 4 Frequency of AAbs panels in breast cancer patients and controls

	methods	panel	cases controls	SN/SP	reference
	ELISA	FKBP52, PPIA, PRDX2, HSPD1 and MUC1	142	93	60.5/77.2	[84]
	PM	HSPD1, P53, Her2-Fc, NY-ESO-1 and HSP70	29	28	82.7%	[32]
	HC: healthy controls; ELISA: enzyme linked immunosorbent assay; WB: western blot; PM:
	protein microarray; SN: sensitivity; SP: specificity; AUC: Area under curve.

Table 5

 5 Comparison of anti-HSP AAbs frequencies in breast cancer

	HSPs	method		sample size (N)		AAb frequency %	reference
			cases	HC	benign	cases	HC	benign
	HSPB1	ELISA	579	53	-	37.8%	1.9%	-	[80]
	HSPD1	WB	40	42	-	47.5%	4.7%	-	[81]
		ELISA	107	93	-	31.8%	4.3%	-	[82]
		ELISA	142	93	-	-	-	-	[84]
		PM	29	28	-	-	-	-	[32]
	HSP70	ELISA	369	53	-	40.9%	35.9%	-	[80]
		PM	29	28	-	-	-	-	[32]
	HSP90	ELISA	125	-	-	36.8%	-	-	[78]
		ELISA	13	22	10	8%	0	0	[83]

Table 6

 6 Frequency of autoantibodies in breast cancer patients, benign subjects and healthy controls.

	TAA	sample size (N)	AAb frequency % Reference/year
		cases	HC
	P53	101	-	7.9%	[86] 1999
		2006	-	14.7%	[87] 2000
		158	-	19%	[88] 2003
		71	205	18.3%	[89] 2003
		144	242	21.5%	[90] 2005
		50	436	34%	[91] 2006
		25	879	16%	[92] 2009
		61	20	35%	[93] 2010
	HER2	20	-	55%	[94] 1994
		107	200	11.2%	[95] 1997
		37	157	7%	[96] 2000
	MUC1	24	-	8.3%	[97] 1994
		40 a		37.5%
		140 b	96	25.7%	[98] 1996
		61 c		18%
	c-myb	72	49	43%	[99] 1991
	fibulin	20	20	75%	[100]
	RPA32	801	65	10.9%	[101]
	lipophilin B	74	20	27%	[102]

Table 7

 7 Frequency of anti-TAA AAbs panels in breast cancer and healthy controls breast carcinoma and 346 normal subjects. It yielded a sensitivity of 92% and specificity of 85% [123]. Another group studied 8 autoantibodies in 41 cancer patients and this panel reaches a sensitivity of 61% and specificity of 89% [130]. However, both studies could cause overfitting bias. Overfitting means that if a multi-markers panel is inappropriately

	panel	methods	cases	controls	AUC	SN/SP	Reference/year
	IMP1, p62, Koc,
	P53, c-MYC, cyclin	ELISA	64	346	-	92%/85%	[123, 124]2003
	B1, and survivin
	survivin and livin	ELISA	46	10	-	52.2%/-	[104] 2005
	p16, P53, and c-myc	ELISA	41	82	-	43.9%/97.6%	[125] 2006
	P53, c-Myc, HER2, NY-ESO-1, and MUC1 BRCA1, BRCA2,	ELISA	97 a 40 b	94	--	64%/85% 45%/85%	[126] 2007
	MUC1, HER2, P53, and IGFBP2	ELISA	184 c	134	-	31%/-	[73] 2008
	P53, HER2, IGFBP-2, and TOPO2α				0.63	-
	ASB-9, SERUMC1, and RELT	ELISA	87	87	0.861	77%/82.8%	[127] 2008
	FKBP52, PPIA,		60 a		0.73	55.2/87.9%
	PRDX2, HSPD1 and	ELISA		93			[84] 2009
	MUC1		82 b		0.80 72.2%/72.6%
	RBP-Jk, HMGN1,
	PSRC1, CIRBP, and	ELISA	59 a	61 b	0.749	86.1%/75%	[128] 2012
	ECHDC1
	GAL3, PAK2,
	PHB2, RACK1 and	ELISA	114	68	0.81	66%/87%	[129] 2013
	RUVBL1
	p62, P53, c-myc,
	survivin, p16, cyclin B1, cyclin D1 and	ELISA	41	82	-	61%/89%	[130] 2013
	CDK2
	FTH1 and hnRNPF	ELISA	150	150	0.816	91.1%/72%	[131] 2013
	a primary breast cancer patients, b patients with ductal carcinoma in situ (DCIS), c advanced-
	stage disease, SN: sensitivity, SP: specificity, ROC -area under ROC curve.
	Among all these AAbs panels, anti-P53 antibodies were screened in half of these studies
	[130, 123, 73, 124-126]. Good performance was obtained by [130, 123]. One of this study

Table 8

 8 Anti-TAA AAbs identified by multiple studies for breast cancer diagnosis

	Tumor	Number	Range of sample size (N)	Range of sensitivity
	antigen	of studies	cases	HC	across studies	reference
						[130, 123, 73, 124-
	P53	14	25-2006	82-346	7.9%-35%	126, 86-93]
	HER2	6	20-144	157-242	7%-55%	[73, 94-96, 90, 126]
	MUC1	5	24-241	93-134	8.3%-37.5%	[97, 98, 126, 73, 84]
	c-myc	4	41-137	82-346	13%-22%	[123-126, 130, 104]
	Survivin	3	41-64	10-346	7.8%-23.9%	[124, 130, 123, 103]
	cyclin B1	3	7-64	27-346	4.7%-42.8%	[124, 130, 123]
	P16	2	41	82	12.2%	[125, 130]
	P62	2	41-64	82-346	7.8%-12.2%	[124, 130, 123]
	IGFBP2	2	80-184	134-200	5%-7%	[103, 73]

Table 9

 9 Immobilization strategies for protein immobilization

	Immobilization	Surface chemistry Attachment site	Advantages	Disadvantages
	chemistry
	Adsorption	Nitrocellulose,	Electrostatic	The simplest	Random
		Poly-L-lysine,	interactions,	immobilization	orientation, high
		agarose, etc.	hydrogen binding,		background
			van derwaals
			interactions
	Covalent binding Maleimide,	Thiol,	Robust	Potential loss of
		hydrazine,	carbohydrate,	immobilisation	activity of
		succinimidyl	amine		immobilized
		ester, epoxide,			proteins
		aldehyde, etc
	Affinity based	Protein A or G,	Fc region, biotin,	Oriented	Pretreatment of
	binding	streptavidin,	GST tag, etc	immobilization	spotted proteins
		glutathione, etc.

Table 10

 10 Panorama of commercial protein microarrays

	Category	Company	Products	Printed proteins	Replication of printed proteins	Price/ slide	Sample slide tested/	Surface	Reference
			FAST Quant	Antibodies
			TH1/TH2	against 9	3	525 €	16	Nitrocellulose	[157, 158]
		Whatman	arrays	cytokines
			FAST Quant	Antibodies
			angiogenesis	against 9	3	525 €	16	Nitrocellulose	[158]
			arrays	cytokines
	Cytokine	R&D system	Human Cytokine Array Panel A	Antibodies against 36 cytokines	2	128 €	1	Nitrocellulose	http://www.rn dsystems.com /Products/ary 005/Citations
	test		Human XL	Antibodies
			Cytokine	against 102	2	186 €	1	Nitrocellulose	No
			Array	cytokines
		RayBiotech	Human Quantibod® Arrays Q1 Cytokine	Antibodies cytokines against 20	4	698 €	16	N.A	[159-162]
			Human Quantibod® Cytokine Arrays Q440	Antibodies against 440 cytokines	4	12900 €	16	N.A	[163]
									http://www.lif
									etechnologies.
									com/fr/fr/hom
									e/life-
									science/protei
	Protein profiling	Invitrogen	ProtoArray® Human Protein Microarray	9,000 unique human proteins	2	1180 €	1	Nitrocellulose	n-biology/protei n-assays-analysis/prote in-
									microarrays/te
									chnical-
									resources/liter
									ature-
									citations.html

.4.2.1 Cytokines microarray Among

	Whatman has developed several commercial protein microarrays to test cytokine:FAST
	Quant human TH1/TH2 arrays, angiogenesis arrays, Human Cytokine II arrays. Each FAST

all these commercial protein microarrays, cytokine microarray is one of the most common kits and has been developed by several companies like Whatman, R&D Systems and RayBiotech, Inc. Cytokines play an important role in the understanding and treatment of diseases in many medical specialities and protein microarray technology is accelerating the rate at which researchers can obtain cytokine expression information.

Quant array contains 8 to 10 monoclonal antibodies with affinities for common human cytokines. The antibodies are arrayed in triplicate on each array. Each slide has 16 microwells and two wells were used to obtain standard curve which was generated by creating dilution series from recombinant antigen mass standards. FAST Quant claims it exhibits sensitivity and reproducibility better than traditional ELISA. However, there are currently relatively few publications using FAST Quant technology. One study compared two multiplex sandwich ELISA procedures (FAST Quant and SearchLight) and a bead based assay (UpState Luminex)

  Lash using ELISA, FAST Quant human angiogenesis and TH1/TH2 arrays to detect cytokines. However, the author did not compare the results obtained from FAST Quant arrays and ELISA, thus the feasibility of this commercial array is which ranges from 20 (Human Cytokine Array Q1) to 440 (Human Cytokine Array Q440). The price ranges from 698€ to 12900€ correspondingly to the number of printed antibodies. Each slide has 16 micro-wells, among which eight wells were used to obtain standard curve and the resting 8 wells were used for sample detection. There are In[182], they found that 94 antigens exhibited enhanced reactivity in cancer patient' sera relative to control sera. Then 4 antigens were selected and validated by using immunoblot analysis and tissue microarrays. Three of them were found to exhibit increased expression in cancer tissues relative to controls. In another study[183], 202 proteins were

	1.4.2.3 Cancer biomarker screening microarray
	Identifying cancer biomarkers can aid in diagnosing disease, estimating prognosis, and
	monitoring treatment. Recently several companies have developed some commercial cancer
	currently lots of publications using different type of Quantibody® Cytokine Arrays, more preferentially immunogenic in ovarian cancer sera compared with healthy controls. They biomarker arrays. RayBiotech Inc. has commercialized array for gastric cancer detection
	detailed information is shown on their website. RayBiotech Inc. has cooperated closely with validated 2 antigens by ELISA and results showed high degree of similarity between the two which is called Human Gastric Cancer Biomarker Array Q1. Antibodies against 5 human
	various academic researches and been involved in many publications, which showed high methods. However, no common biomarkers were found when we compared the top 15 gastric cancer biomarkers (CA19-9, CA72-4, CEA, Pepsinogen 1 and Pepsinogen 2) were
	quality of their commercialized products. However, results obtained by other independent antigens screened by these two studies. Difference on sample population (30 cancer cases for arrayed on glass support in quadruplicate. However, their efficiency is unknown as there are
	research were not always satisfying. One study analyzed cytokine levels in tissue lysates [182] and 51 cases for [183]), ProtoArray® Human Protein Microarray version (version 3.0 currently no publications using this product.
	using 4 different multiplex ELISA-based immunoassay arrays. They include Quansys for [182] and version 4.0 for [183]) maybe could explain the discrepancy. However, it also
	BioSciences (microplate-based), Aushon Biosystems SearchLight (microplate-based), showed that discrepancy exists between different laboratories even when they used products
	One of them is ProtoArray® Human Protein Microarray developed by Invitrogen. The
	newest version 5.0 contains over 9,000 unique human proteins individually purified and
	arrayed in duplicate under native conditions on nitrocellulose coated glass slide. This platform showed that Panorama® antibody microarray is a powerful tool for profiling disease-state
	enables rapid profiling of thousands of biochemical interactions in as little as one day. proteins and finding novel biomarkers of diseases, however, this array has been discontinued
	According to their website, more than 110 publications utilized this product in different by Sigma-Aldrich due to lacking of sale.
	disease states including transplantation [175-177], various cancers [178-184], and

unknown

[158]

.

R&D system also developed two Human Cytokine Array kit. One kit is called XL Cytokine Array Kit and it contains 102 cytokine antibodies. R&D system claims that this array could detect multiple cytokines, chemokines, growth factors and other soluble proteins in cell culture supernatants, however, no publications were reported on this product. The other kit is called Panel A and it contains 36 different cytokine antibodies. Antibodies against human cytokines, chemokines, and acute phase proteins were spotted in duplicate on nitrocellulose membranes. Captured proteins are finally visualized using chemiluminescent detection reagents. This kit has been validated for analyte detection in cell culture supernatants, cell lysates, tissue lysates, serum, and plasma. Currently, more than 63 publications were reported on this product. Results showed that it is a powerful tool for cytokine screening in various disease conditions, like cancer [168-171] and inflammatory

[172]

. Furthermore, some studies validated the results by ELISA and found that ELISA data were consistent with and supported the data obtained from the Human Cytokine Array Panel A [173]. However, although array could determine the expression of multiple cytokines in a single sample, one membrane can only detect one sample, making large sample screening extra expensive. RayBiotech Inc. has developed various series of cytokine Arrays including Quantibody®, L-Series, C-series, G-Series and E-Series. Among all these series, Quantibody® arrays were widely used by academic researches. So a detailed description about Quantibody® cytokine arrays will be presented as a representative in the following. Overall, 20 human Quantibody® cytokine arrays have been developed. The difference between different arrays is the size of printed antibodies, Milliplex MAP Sample (bead-based), and a RayBiotech Inc. (slide-based) kit. Overall, the Quansys Biosciences and SearchLight arrays screened several elevated cytokines, being more sensitive than traditional single ELISA kits. However, the Milliplex bead array technique and the RayBiotech slide technology did not measure any level of cytokines due to lower sensitivity [174]. Despite of this, we cannot conclude that RayBiotech slide is not suitable for cytokine screening, however, the variability often exists and validation by independent laboratories is needed. 1.4.2.2 Protein profiling microarray Profiling of thousands of biochemical interactions can have wide applications including novel disease biomarker identification, drug target discovery and therapeutic antibody development. Several companies have developed commercialized products which are widely used by academic researches. autoimmune diseases [185-187]. However, discrepancy was observed when we compared independent similar researches. For example, both Michael E. Hudson et al. and Sacha Gnjatic et al. used ProtoArray® Human Protein Microarray to screen biomarkers in ovarian cancer. provided by the same companies. ProtoArray® Human Protein Microarray is a powerful tool for biomarker pre-screening, while further validation by classic methods like ELISA is needed. Sigma-Aldrich has also developed Panorama® Antibody Arrays for protein expression profiling, among which Cell Signaling Kit was the mostly used by academic researches. The Cell Signaling array contains 224 different antibodies each spotted in duplicate on nitrocellulose coated glass slides. These antibodies represent biological pathways including apoptosis, cell cycle, neurobiology, cytoskeleton, signal transduction, and nuclear proteins [188]. It has been used to profile differential protein expression between normal and cancer patients, thus identifying novel potential cancer biomarkers, like in breast cancer [189-191], colorectal cancer [192], prostate cancer [193], lung cancer [194]. For breast cancer research, Julio E. Celis et al. used Panorama® Antibody Arrays to screen mammary adipose tissue and numerous proteins were identified, including signaling molecules, hormones, cytokines, and growth factors. Furthermore, these proteins were validated by immunoblotting [190]. In [191], they analyzed breast cancer cell line in order to find novel predictive biomarkers for target therapy. Relevant proteins were also validated by western blotting. All these researches Arrayit has developed OvaDx® for ovarian cancer diagnosis. Arrayit declaims that this test could monitor the response of the immune system during early stages of ovarian tumor development through measuring about 100 biomarkers in serum samples. It has high sensitivity (79.7%) and specificity (100%) for all types and stages of ovarian cancer. It is therefore an effective elective test for screening women at elevated risk for ovarian cancer.

  Several other companies have also developed similar commercial protein microarrays for research use only. However, assays sold as "for Research Use Only" are not regulated by the U.S. Food and Drug Administration or the equivalent European agencies as part of Health

have developed allergen testing microarrays. ImmunoCAP ISAC is one of them developed by Thermo Fisher Scientific. It is a miniaturized immunoassay platform where allergen components are covalently immobilized in triplicates, on a polymer coated slide. Each slide contains 4 microarrays giving results for 4 different samples per slide. It allows the measurement of IgE antibodies to a fixed panel of 112 components from 51 allergen sources in a single step. ImmunoCAP ISAC has been used by several independent academic researches [164-167]. However, the sensibility of ImmunoCAP ISAC for latex allergen detection is lower compared with conventional methods [167]. Moreover, further improvements in threshold and better interpretation algorithms are needed to fully capitalize on the potential of microarray [166].

Arrayit has also developed protein microarray allergy tests. Allergens printed on standard glass substrate slides through covalent binding for testing 123 IgE and 101 IgG. Likewise, there is no publication for the products produced by Arrayit.

  Then they were stored under 2 conditions: dry at 4°C, and in blocking solution at 4°C. Results showed that all slides tested showed an unexpected increase of signal intensities after 2 weeks of storage. Furthermore, for all non-gel-coated surfaces, immobilized antibodies showed no significant loss of signal intensity over time, under these conditions tested. While for gel-coated slides, they showed higher signal intensities when stored dry at 4°C compared to those in blocking solution[154]. Kusnezow et al. also found that dry condition could keep better activity of printed antibody microarray.They compared storage conditions of printed antibody onto APTES and GPTS surfaces.Results showed that all microarrays could be used for at least 2 months of storage without any apparent deterioration of the performance parameters. As a matter of fact, an increase of signal intensities was observed similarly to the results obtained with the non-printed slides

Angenendt et al. immobilized 5 different antibodies on 2 types of surfaces: gel-coated surfaces and non-gel-coated glass or plastic surfaces. [205]. Based on this, Wu et al. also stored printed antibody microarrays at 4°C sealed under nitrogen for 1 month. Antibodies' activity decreased after 1 month of storage but reasonable high intensity can be maintained, especially when antibodies were printed with PVA additives [206]. In addition to antibody microarray, protein-protein interaction arrays and enzyme arrays were studied by Nath et al. [207]. They used 5 probe-target protein pairs and 3

•

  Firstly, although numerous candidate biomarkers have been reported as shown in the literature overview, only few biomarkers have been approved to be used in clinic. Furthermore, biomarkers used in routine are not effective for early diagnosis

of breast cancer because of low sensitivity and specificity. Among potential biomarkers of interest, autoantibodies (AAbs) against HSPs seem to be good candidate biomarkers for breast cancer diagnosis and prognosis. Moreover, protein microarrays are powerful tools for the validation of candidate biomarkers. So the first objective is to develop customized antigen microarrays to profiling anti-HSPs antibodies in breast cancer serum.

• Secondly, uPA and PAI-1 are strong prognostic and predictive tissue biomarkers and have the highest level-of-evidence (LOE-1) in breast cancer. High level of uPA and PAI-1 are associated with high risk of recurrence and chemotherapy will contribute great benefit for patients. However, it is limited by the detection method-ELISA. ELISA requires at least 300 mg of fresh or frozen tissue. Protein microarray could overcome this limitation because it utilizes tiny sample volume.

So the second purpose is using antibody microarray to titrate uPA and PAI-1 in breast tumor tissue. We will also evaluate the performance of our protein microarray compared to commercial ELISA kit.

• Last but not the least, because protein microarrays is influenced by various factors including storage conditions, concentration of spotted proteins, incubation time, composition of buffer, etc. So

we will optimize assay conditions of our customized protein microarrays (antigen microarray and antibody

	86. Dalifard I, Daver A, Larra F (1999) Cytosolic p53 protein and serum p53 autoantibody
	evaluation in breast cancer. Comparison with prognostic factors. Anticancer Res 19
	(6B):5015-5022.
	87. Soussi T (2000) p53 Antibodies in the sera of patients with various types of cancer: a
	review. Cancer Res 60 (7):1777-1788.
	88. Sangrajrang S, Arpornwirat W, Cheirsilpa A, Thisuphakorn P, Kalalak A, Sornprom A, 2.1 Introduction
	Soussi T (2003) Serum p53 antibodies in correlation to other biological parameters of breast 2.2 Materials and methods
	cancer. Cancer Detect Prev 27 (3):182-186. 2.2.1 Materials
	89. Shimada H, Ochiai T, Nomura F (2003) Titration of serum p53 antibodies in 1,085 2.2.2 Serum samples
	patients with various types of malignant tumors: a multiinstitutional analysis by the Japan p53 2.2.3 Surface functionalization of glass slides
	Antibody Research Group. Cancer 97 (3):682-689. 2.2.4 Optimization of protein immobilization condition
	2.2.5 Multiplex immunoassays on micro-structured protein microarray
	2.2.6 Fluorescence scanning
	2.2.7 Data analysis
	2.3 Results
	2.3.1 Optimization of tumor antigen microarray conditions
	2.3.2 Detection of autoantibodies against HSPs and P53 in breast cancer sera by
	multiplex immunoassays on antigen microarray
	2.3.2.1 Detection of autoantibodies against HSPs and P53 in breast cancer sera and
	healthy control sera
	2.3.2.2 Association of anti-HSPs autoantibodies profile with tumor stage
	2
	95. Disis ML, Pupa SM, Gralow JR, Dittadi R, Menard S, Cheever MA (1997) High-titer
	HER-2/neu protein-specific antibody can be detected in patients with early-stage breast
	cancer. J Clin Oncol 15 (11):3363-3367.
	96. Disis ML, Knutson KL, Schiffman K, Rinn K, McNeel DG (2000) Pre-existent immunity
	to the HER-2/neu oncogenic protein in patients with HER-2/neu overexpressing breast and
	ovarian cancer. Breast Cancer Res Treat 62 (3):245-252.

90. Gao RJ, Bao HZ, Yang Q, Cong Q, Song JN, Wang L (2005) The presence of serum anti-p53 antibodies from patients with invasive ductal carcinoma of breast: correlation to other clinical and biological parameters. Breast Cancer Res Treat 93 (2):111-115. 91. Muller M, Meyer M, Schilling T, Ulsperger E, Lehnert T, Zentgraf H, Stremmel W, Volkmann M, Galle PR (2006) Testing for anti-p53 antibodies increases the diagnostic sensitivity of conventional tumor markers. Int J Oncol 29 (4):973-980. 92. W Min, M Chen, C Qing, C Xin-Wei, Wei-Seng Z (2010) Serum p53 protein and anti-p53 antibodies are associated with increased cancer risk: a case-control study of 569 patients and 879 healthy controls. Mol Biol Rep 37 (1):339-343. 93. Kulic A, Sirotkovic-Skerlev M, Jelisavac-Cosic S, Herceg D, Kovac Z, Vrbanec D (2010) Anti-p53 antibodies in serum: relationship to tumor biology and prognosis of breast cancer patients. Med Oncol 27 (3):887-893. 94. Disis ML, Calenoff E, McLaughlin G, Murphy AE, Chen W, Groner B, Jeschke M, Lydon N, McGlynn E, Livingston RB, et al. (1994) Existent T-cell and antibody immunity to HER-2/neu protein in patients with breast cancer. Cancer Res 54 (1):16-20. 97. Kotera Y, Fontenot JD, Pecher G, Metzgar RS, Finn OJ (1994) Humoral immunity against a tandem repeat epitope of human mucin MUC-1 in sera from breast, pancreatic, and colon cancer patients. Cancer Res 54 (11):2856-2860.

.4 Discussion and Conclusions References 2.1 Introduction Heat

  shock proteins (HSPs) are induced by stress conditions such as heat shock, decrease in pH, hypersalinity, alcohols, heavy metals, oxidative stress, inhibitors of energy metabolism and fever or inflammation. They are molecular chaperones that act to assist other proteins'

Table 1

 1 All human samples were prospectively collected between 2005 and 2007 at the CRLC Val d'Aurelle Cancer Institute (Montpellier, France) at the time of cancer diagnosis after obtaining written informed consent. Blood samples were centrifuged at 1250g for 5min, and the serum was stored at -80°C. For the multiplex immunoassay, 76 serum samples were examined: 26 healthy controls with negative mammograms, negative physical breast exams for at least 4 years, and no history of prior malignancy, and 50 patients who underwent surgery and had a histopathologic diagnosis of breast cancer. The tumor stage of breast cancer patients were summarized in Table1. Tumor stage of breast cancer patients

	Tumor stage	Number of patients
	Stage I	24
	Stage II	14
	Stage III	10
	Unknown	2
	Total	50

Table 2

 2 Optimal conditions for efficient immobilization of HSPs and P53 for screening anti-HSP and anti-P53 antibodies in breast cancer serum.

			Optimal immobilization
	Immobilized tumor antigen	Optimal surfaces	concentration
	HSPB1	chitosan	0.05mg/mL
	HSPD1	COOH	0.05mg/mL
	HSP70	chitosan = COOH	0.05mg/mL
	P53	COOH > chitosan	0.1mg/mL
	HSP90	chitosan > COOH	0.05mg/mL
	HSPA5	COOH > chitosan	0.05mg/mL
	HSP90B1	COOH > chitosan	0.05mg/mL
	HSP110	COOH > chitosan	0.05mg/mL

.2 Detection of autoantibodies against HSPs and P53 in breast cancer serum by multiplex immunoassays on antigen microarray 2.3.2.1 Detection of autoantibodies against HSPs and P53 in breast cancer serum and healthy control serum

Table 3

 3 The area under receiver operating characteristic curve (AUC) of individual antibody and combination of 8 antibodies to discriminate breast cancer patients from healthy controls

	Anti-HSP antibody	AUC (95% CI)
	Anti-HSPB1 antibody*	0.631 (0.528-0.743)
	Anti-HSPD1 antibody**	0.683 (0.581-0.773)
	Anti-HSP70 antibody**	0.732 (0.663-0.817)
	Anti-P53 antibody*	0.581 (0.459-0.695)
	Anti-HSP90 antibody**	0.710 (0.621-0.837)
	Anti-HSPA5 antibody*	0.723 (0.567-0.836)
	Anti-HSP90B1 antibody**	0.728 (0.608-0.837)
	Combination of 7 antibodies	0.978 (0.938-1.000)

*The detection of antibodies was obtained on chitosan surface, **the detection of antibodies was obtained on COOH surface.

Table 4

 4 Frequency of single anti-HSP autoantibody detected in breast cancer patients and healthy controls. determination, only for similar sample size (less than 50 breast cancer cases and about 25 healthy controls). However, if sample size if different (2 times more in ELISA study), then the frequency of anti-HSP90 was very different suggesting that the size of the sample is a very important parameter in the reliability of the study. For the 2

	HSPs	Methods	Sample size	AAb frequency %	p-value	Reference
			Cases	HC	Cases	HC
	HSPB1	ELISA	579	53	37.8%	1.9% p<0.001 [6]
		PM	50	26	8%	0	0.049*	Our study
	HSPD1	WB	40	42	47.5%	4.7% p<0.01	[8]
		ELISA	107	93	31.8%	4.3% p<0.0001 [9]
		PM	50	26	14%	3.8% 0.01**	Our study
	HSP70	ELISA	369	53	40.9%	35.9% N/A	[6]
		PM	50	26	34%	0	0.002** Our study
	HSP90	ELISA	125	N/A	36.8%	N/A	N/A	[7]
		ELISA	13	22	7.7%	N/A	N/A	[10]
		PM	50	26	4%	0	0.002** Our study
	HC: healthy controls; ELISA: enzyme linked immunosorbent assay; PM: protein microarray;
	WB: western blot; N/A: not available; * Results obtained on chitosan surface, ** Results
	obtained on COOH surface.

Only 4 anti-HSPs autoantibodies studied in our work were evaluated by others. Results obtained for anti-HSP70 were very close between our antigen microarray and ELISA, even if sample size were very different. The frequency of anti-HSP90 found in our study was also in accordance with ELISA

  6. Add standard solutions: adding 100 µl uPA or PAI-1 (from Femtelle kit) into wells captured with anti-uPA antibody or anti-PAI-1 antibody respectively. Cover the plate and incubate for 1 hour at RT. Both uPA and PAI-1 have 6 concentrations.7. Remove the solution and wash the plate with 200 µl per well wash buffer for 3 x 5 minutes.

	8. Add diluted (1:3) biotinylated detection antibody (from Femtelle kit) to the plate, cover and
	incubate for 1 hour at RT.
	9. Remove the solution and wash the plate with 200 µl per well wash buffer for 3 x 5 minutes.
	10. Add 100 µl diluted enzyme conjugate (from Femtelle kit: 1 µl enzyme conjugate in 1ml
	enzyme conjugate diluent) to the plate, cover and incubate for 1 hour at RT.
	11. Remove the solution and wash the plate with 200 µl per well wash buffer for 3 x 5
	minutes.

Table 1

 1 Optimal conditions for the immobilization of anti-uPA antibody and anti-PAI-1 ScFv antibody.

	Antibody/spotted concentration	Surface Spotting buffer LOD (ng/ml) Dynamic range
	anti-uPA antibody at 6.6 µM	COOH	PBS	0.2	0.2 -1.5ng/ml
		NHS	PBS	0.2	0.2 -2ng/ml
		chitosan	PBS	0.2	0.2 -1.5ng/ml
	anti-PAI-1 antibody (scFv) at 10 µM	COOH	carbonate	2	2 -20 ng/ml
		NHS	carbonate	2	2 -20 ng/ml
		chitosan	PBS	2	2 -20 ng/ml
	3.3.

2 Quantification of uPA in breast tumor tissue extracts 16

cytosolic extracts were obtained from frozen breast tumor tissues by 10% Triton X-100 (recommended by Femtelle kit). The concentration of uPA in each cytosolic extracts was determined by Femtelle kit and ranked from 0.4 ng/ml to 8 ng/ml (see Table

2

).

Table 2

 2 The concentration of uPA in 16 cytosolic extracts determined by Femtelle kit

	No. of patient Concentration of uPA (ng/ml)
	1	0.4
	2	0.9
	3	1.4
	4	1.5
	5	2.1
	6	2.3
	7	2.4
	8	3.1
	9	3.3
	10	3.6
	11	3.9
	12	4.3
	13	4.6
	14	6.4
	15	7
	16	8

Table 2

 2 Incubation process for protein microarray reproducibility

	Spotted proteins	Biological recognition step	Detection step	Detection step
		Anti-P53 Ab-biotin:	Strep-F555:
	P53	0.5µM	0.19 µM	Buffer
			Strep-F555:
	BSA-biotin	Buffer	0.19 µM	Buffer
			Anti-p53 Ab-biotin:	Strep-F555:
	Anti-P53 antibody	P53: 0.05 µM	0.5µM	0.19 µM
	Streptavidin-F555	Buffer	Buffer	Buffer

Table 3

 3 Experimental time in the protocols before and after optimization

	Factors	Initial protocol Optimized protocol
	Blocking time (hr)	2	1

Table 6

 6 Repeatability and reproducibility of protein microarrays. Intra-slide and inter-slides

	coefficient of variation (CV) of P53/anti-P53 system studied onto flat and microstructured
	glass slides functionalized with COOH, NHS and chitosan.
			COOH	NHS	Chitosan
	P53/surface	Flat	micro-structured	Flat	micro-structured	Flat	micro-structured
	Intra CV	20%-22% 21%-24% 10%-13%	7%-13%	15%-29%	4%-12%
	Inter CV	14%	5%	6%	21%	28%	9%

Table 1 ,

 1 the first column is the sample size. In total, we have tested 76 samples, including 26 healthy controls and 50 breast cancer patients. The second column is the value obtained from experiment; we also used these values as cut off value. The third column is the sample characteristic, either cancer or healthy control. Cancer patients were clinical diagnosed and we know this information before we tested. For the last two columns, one is the value of x axis: 1-specificity; the other is y axis: sensitivity.

Table 1

 1 Results of HSPA5 immobilized on chitosan surface for all samples

	No. (26HC &	Value/ Cut off	Sample	X axis:	Y axis:
	50 BC)		characteristic	1-specificity	Sensitivity
	1	5.0	Cancer	1-(26/26)=0	1/50=2%
	2	4.8	Cancer	1-(26/26)=0	2/50=4%
	3	4.1	Cancer	1-(26/26)=0	3/50=6%
	….	….	Cancer	1-(26/26)=0	….
	13	2.3	Cancer	1-(26/26)=0	13/50=26%
	14	2.2	Healthy control	1-(25/26)=4%	13/50=26%
	15	2.1	Cancer	1-(25/26)=4%	17/50=34%
	….	….	Cancer	….	….
	20	1.8	Cancer	1-(25/26)=4%	20/50=40%
	21	1.7	Healthy control	1-(24/26)=8%	20/50=40%
	….	….	….	….	….
	76	0.4	Healthy control 1-(0/26) =100%	50/50 = 100%
	HC: Healthy controls; BC: breast cancer.

  sensibilité sans réduire la spécificité du test. D'autre part, les anticorps anti-p53 circulant sont associés à un mauvais pronostic avec une survie courte. carcinoma premier stade, c cancer en stade avancé HER2 est un récepteur de facteur de croissance épidermique (EGFR) qui est amplifié et surexprimé dans 20%-30% des cancers du sein. Un test positif à HER2 est associé à un mauvais pronostic dû à la forte incidence des métastases et à la résistance aux chimiothérapies conventionnelles ou endocriniennes[81].

	grand nombre de chimies de surface pour pouvoir ensuite immobiliser de manière robuste les
	protéines tout en conservant leur intégrité, leur conformation originelle et leur fonction
	biologique [93]. [94].
				Taille de l'échantillon
	TAA Du point de vue commercial, on trouve une large variété de lames disponibles avec des (N) AAb frequence % Reference/année
	cases Les Anti-HER2 aussi sont détectés chez les patientes atteintes de cancers du sein. Une Sains chimies de surface différentes telles que des lames en nitrocellulose FAST, hydrogel,
		p53		101		-	7.9%		[56]
	2006 étude conduite sur une période de 6 ans ( de 1994 à 2000) indique une présence significative -14.7% [55] SuperAldehyde and epoxy-silane ES, des poly-L-lysine, des surfaces aldehyde ,
	158 des anticorps anti-HER2 chez les patientes au premier stade des cancers du sein comparée à -19% [57] polyacrylamide, PolyEthylenGlycol-epoxy or dendrimères …
	Taille de l'échantillon (N) 71 205 des sujets sains [63, 64] et un niveau beaucoup plus élevé est noté pour des stades avancés AAb frequence % 18.3% [58]
	Hsp [65]. Ces études suggèrent qu'une réponse immune humorale aux HER2 pourrait jouer un méthode cancer sain benin cancer sain benin P valeur reference 144 242 21.5% [59] Il existe une littérature foisonnante concernant les stratégies d'attachement des protéines
	Hsp27 rôle dans la limitation de la progression tumorale. ELISA 579 53 -37.8% 50 436 34% sur une surface, cette étape clé déterminant les propriétés du microarrays. Le tableau ci-1.9% -p<0.001 [50] [60]
	Hsp70 Hsp90 dessous rassemble les différentes modalités d'interactions surface/protéines et les ELISA 369 53 -40.9% 35.9% --[50] ELISA 125 --36.8% ---61 20 35% [62] [49] 25 879 16% [61]
	Hsp60 avantages/inconvénients des différents modes d'immobilisation [95-97]. WB 40 42 -47.5% 4.7% -HER2 20 -55% [63]	p < 0.01	[51]
	Hsp60	ELISA	107	107	93	-200	31.8% 11.2%	4.3%	-[64]	p<0.0001	[52]
	Hsp90	ELISA	13	37	22	10 157	8%	7%	0	0 [65]	-	[53]
		MUC1		24		-	8.3%		[66]
				40 a			37.5%
				140 b		25.7%
				61 c		96		18%		[67]
		c-myb		72		49		43%		[68]
		fibulin		20		20		75%		[69]
		RPA32		801		65	10.9%		[70]
	Au delà des hsps, d'autres antigènes tumoraux ont été dirigés contre les auto-anticorps. 74 27% A coté des 3 auto anticorps présentés ci dessus, d'autres molécules telles que c-myb,
	[54]. Le tableau suivant recense un certain nombre d'études visant à identifier les auto-lipophilin B 35 c 20 37.1% [71] fibulin, RPA32, lipophilin B, cyclin B1, survivin, livin, endostatin, GIPC-1, insulin-like
	anticorps tumoraux dans les sera de cancer du sein, mais un petit nombre seulement (anti-cyclin B1 7 27 42.8% [72] growth factor binding protein 2 (IGFBP-2), AHSG, SPAG9, SOX2 and p90/CIP2A etc…
	survivin p53, anti-Her2/neu, anti-MUC1) ont fait l'objet d'études plus détaillées. 23.9% livin 46 10 32.6% [73] sont également impliquées dans les cancers du sein à travers les différents mécanismes de la
	36 b cancérisation. Mais la fréquence de leur détection varie fortement (de 5% à 80%). Ces 66.6%
	L'antigène tumoral p53 est surexprimé dans les cellules cancéreuses et induit la production endostatin 59 c 24 42.4% [74] grandes variations pourraient résulter de plusieurs facteurs tells que l'hétérogénéité de la
	auto anticorps anti-p-53. In 2000, Soussi compulse la bibliographie de 1979 à 1999 GIPC1 22 10 77% [75] tumeur, la taille, la qualité et l'origine des échantillons des méthodes et protéines utilisées…
	concernant les auto-anticorps anti-p53 AAbs dans les sera de patients de tout type de cancer. IGFBP2 80 200 5% [76] etc.
	Une quinzaine d'études a permis d'identifier l'anti-p53 dans les cancers du sein avec une AHSG 81 73 79.1% [77]
	SPAG9 fréquence allant de 2.8% à 47.5%. Si l'on considère l'ensemble de ces études, l'anti-p53 100 50 80% [78] 282 Un résumé des travaux basés sur des tests ELISA et utilisant des panels plus ou moins 18.4% AAbs a été détecté dans 14.7% de patientes (296/2006) avec une différence significative des SOX2 78 a 194 6.4% [79] importants (de 2 à 10 biomarqueurs) est présenté dans le tableau ci-dessous.
		p90/CIP2A	168		88	19.1%		[80]

Ainsi pris indépendamment, le potentiel diagnostique ou pronostique de chaque anti-hsp est très faible. Cependant, l'utilisation de nouveaux outils de détection multiplexée telle que les microarrays permet d'une part des études à grande échelle (large panel de biomarqueurs, cohorte importante) et d'autre part d'augmenter considérablement la sensibilité et la spécificité du diagnostic jusqu'à des valeurs de plus de 80%

[20]

.

sujets sains (P < 0.0001)

[55]

. Ces études montrent que les anticorps anti-p53 ont une forte spécificité (supérieur à 95%) mais une faible sensibilité (en moyenne 20.8%). Aussi, il est impératif de combiner la recherche d'anticorps anti-avec d'autres biomarqueurs pour augmenter la Tableau de Fréquence des auto-anticorps dans les patients du cancer du sein : a tumeurs bénines, b Les Mucines (MUC) sont des glycoprotéines de fort poids moléculaire exprimées à la surface cellulaire. MUC1 a été trouvé de manière abondante dans les cancers du sein mais aucune corrélation avec le stade de la maladie n'a pu être faite

[66]

. Par contre, les anticorps Anti-MUC1 ont été détectés beaucoup plus souvent pour des tumeurs bénignes que pour des cancers du sein. Ainsi, une corrélation négative a été observée entre la présence d'anti-MUC1 et le développement de la maladie. Ceci suggère qu'une réponse immune humorale naturelle aux MUC1 serait protectrice d'une progression de la maladie tandis qu'un manque de réponse immune serait associé à un pronostic défavorable

[67]

.

Table 10 .

 10 Les microarrays commercialisés Soulignons par exemple l'intérêt du microarray ProtoArray® Human Protein Microarray développé par Invitrogen qui contient plus de 9 000 protéines humaines . Selon leur site, plus de 110 publications utilisent ce produit pour tester des maladies différentes comme les transplantations [110-112], différents cancers [113-119], et des maladies auto immunes [120-122]. Cependant, des dispersions sont observées lorqu'on compare les résultats issus de différents laboratoires mais utilisant les mêmes produits. Aussi, le ProtoArray® Human Protein Microarray est un outil puissant pour faire un premier screening de biomarqueurs mais une validation est ensuite nécessaire avec des méthodes traditionnelles comme l'ELISA. Sigma-Aldrich a aussi développé une puce Panorama® Antibody Arrays pour analyser les profil d'expression des protéines [123]. Celle-ci a permis d'identifier de nouveaux biomarqueurs potentiels aussi bien dans le cancer du sein [124-126], le cancer colorectal [127], le cancer de la prostate [128], le cancer du poumon [129]. Les protéines d'interêt ont aussi été validées par Western Blot. Malheureusement, ce microarray n'est plus disponible à cause du manque de ventes.

  Table des Fréquences de chaque auto-anticorps anti-HSP détecté sur les cancers du sein et contrôles sains.En conclusion, cette étude a permis de démontrer que les supports de microarrays microstructurés développés au laboratoire, sont un outil de criblage très puissant. En effet, ils ont permis de déterminer rapidement les meilleures conditions d'immobilisation (chimie de surface, concentration) d'antigènes sondes pour la détection d'anticorps. Ces supports microstructurés ont également permis d'élaborer des puces à antigènes à façon (sélection des meilleures conditions pour chaque antigène sonde) afin d'établir les profils d'expression d'anticorps anti-hsps présents dans le sérum de patientes atteintes du cancer du sein. Nous avons alors montré que la détection multiplexe de 7 biomarqueurs permet d'augmenter significativement la sensibilité et la spécificité d'un test clinique à valeur diagnostique.

	HSPs	Méthodes	Echantillonnage	AAb fréquence %	p-value	Reference
			cancer	sain	cancer	sain
	HSPB1	ELISA	579	53	37.8%	1.9% p<0.001 [50]
		Microarray 50	26	8%	0	0.049*	Our study
	HSPD1	Western B 40	42	47.5%	4.7% p<0.01	[51]
		ELISA	107	93	31.8%	4.3% p<0.0001 [52]
		Microarray 50	26	14%	3.8% 0.01**	Our study
	HSP70	ELISA	369	53	40.9%	35.9% PE	[50]
		Microarray 50	26	34%	0	0.002** Our study
	HSP90	ELISA	125	PE	36.8%	PE	PE	[49]
		ELISA	13	22	7.7%	PE	PE	[53]
		Microarray 50	26	4%	0	0.002** Our study

PE:pas exploitable; * Résultats obtenus sur chitosan, ** Résultats obtenus sur COOH

Scientific meeting communications (7 oral presentations and 6 posters)

Acknowledgements

It was a great pleasure for me to make a thesis in the team of chemistry and nanobiotechnology in Lyon Institute of Nanotechnology (INL), and I want to thank a lot of people for these three years.

microarray). By optimizing these factors, the performance of protein microarray can be greatly improved.

Chapter 2

Anti-heat shock proteins autoantibodies profiling in breast cancer serum using customized protein microarrays solution (1mg/ml in DMSO) and NHS-activated carboxymethyl dextran solution (1mg/ml in MES buffer), respectively.

Design of protein immobilization on flat glass slides

HSPB1, HSPD1, HSP70, HSP90, HSPA5, HSP90B1, HSP110 and P53 were spotted (sciFLEX-ARRAYER S3, Scienion, Germany) onto chemically functionalized flat glass slides according to Figure 2. Each field contains one protein spotted at different concentrations (0.005mg/ml, 0.01mg/ml, 0.05mg/ml and 0.1mg/ml). For each concentration, 8 replicates were spotted. Streptavidin-F555 (0.01mg/ml) and buffer solution were spotted as reference for surface chemistry quality and negative controls, respectively. According to our previous results, carbonate buffer (pH=9.6) was used as spotting buffer on chitosan surface; acetate buffer (pH=4.5) was used on the other surfaces (COOH, NHS, APDMES, CMD and MAMVE surfaces). After spotting, proteins were allowed to react with surfaces under saturated water vapors overnight at 4°C. Then slides were washed sequentially for 2 × 5 min with PBS, for 5 min with PBS-T (0.1%), and blocked with 10% BSA/PBS-T (0.1%) solution for 2h at room temperature (R.T.) to limit unspecific adsorption. Then slides were washed for 3 × 5 min with PBS-T (0.1%) and dried by centrifugation 3min at 1300rpm. Slides were then incubated with 0.1 µM purified antibodies diluted in 4% BSA/PBS-T 0.1%. These antibodies include biotin-labeled antibodies against HSPB1, HSPD1, HSP70 and P53 and non-labeled antibodies against HSP90, HSPA5, HSP90B1 and HSP110. Each field was incubated with one antibody solution and left to react for 1h at R.T. in saturated water vapors; then slides were washed for 3 × 5 min with PBS-T 0.1% and dried.

Chapter 3 Antibody microarray for the quantification of uPA and PAI-1 in breast tumor tissue

Concerning anti-PAI-1 antibodies, among all three antibodies tested, only anti-PAI-1 scFv was able to recognize PAI-1, as it was observed on our antibody microarray. Moreover, as previously described for anti-uPA, the recognition activity of anti-PAI-1 in ELISA is lower than those of the Femtelle kit. Thus, in immunoassay development, the choice of antibody is critical. High affinity between antibody and antigen is indispensable. Moreover, optimization of all the process including concentration, incubation solutions and time, is a required step to get best performance. For antibody microarray, surface chemistry is also a key parameter to improve the immunoassay. Among the 6 surface chemistries tested, COOH, NHS and chitosan surfaces showed best performance for the immobilization of anti-uPA and anti-PAI-1 scFv antibodies. Therefore, these 3 surfaces were selected for further experiments.

Influence of the concentration of captured antibodies

The concentration of capture antibody greatly influenced the lowest limit of detection (LOD) of antibody microarray. The threshold value of LOD was defined as mean of fluorescence signal (SNR) of buffer plus 3 S.D., where S.D. represents standard deviation. Thus, the lowest detected concentration displaying a SNR value higher than the threshold value, is defined as LOD. Moreover, spotting concentration influenced the dynamic range of detection. Dynamic range is a critical factor to evaluate the performance of assay. Generally, wider dynamic range corresponds to higher ability to test concentrations of uPA and PAI-1 in breast tumor tissue extraction.

Anti-uPA antibody (from Thermo Scientific) was spotted at 2 concentrations (3 µM and 6.6 µM) on the 3 selected surface chemistries, and detection of uPA was evaluated as a function of the concentration. Results obtained on chitosan surface are presented in Figure 8. As can be see, the detection of uPA is better when the spotted concentration of anti-uPA antibody is higher, reaching 0.2 ng/ml as LOD. Furthermore, spotting concentration affected the dynamic range of uPA detection. At the highest spotted antibody concentration (6.6 µM), the dynamic range was wider: 0.2 ng/ml-1.5 ng/ml. Therefore, in order to obtain high sensitivity of detection, high concentration of immobilized anti-uPA antibody is necessary.

(Figure 15 d ande). Furthermore, fluorescent signal (SNR) didn't reach a plateau at 2ng/ml, therefore, higher uPA concentration could be tested on NHS surface in order to increase the dynamic range. Furthermore, we compared correlation coefficient (R 2 ) of ELISA kit and our antibody microarray. Correlation coefficient (R 2 ) defines the association between x and y. An R 2 value of exactly 1 indicates the all data points lie exactly on a straight line. Generally, a correlation greater than 0.8 is described as strong, whereas a correlation less than 0.5 is described as weak. The R 2 of ELISA kit is 0.9999 (Figure 15a), very close to 1. In comparison, the R 2 obtained from our customized antibody microarray was also high; it was 0.9945 when the spotting concentration of anti-uPA antibody was at 6.6 µM on NHS surface (Figure 15 e). Table 3 summarized LOD, dynamic range of uPA detection and its corresponding SNR range on COOH, NHS and chitosan surface when the concentration of spotted anti-uPA antibody was at both 3µM and 6.6 µM. Among all three surfaces tested, NHS surface had the widest dynamic range for uPA detection at both two spotted concentrations of anti-uPA antibody. Moreover, the dynamic range is wider on chitosan surface when the concentration of spotted anti-uPA antibody is higher (at 6.6 µM) but fluorescent signals were weaker.

Materials

The proteins used were as followers: HSPB1, HSPD1, HSP70, HSP90, HSPA5, HSP90B1, HSP110, p53, mouse-anti human anti-HSPB1 antibody-biotin, mouse-anti human anti-HSPD1 antibody-biotin, mouse-anti human anti-HSP70 antibody-biotin, mouse-anti human anti-HSP90 antibody, mouse-anti human anti-HSPA5 antibody, mouse-anti human anti-HSP90B1 antibody, mouse-anti human anti-HSP110 antibody, mouse-anti human anti-p53 antibody-biotin, F555-labeled streptavidin and Cy3-labeled goat anti-mouse antibody IgG.

Detailed information of these proteins can be referred in part 2.

Surface functionalization of flat and microstructured glass slides

The details of surface functionalization of glass slides can be referred in part 2.2.3. Glass slides were functionalized with the 6 chemistries including COOH, NHS, chitosan, APDMES, MAMVE and CMD surfaces.

Elaboration and processing of protein microarray

Antigen proteins were spotted (between 4 to 8 replicates for each protein) on functionalized glass slides at different concentrations (0.01 mg/ml, 0.025 mg/ml, 0.05 mg/ml and 0.1 mg/ml) in acetate buffer (pH=4.6), using sciFLEX-ARRAYER S3 (Scienion, Germany). Streptavidin-

Stability of chemically functionalized glass slides

Chemically functionalized glass slides were stored under dry condition and then evaluated for chemical stability by contact angle measurement and by their ability to immobilize probe molecules (oligonucleotides (ODN) and proteins).

Contact angle measurement

Surface energy of chemically functionalized flat glass slides was followed by contact angle measurements (Digidrop Goniometer, GBX, France) as a function of time (1 month, 2 months, 3 months and 4 months). De-ionized water, ethylene-glycol and diiodomethane were used in all measurements. To minimize the experimental error, the contact angle was measured at three random locations for each sample and the average value was calculated.

Surface energy was determined according to Owens-Wendt model [16]. After spotting, proteins were allowed to react with functionalized surfaces under saturated water vapors overnight at 4°C. Then slides were washed sequentially for 15 min with 2% BSA (PBS-T 0.1%), for 2 × 10 min with PBS-T 0.1%, and then 10 seconds in DI water and dried for scanning.

Immobilization of probe molecules

Storage of spotted slides

In a first test, micro-structured glass slides were functionalized with both NHS and chitosan. P53 and HSPD1 were spotted in acetate buffer (pH=4.6) at 0.1 mg/ml; streptavidin-Cy3 (0.1mg/ml) and acetate buffer were spotted as quality and negative control, respectively, as shown in Figure 2. After spotting, proteins were allowed to react with functionalized surfaces under saturated water vapors overnight at 4°C. Then slides were washed sequentially for 2 × 5 min with PBS, for 5 min with PBS-T, and then dried by centrifugation for 3 min at 1300 rpm. Slides were stored for 1 month at 4°C without blocking or after blocking with 10%

BSA/PBS-T solution for 2hrs at room temperature to limit unspecific adsorption. Storage conditions tested were: dry under nitrogen atmosphere, in 5% threhalose solution and in 50% glycerol solution. Concerning the influence of the concentration of streptavidin-F555 on the detection level, Figure 13 showed the performance of HSP70/anti-HSP70 detection on APDMES surface using 2 streptavidin-F555 concentrations (0.05 µM and 0.19 µM). Fluorescence intensity of buffer was lower when the concentration of streptavidin-F555 was lower (0.05 µM) indicating that less unspecific adsorption was obtained. Thus to the detection signal (SNR) of HSP70/anti-HSP70 antibody interaction was higher. Same results were obtained for other proteins (HSPB1, HSPD1 and P53) on APDMES surface. In contrast, no significant difference was observed when the 4 antigens (HSPB1, HSPD1, HSP70 and P53) were immobilized on COOH and NHS surfaces. This could be due to higher unspecific adsorption of streptavidin-F555 on APDMES surface compared to COOH and NHS surfaces. Therefore, in order to reduce the unspecific adsorption, low concentration of strep-F555 (0.05 µM) will be used in further experiment.

Figure 13

Influence of the concentration of streptavidin-F555 on the performance of HSP70/anti-HSP70 detection; 2 streptavidin-F555 concentrations (0.05µM and 0.19µM) were tested.

At last, the concentration of second antibody (IgG-Cy3) could also influence the detection level of antigen/antibody recognition. Figure 14 showed the performance of HSP90/anti-HSP90 antibody recognition on COOH surface using 2 concentrations of IgG-cy3 (0.01 µM and 0.05 µM). As previously observed for streptavidin-F555, at high IgG-Cy3 concentration (0.05 µM), unspecific binding is higher (high fluorescence signal of buffer) leading to lower specific signal (SNR). Same results were obtained for other proteins (HSPA5, HSP90B1 and HSP110) on COOH, NHS and APDMES surfaces. Therefore, in order to reduce the unspecific binding, low concentration of IgG-Cy3 (0.05 µM) will be used in further experiment.

Conclusions

The aim of our study is to develop efficient protein microarray to screen biomarkers in breast cancer patients, thus providing diagnostic, prognostic and predictive value for each patient.

An overview of recent literature shows that a large amount of biomarkers (> 1200 molecules) are presented as candidates of high potential to develop molecular diagnosis for cancer detection. Thus, after a selection of numerous biomarkers involved in breast cancer, specific technology was developed to make customized microarrays based on microstructured glass slides. Two types of microarrays were elaborated.

We used antigen microarray to screen autoantibodies against heat shock proteins (HSPs) in breast cancer patients for providing diagnostic and prognostic value. In order to obtain efficient microarray performance, we firstly optimized various factors which influence the performances of protein microarray, including surface chemistry, spotting concentration, etc.

Among the 6 surface chemistries tested, two of them (COOH and chitosan) showed good performances for the immobilization of HSPs; therefore, these two surfaces were selected for screening the antibodies against HSPs in breast cancer serum. In total, 50 breast cancer patients and 26 healthy controls were tested. Our results showed that combining multiplex detection of anti-HSPs antibodies could achieve AUC of 0.978. It could discriminate breast cancer patients from healthy controls with sensitivity 86% and specificity 100%. Compared with literature, our antibody panel showed better performance for discriminating breast cancer patients from healthy controls. Furthermore, our data analysis method is more complete and comprehensive. Various studies only provide data on sensitivity without specificity, which is not complete. In contrast, we analyzed the AUC of the performance of our antibody panel and we could provide both sensitivity and specificity of our antibody panel.

Secondly, we used antibody microarray to test the concentration of urokinase type plasminogen activator (uPA) and its main inhibitor plasminogen activator inhibitor 1 (PAI-1)

in breast tumor tissue. High levels of uPA and PAI-1 are associated with high risk of recurrence and benefit of chemotherapy for breast cancer patients; therefore, they are good prognostic and predictive biomarkers for breast cancer. In order to obtain efficient antibody microarray, we firstly optimized the immobilization process. We have tested 3 antibodies against PAI-1; however, only one scFv antibody worked on one surface (COOH surface). En ce qui concerne les anticorps anti-uPA, la concentration de dépôt est de 6.6 µM en tampon PBS 1X (pH=7.4). La LOD de uPA obtenue dans ces conditions est de 0.2 ng/mL avec une gamme dynamique de 0.2 -2 ng/mL. La concentration de l'anticorps de détection anti-uPA biotinylé, préparé à partir du kit Femtelle, a également été optimisée.

Compte-tenu de ces résultats, des biopuces à anticorps anti-uPA ont été élaborées sur les