J Sven

D Mieog

Merlijn Hutteman

Joost R Van Der Vorst

Peter J K Kuppen

Ivo Que

Jouke Dijkstra

Eric L Kaijzel

Frans Prins

Clemens W G M Löwik

Vincent T H B M Smit

Cornelis J H Van De Velde

Dr Alexander L Vahrmeijer
email: a.l.vahrmeijer@lumc.nl

Image-Guided Tumor Resection using Real-Time Near-Infrared Fluorescence in a Syngeneic Rat Model of Primary Breast Cancer

Keywords: Near-Infrared Fluorescence Imaging, Image-Guided Surgery, Breast Conserving Surgery, Incomplete Tumor Resection, Cathepsin FFPE = formalin fixed paraffin embedded, NIR = near-infrared

Tumor involvement of resection margins is found in a large proportion of patients who undergo breast-conserving surgery. Near-infrared (NIR) fluorescence imaging is an experimental technique to visualize cancer cells during surgery. To determine the accuracy of real-time NIR fluorescence imaging in obtaining tumor-free resection margins, a proteaseactivatable NIR fluorescence probe and an intraoperative camera system were used in the EMR86 orthotopic syngeneic breast cancer rat model. Influence of concentration, timing and number of tumor cells were tested in the MCR86 rat breast cancer cell line. These variables were significantly associated with NIR fluorescence probe activation. Dosing and tumor size were also significantly associated with fluorescence intensity in the EMR86 rat model, whereas time of imaging was not. Real-time NIR fluorescence guidance of tumor resection resulted in a complete resection of 17 out of 17 tumors with minimal excision of normal healthy tissue (mean minimum and a mean maximum tumor-free margin of 0.2 ± 0.2 mm and 1.3 ± 0.6 mm, respectively). Moreover, the technique enabled identification of remnant tumor tissue in the surgical cavity. Histological analysis revealed that the NIR fluorescence signal was highest at the invasive tumor border and in the stromal compartment of the tumor. In conclusion, NIR fluorescence detection of breast tumor margins was successful in a rat model. The present study suggests that clinical introduction of intraoperative NIR fluorescence imaging has the potential to increase the number of complete tumor resections in breast cancer patients undergoing breast-conserving surgery.

INTRODUCTION

Incomplete tumor resections are an important clinical problem in breast cancer surgery.

Tumor involvement of resection margins is found in 5-40% of patients who undergo breastconserving surgery and these patients require additional surgery or intensified radiotherapy [START_REF] Mai | Resection margin status in lumpectomy specimens of infiltrating lobular carcinoma[END_REF][START_REF] Chagpar | Lumpectomy margins are affected by tumor size and histologic subtype but not by biopsy technique[END_REF][START_REF] Smitt | Association of clinical and pathologic variables with lumpectomy surgical margin status after preoperative diagnosis or excisional biopsy of invasive breast cancer[END_REF][START_REF] Rizzo | The effects of additional tumor cavity sampling at the time of breast-conserving surgery on final margin status, volume of resection, and pathologist workload[END_REF]. Furthermore, additional biopsies of the surgical cavity after primary resection have been shown to contain residual disease in 10% of patients with tumor-free specimen margins [START_REF] Hewes | Importance of routine cavity sampling in breast conservation surgery[END_REF]. As a result, 5-year isolated local recurrences rates of 6.7-11% are reported in patients with tumor-free specimen margins treated with breast-conserving surgery and radiotherapy [START_REF] Clarke | Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials[END_REF]. The occurrence of local relapse reduces the 15-year breast cancer specific and overall survival [START_REF] Clarke | Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials[END_REF]. Consequently, increase of the radical resection rate will likely improve breast cancer outcome. Intraoperative real-time visualization of cancer cells is a promising method to achieve that goal [START_REF] Nyirenda | Preclinical evaluation of nuclear morphometry and tissue topology for breast carcinoma detection and margin assessment[END_REF].

Near-infrared (NIR) fluorescence imaging is an experimental technique that can be used to visualize cancer cells during surgery. In current surgical practice, surgeons can only rely on palpation and visual inspection. Therefore, the use of NIR fluorescence imaging can be of great value, as already demonstrated in patients with glioma and liver cancer [START_REF] Stepp | ALA and malignant glioma: fluorescence-guided resection and photodynamic treatment[END_REF][START_REF] Ishizawa | Real-time identification of liver cancers by using indocyanine green fluorescent imaging[END_REF][START_REF] Nguyen | Real time intraoperative confocal laser microscopy-guided surgery[END_REF].

Advantages of NIR fluorescence light (700-900nm) include high tissue penetration (up to several centimeters deep) and low autofluorescence providing sufficient signal-tobackground ratio [START_REF] Frangioni | New technologies for human cancer imaging[END_REF]. Moreover, as the human eye is insensitive to NIR wavelengths, the use of NIR light will not interfere with the surgical field.

NIR fluorescence probes can target tumor cells through several mechanisms. For example, fluorophores can be conjugated to a tumor-specific antibody (e.g. directed to the Her-2/neu receptor), labeled to glucose derivates in order to visualize elevated metabolic rate, or autoquenched fluorophores can be activated by enzymatic cleavage in order to become fluorescent. The latter is of particular interest as certain enzyme systems are upregulated by a wide variety of cancer types, thus providing a more universally applicable NIR fluorescence probe. Proteolytic enzymes and in particular cathepsins from the cysteine protease family are a good candidate as they play essential roles in tumor growth, angiogenesis, resistance to apoptosis, and invasion [START_REF] Mohamed | Cysteine cathepsins: multifunctional enzymes in cancer[END_REF][START_REF] Gocheva | Distinct roles for cysteine cathepsin genes in multistage tumorigenesis[END_REF]. A member of this family, cathepsin B, is commonly active in the tumor microenvironment in various human cancers including breast cancer [START_REF] Mohamed | Cysteine cathepsins: multifunctional enzymes in cancer[END_REF][START_REF] Parker | Primary tumour expression of the cysteine cathepsin inhibitor Stefin A inhibits distant metastasis in breast cancer[END_REF][START_REF] Harbeck | Prognostic impact of proteolytic factors (urokinasetype plasminogen activator, plasminogen activator inhibitor 1, and cathepsins B, D, and L) in primary breast cancer reflects effects of adjuvant systemic therapy[END_REF][START_REF] Lah | Stefins and lysosomal cathepsins B, L and D in human breast carcinoma[END_REF]. Upregulated expression of cathepsin B is found in tumor, endothelial and immune cells, in particular macrophages [START_REF] Gocheva | Distinct roles for cysteine cathepsin genes in multistage tumorigenesis[END_REF]. Cathepsin B overexpression in human breast carcinomas is associated with poor differentiation, lymph node involvement, absence of estrogen receptor expression and impaired overall survival [START_REF] Lah | The Expression of Lysosomal Proteinases and Their Inhibitors in Breast Cancer: Possible Relationship to Prognosis of the Disease[END_REF][START_REF] Foekens | Prognostic significance of cathepsins B and L in primary human breast cancer[END_REF].

The protease-activatable NIR fluorescence probe ProSense (VisEn Medical, Woburn, MA) has been shown to detect a variety of tumors in nude or transgenic mice [START_REF] Weissleder | In vivo imaging of tumors with protease-activated near-infrared fluorescent probes[END_REF][START_REF] Kirsch | A spatially and temporally restricted mouse model of soft tissue sarcoma[END_REF][START_REF] Bremer | Imaging of differential protease expression in breast cancers for detection of aggressive tumor phenotypes[END_REF][START_REF] Von Burstin | Highly sensitive detection of early-stage pancreatic cancer by multimodal near-infrared molecular imaging in living mice[END_REF][START_REF] Sheth | Improved detection of ovarian cancer metastases by intraoperative quantitative fluorescence protease imaging in a pre-clinical model[END_REF][START_REF] Alencar | Colonic adenocarcinomas: near-infrared microcatheter imaging of smart probes for early detection--study in mice[END_REF][START_REF] Bogdanov | Cellular activation of the self-quenched fluorescent reporter probe in tumor microenvironment[END_REF][START_REF] Niedre | Early photon tomography allows fluorescence detection of lung carcinomas and disease progression in mice in vivo[END_REF][START_REF] Ntziachristos | Fluorescence molecular tomography resolves protease activity in vivo[END_REF][START_REF] Bremer | Optical imaging of spontaneous breast tumors using protease sensing 'smart' optical probes[END_REF][START_REF] Gounaris | Live imaging of cysteine-cathepsin activity reveals dynamics of focal inflammation, angiogenesis, and polyp growth[END_REF][START_REF] Grimm | Use of gene expression profiling to direct in vivo molecular imaging of lung cancer[END_REF][START_REF] Nguyen | Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival[END_REF].

However, as tumor progression and metastasis are regulated by the surrounding microenvironment, it is important to use syngeneic animal models that allow appropriate crosstalk at the invasive tumor border to study probes that are activated by proteolytic activity. Moreover, the use of a larger animal model such as the rat offers more challenges in terms of tissue penetration of NIR fluorescence probes.

Therefore, the aim of this study was to assess the technique of NIR fluorescence imaging in a syngeneic breast cancer rat model using ProSense and to determine the accuracy of intraoperative tumor detection to obtain an adequate tumor-free resection margin.

MATERIAL AND METHODS

Breast cancer cell line and culture conditions:

The MCR86 cell line is a rapidly growing, syngeneic breast cancer cell line derived after subcutaneous transplantation of macroscopic lung tumors, which developed in a female WAG/Rij rat after intravenous inoculation of MCR83 breast cancer cells [START_REF] Van Dierendonck | Surgically induced cytokinetic responses in experimental rat mammary tumor models[END_REF]. Tumor cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine (Gibco, Invitrogen Ltd, Carlsbad, CA), 10% heat-inactivated fetal calf serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin sulphate.

Breast cancer model and tumor induction:

The related EMR86 model is a transplantable, hormone-dependent, metastasizing mammary carcinoma that originated in a female WAG/Rij rat bearing a subcutaneously implanted estrogen pellet and is developed by our research group (Fig. 1a) [START_REF] Wijsman | A prolactin-dependent, metastasising rat mammary carcinoma as a model for endocrinerelated tumour dormancy[END_REF]. Tumors are only induced and maintained in rats carrying estrogen pellets, whereas tumors transplanted into non-estrogenized animals do not grow out. Removal of the estrogen pellet induces apoptosis and tumor regression (Fig. 1b).

EMR86 tumors are histologically classified as high-grade invasive ductaltype adenocarcinomas, with both a cribiform and a solid growth pattern. In large tumors areas, comedo type necrosis can be appreciated. Tumors have a stromal compartment of approximately 30% depending on tumor size (Fig. 1c). EMR86 tumor cells show strong nuclear expression of the estrogen and progesterone receptor in more than 90% of cells, but stain negative for HER2/neu receptor (Fig. 1d-f). Therefore, EMR86 tumors closely resemble the luminal A molecular subtypethe most prevalent subtype of human breast cancer [START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF][START_REF] Sihto | Molecular subtypes of breast cancers detected in mammography screening and outside of screening[END_REF].

For tumor induction, fresh EMR86 tumor fragments of 0.5-1 mm 3 were implanted in the mammary fat pad at four sites of female WAG/Rij rats (Charles River, Maastricht, the Netherlands) aging 4-6 months. (A stable cell line from EMR86 tumor has not yet been established successfully, therefore, tumor transplantation is used.) During the same session, an estrogen pellet was implanted subcutaneously in the intrascapular region of the neck. The in-house generated pellets consist of 2 mm by 3 mm silicone tubes containing 1.5 mg 17 βestradiol on a 1:3 cholesterol/paraffin basis. Tumor volumes were estimated twice weekly using digital calipers by measuring three orthogonal diameters of the tumor and multiplying this product by π/6. All rats were housed in the animal facility of the Leiden University Medical Center. Pellet food and fresh tap water were provided ad libitum. The weight of the animals was followed throughout the experiment to monitor their general health state. The Animal Welfare Committee of the Leiden University Medical Center approved the study.

The study was conducted in concordance with the "Guidelines for the Welfare of Animals in Experimental Neoplasia" (Second Edition, 1997) available online at http://www.ncrn.org.uk/csg/animal_guides_text.pdf.

NIR fluorescence probe:

The commercially available, protease-activatable NIR fluorescence probes ProSense680 and ProSense750 were used (VisEn Medical). The probes consist of a synthetic graft polymer composed of poly-L-lysine that is sterically protected by multiple methoxypolyethylene glycol side chains and to which multiple fluorophores are attached [START_REF] Weissleder | In vivo imaging of tumors with protease-activated near-infrared fluorescent probes[END_REF]. In this non-activated state, the fluorophores are positioned in close proximity to one another, which results in mutual energy transfer and thus inhibition of fluorescence emission. After enzymatic cleavage of the backbone, the fluorophores are released and regain their fluorescent characteristics. A number of cysteine proteases are involved in this process. Cathepsin B, and to a lesser degree cathepsin K, L, and S, has been demonstrated to be a major contributor to cleavage and activation of ProSense [START_REF] Weissleder | In vivo imaging of tumors with protease-activated near-infrared fluorescent probes[END_REF]. ProSense680 and ProSense750 have peak absorption of 680 nm and 750 nm, respectively. ProSense680 was selected for intraoperative studies and fluorescence microscopy because of the better matching of the laser of the intraoperative camera system with the peak excitation of ProSense680. ProSense750 was used for cell line experiments and non-invasive animal experiments because of the better spectral separation of autofluorescence signal.

Intraoperative NIR fluorescence camera system: The Fluobeam intraoperative NIR fluorescence camera system (Fluoptics, Grenoble, France) used in this study has been described previously by our group. [START_REF] Mieog | Novel Intraoperative Near-infrared Fluorescence Camera System For Optical Image-guided Cancer Surgery[END_REF][START_REF] Keramidas | Intraoperative near-infrared image-guided surgery for peritoneal carcinomatosis in a preclinical experimental model[END_REF] Briefly, the system is composed of a class 3B laser (100mW) emitting at 690 nm resulting in an illumination power of 2.6 mW/cm 2 .

Filtered white light (350-650 nm) provides an irradiance of 7x10 3 lx at the focus level. The emitted fluorescence is collected through a high pass filter (> 700 nm) by a 12 bits CCD camera resulting in a system spatial resolution of 0.17 mm/pixel.

Experimental design:

Cell line experiments: For fluorescence measurements, tumor cells were harvested with a solution of 0.25% (w/v) EDTA and 0.25% (w/v) trypsin in Hanks' Buffered Salt Solution (Sigma-Aldrich, St. Louis, MO), washed three times in 0.9% phosphate buffered saline and 200 µL complete medium suspensions were made and transferred on a 96-well acrylate plate (Greiner Bio-one, Alphen aan de Rijn, the Netherlands, #655090; suitable for fluorescence measurements) and kept at 37ºC and 5% CO 2 . At day 2, cells were washed and autoquenched ProSense750 (22.5 to 180 nM, 200 µl) was added. At day 3, cells were washed and 200 µl complete medium was added. Also, time-dependent studies were performed, during which ProSense750 was added at the indicated time-points (8 to 48 h).

Fluorescence intensity was measured using the Odyssey NIR fluorescence scanning device (LI-COR Biosciences, Lincoln, NE). Overlying grids were drawn and fluorescence intensity was measured for each well using the Odyssey software (Version 2.1).

Animal experiments: Throughout injection, imaging and surgical procedures, rats were anaesthetized with inhalation of 2% mixture of isoflurane in oxygen. The rats were constantly monitored for the rate of the respiration and depth of anesthesia. Before imaging, rats were shaved to reduce absorption of the optical signal. A total of 20 rats bearing 77 primary mammary tumors varying in size from 0.01 to 1.8 cm 3 were used in this study.

In a dose-dependent and time-dependent experiment, tumor-bearing rats (N = 9) were randomly assigned over three ProSense750 dose groups and intravenously injected with 2.5, 5 or 10 nmol ProSense750 (150 µl). Whole-body fluorescence was measured 24 h and 48 h after administration of ProSense750 using the IVIS Spectrum (Caliper LifeSciences, Hopkinton, MA), which allowed separation of the ProSense750 signal from the background fluorescence by means of spectral unmixing [START_REF] Mansfield | Visualization of microscopy-based spectral imaging data from multi-label tissue sections[END_REF]. Acquisition settings were kept constant for the different dose groups and on the two consecutive days. Total photon counts per second were measured for each tumor using the Living Image software (Version 3.0, Caliper LifeSciences) and divided by the tumor volume as assessed by digital caliper measurement.

In an intra-operative experiment, tumor-bearing rats (N = 7) were operated under direct fluorescence guidance 24 h after intravenous administration of 10 nmol ProSense680 (150 µl). NIR fluorescence intensity of exposed tumors and surrounding tissues was measured with the Fluobeam intraoperative camera system. Tumor-to-background ratios were calculated by drawing regions of interest at the tumor and at the surrounding tissue by visual interpretation and subsequent measurement of fluorescent intensity using the opensource software ImageJ [39]. Merged visible light and NIR fluorescence light images were created using Adobe Photoshop CS3 Software (Version 10.0.1, Adobe Systems Inc., San Jose, CA). In order to determine sensitivity and specificity of the intraoperative NIR fluorescence technique, an attempt was made to completely remove all tumor tissue while removing as little as possible of the normal surrounding mammary fat pad tissue strictly based on the fluorescence signal in 5 of these 7 rats. Tumors were removed by sharp dissection. Excised tumors were inked with India ink, sliced in two or three parts depending on the size of the tumor and fixed overnight in 4% buffered formalin and embedded in paraffin (FFPE) blocks, mimicking the standard clinical workflow. After resection of the primary tumor, the surgical cavity was inspected with the Fluobeam to detect any remnant fluorescent tissue. After resection of all remnant fluorescent spots, random biopsies were taken of the surgical cavity from every quadrant in order to determine specificity of the technique. In one additional rat an irradical resection was performed intentionally to test the Fluobeam's ability to detect remnant tumor tissue. All specimens were fixed in formalin as described above. FFPE tumor sections of 4 µm were air-dried and stained with hematoxylin and eosin (H&E). The tumor size, the minimum and maximum tumor-free margin and the presence of tumor in the random biopsies were determined by an experienced breast pathologist (V.T.H.B.M.S.).

Fluorescence microscopy:

Cell line experiments: Time-dependent microscopic analysis of ProSense680 activation by cultured MCR86 cancer cells was performed using the LSM510 Zeiss confocal microscope (Jena, Germany, 40x/0,75w Ph2 ACHROPLAN objective). A 633 nm laser was used for fluorescence excitation and a 650 nm Long Pass for emission. Cells were cultured in 3.5 cm petri dishes incubated with 33.3 nM ProSense680 in 3 ml medium. Cells were kept at 37ºC and imaged for 4.5 hours.

Ex vivo tumor imaging:

Freshly excised tumors with a wide rim of surrounding normal mammary tissue of rats injected with 10 nmol ProSense680 (N = 3) were halved. From one half, a 2 mm section was analyzed using the Odyssey scanning device at 21 µm resolution.

Tumor border was defined as the outer rim of the tumor and its width was approximately 15% of the tumor diameter. The other half was snap-frozen on dry ice and stored at -80ºC.

Unfixed 20 µm sections were measured for fluorescence using the Odyssey scanning device at 21 µm resolution. Processing of sections was performed under reduced light conditions to prevent photobleaching. Subsequently, the tissue sections were stained with H&E. The fluorescence image and the H&E image were merged using Adobe Photoshop enabling detailed analysis of the NIR fluorescence distribution along with the histological context.

Statistical analysis:

Statistical analyses and generation of graphs were performed using GraphPad Prism software (Version 5.01, La Jolla, CA). Continuous variables were analyzed using the (paired) t-test for comparison of two groups and one-way analysis of variance (ANOVA) for comparison of more than two groups. To test the effect of two independent variables two-way ANOVA was used. Trend analysis and one-tailed planned comparisons between adjacent groups were conducted. When the assumption of homogeneity of variance was violated (Levene's test), the Brown-Forsythe F-ratio was reported. Pearson's correlation coefficients R were calculated for correlation analyses. All statistical tests were two-tailed and P < 0.050 was considered significant.

RESULTS

In vitro activation of ProSense by breast cancer rat cell line:

The autoquenched NIR fluorescence probe ProSense680 was activated by MCR86 breast cancer cells within two hours (Online Resource 1). Microscopic analysis revealed an intracellular localization of activated ProSense680 (Fig. 2a-b). Both incubation time (F(3, 36)=1615, P < 0.0001) and concentration of ProSense750 (F(3, 36)=3704, P < 0.0001) significantly influenced ProSense750 activation as measured by fluorescence intensity (two-way ANOVA, Fig. 2c).

Also, there was an interaction between incubation time and ProSense750 concentration (F(9, 36)=230.4, P < 0.0001), indicating that the difference in incubation time within ProSense750 concentration groups influenced ProSense750 activation. Furthermore, fluorescence intensity was highly correlated with number of MCR86 cells (F(4, 10.97) = 39.13, P < 0.0001, R = 0.904; Fig. 2d).

In vivo activation of ProSense by syngeneic rat model of primary breast cancer: EMR86

breast tumors were successfully imaged percutaneously using the IVIS Spectrum after intravenous administration of ProSense750 (Fig. 3a-b). To test the effect of ProSense750 dose and time of imaging on fluorescence intensity, nine rats (N = 35 mammary tumors, mean volume = 0.38 0.36 cm 3 ) were randomly assigned to three ProSense750 dose groups and were imaged 24 h and 48 h post-injection. In concordance with the in vitro data, ProSense750 dose significantly influenced fluorescence intensity (F(2,32) = 3.56, P = 0.04, two-way ANOVA, Fig. 3c). Of note, a substantial part of the tumors could not be identified in the 2.5 nmol dose group (24 h: 4 of 11 tumors, 48 h: 7 of 11 tumors) and the 5 nmol dose group (24 h: 2 of 12 tumors, 48 h: 3 of 12 tumors), whereas in the 10 nmol dose group all tumors were identified. In contrast to the in vitro data, time of imaging did not influence fluorescence intensity (F(2,32) = 2.47, P = 0.13, two-way ANOVA, Fig. 3c). Furthermore, there was no interaction between time of imaging and ProSense750 dose (F(2,32) = 1.06, P = 0.36), indicating that the difference in time of imaging within dose groups did not influence fluorescence intensity. Based on these results, a dose of 10 nmol was selected for further in vivo testing. Imaging 24 h after administration of ProSense was selected for practical purposes. With these settings, fluorescence intensity was significantly correlated with tumor volume (R = 0.934, P < 0.0001; Fig. 3d), which was in concordance with the in vitro data.

Intraoperative NIR fluorescence-guided resection of primary breast cancer: Using the Fluobeam intraoperative camera system, all primary breast tumors (N = 26 tumors, 7 rats) were successfully identified 24 h after intravenous administration of 10 nmol ProSense680 (Fig. 4a) as shown in Online Resource 2. The technique provided a clear demarcation of tumor and surrounding mammary fat pad tissue with a mean tumor-to-background ratio of 2.35 ± 0.37 (paired t-test, t = 14.95, P < 0.0001, Fig. 4b). To determine the accuracy of tumor margin detection of the intraoperative NIR fluorescence technique, 17 tumors (N = 5 rats) were resected completely under direct, real-time NIR fluorescence guidance, while removing as little as possible of the normal surrounding mammary fat pad tissue and processed for histopathological analysis (Fig. 4c). All 17 tumors were completely excised with a mean minimum and a mean maximum tumor-free margin of 0.2 ± 0.2 mm and 1.3 ± 0.6 mm, respectively (Table 1). Mean pathological tumor size was 5.0 ± 2.1 mm. In two cases, after resection of the primary tumor, remnant fluorescent tissue was detected in the surgical cavity with Fluobeam (Table 1). One specimen contained a lymph node with metastatic involvement and the other contained a reactive lymph node with abundant macrophage influx but no tumor involvement. This false-positive finding can be explained by the fact that macrophages show high cathepsin B expression [START_REF] Gocheva | Distinct roles for cysteine cathepsin genes in multistage tumorigenesis[END_REF]. After resection of all fluorescent spots, random biopsies were taken of the surgical cavity from every quadrant.

None of the random biopsies (N = 64, 5 rats) contained histologically any tumor cells. These results indicate an excellent accuracy of the technique.

In one additional rat, an irradical resection was performed intentionally in order to show the Fluobeam's ability to detect remnant tumor tissue. As shown in Online Resource 3, remnant tumor tissue could be detected and subsequently resected under direct NIR fluorescence guidance.

Ex vivo NIR fluorescence microscopy:

In order to determine the histological localization of ProSense680, tumors were excised with a wide rim of normal mammary tissue 24 h after intravenous administration of 10 nmol ProSense680 (N = 12 tumors, 3 rats; Fig. 5a).

Fluorescence imaging of 2 mm thick, fresh tumor slices revealed that the NIR fluorescence intensity was 1.6 ± 0.3 times higher at the border of the tumor than at its center (paired t-test, t = 4.99, P = 0.0005, N = 12 tumors; Fig. 5b). In order to obtain more detail about the histological localization of ProSense680, unfixed 20 µm frozen tissue sections were measured for fluorescence using the Odyssey and subsequently stained with H&E (Fig. 5c).

These results showed that NIR fluorescence is mainly located in the stromal compartment of the breast tumors and in particular at the tumor border.

DISCUSSION

In the current study, we demonstrated the feasibility of real-time intraoperative NIR fluorescence identification of breast tumors in a syngeneic orthotopic breast cancer rat model using the protease-activatable probe ProSense. In both the cell line and animal experiments, fluorescence intensity was strongly correlated with number of tumor cells, tumor size and ProSense dose. In contrast to the in vitro data, time of imaging (24 h vs. 48 h after administration of ProSense) did not significantly influence fluorescence intensity of breast tumors. This is likely a result of the relatively long blood half-life of ProSense (half-life in Previous studies in which NIR fluorescent and protease-activatable probes were tested have utilized xenograft or transgenic mouse models [START_REF] Weissleder | In vivo imaging of tumors with protease-activated near-infrared fluorescent probes[END_REF][START_REF] Kirsch | A spatially and temporally restricted mouse model of soft tissue sarcoma[END_REF][START_REF] Bremer | Imaging of differential protease expression in breast cancers for detection of aggressive tumor phenotypes[END_REF][START_REF] Von Burstin | Highly sensitive detection of early-stage pancreatic cancer by multimodal near-infrared molecular imaging in living mice[END_REF][START_REF] Sheth | Improved detection of ovarian cancer metastases by intraoperative quantitative fluorescence protease imaging in a pre-clinical model[END_REF][START_REF] Alencar | Colonic adenocarcinomas: near-infrared microcatheter imaging of smart probes for early detection--study in mice[END_REF][START_REF] Bogdanov | Cellular activation of the self-quenched fluorescent reporter probe in tumor microenvironment[END_REF][START_REF] Niedre | Early photon tomography allows fluorescence detection of lung carcinomas and disease progression in mice in vivo[END_REF][START_REF] Ntziachristos | Fluorescence molecular tomography resolves protease activity in vivo[END_REF][START_REF] Bremer | Optical imaging of spontaneous breast tumors using protease sensing 'smart' optical probes[END_REF][START_REF] Gounaris | Live imaging of cysteine-cathepsin activity reveals dynamics of focal inflammation, angiogenesis, and polyp growth[END_REF][START_REF] Grimm | Use of gene expression profiling to direct in vivo molecular imaging of lung cancer[END_REF][START_REF] Nguyen | Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival[END_REF]. A limitation of many animal models of breast cancer is that, compared to the human situation, less normal mammary tissue is present in relation to tumor size. Therefore, these tumors are easily resected by removing all breast tissue. However, this approach does not resemble the principles underlying breast-conserving surgery. This study was performed using an orthotopic breast cancer model that is syngeneic to immunocompetent female WAG/Rij rats.

In syngeneic models, tumors are grown in homologous species and in the strain in which the tumor has originated. Therefore, these models are more representative of the natural tumor-host interaction. Although no preclinical tumor model will contain all features of the complex biology of human cancer, this syngeneic model has several strengths, including its hormone-sensitivity, cribiform growth pattern and its ability to induce regression by estrogen pellet removal. Moreover, this model captures several important features of luminal A hormone-dependent, HER2/neu negative human breast cancer, which is the most predominant subtype of breast cancer [START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF][START_REF] Sihto | Molecular subtypes of breast cancers detected in mammography screening and outside of screening[END_REF]. Another intrinsic limitation of animal models of surgical interventions is the potential occurrence of performance bias, because researchers carrying out the intervention can not be blinded to the allocated treatment. This form of bias may lead to flawed results. [START_REF] Juni | Systematic reviews in health care: Assessing the quality of controlled clinical trials[END_REF] To surpass these limitations in our study, a one-arm study design was chosen using small breast tumors (5.0 ± 2.1 mm). Tumors were excised based on the NIR fluorescence signal and examined by a breast pathologist using standard clinical methodology. Using this approach, all tumors were completely resected with a mean maximum tumor-free resection margin of 1.3 ± 0.6 mm, indicating that a minimal amount of normal mammary tissue was resected. Although syngeneic models provide a relevant tumorhost interaction, their major drawback is that the tumor cells are rodent, and therefore express the rodent homologues of the desired targets. However, the main target of ProSense, the cysteine protease family (most particular cathepsin B) is strongly conserved amongst mammals [START_REF] Mohamed | Cysteine cathepsins: multifunctional enzymes in cancer[END_REF]. Upregulation of cathepsin B has been confirmed extensively in human breast cancer [START_REF] Mohamed | Cysteine cathepsins: multifunctional enzymes in cancer[END_REF][START_REF] Parker | Primary tumour expression of the cysteine cathepsin inhibitor Stefin A inhibits distant metastasis in breast cancer[END_REF][START_REF] Harbeck | Prognostic impact of proteolytic factors (urokinasetype plasminogen activator, plasminogen activator inhibitor 1, and cathepsins B, D, and L) in primary breast cancer reflects effects of adjuvant systemic therapy[END_REF][START_REF] Kuester | The cathepsin family and their role in colorectal cancer[END_REF]. It is therefore expected that this kind of protease-activated NIR fluorescence probes will be applicable for intraoperative NIR fluorescence imaging in a large proportion of breast cancer patients.

Analysis of the histological localization of activated ProSense demonstrated that the invasive tumor border exhibited the most intense NIR fluorescence signal. This observation is in concordance with immunohistological analysis of cathepsin B reported by others [START_REF] Parker | Primary tumour expression of the cysteine cathepsin inhibitor Stefin A inhibits distant metastasis in breast cancer[END_REF][START_REF] Alencar | Colonic adenocarcinomas: near-infrared microcatheter imaging of smart probes for early detection--study in mice[END_REF] and is in line with the pathophysiological role of cathepsins as reviewed by Gocheva [START_REF] Gocheva | Cysteine cathepsins and the cutting edge of cancer invasion[END_REF]. Cathepsins promote tumor invasion through several possible mechanisms. First, they can directly cleave components of the extracellular matrix and basement membrane, essentially clearing a path for the migration of tumor cells away from the primary tumor.

Second, at the cell membrane, cathepsins can direct a proteolytic cascade in which they activate other proteases such as matrix metalloproteinases and urokinase plasminogen activator, which in turn promotes tumor invasion. Third, cleavage of the cell adhesion protein, E-cadherin, at the cell surface can disrupt adherens junctions and thus facilitate cancer cell migration and invasion [START_REF] Gocheva | Cysteine cathepsins and the cutting edge of cancer invasion[END_REF]. Apart from tumor border, NIR fluorescence was higher in the stromal compartment of the tumors. This finding is in concordance with Gounaris et al., who demonstrated that CD11 + tumor-infiltrating macrophages accounted for 75% of the ProSense signal at FACS analysis [START_REF] Gounaris | Live imaging of cysteine-cathepsin activity reveals dynamics of focal inflammation, angiogenesis, and polyp growth[END_REF]. In summary, as complete resection of breast tumors requires adequate visualization of tumor margins, the increased activity of proteolytic enzymes at the invasive tumor border provides an excellent target for intraoperative NIR fluorescence-guided surgery.

Future clinical studies will have to provide proof-of-principle of intraoperative NIR fluorescence tumor detection. Currently, a number of intraoperative NIR fluorescence imaging systems are clinically available and have already been used for sentinel lymph node mapping. [START_REF] Troyan | The FLARE intraoperative nearinfrared fluorescence imaging system: a first-in-human clinical trial in breast cancer sentinel lymph node mapping[END_REF][START_REF] Hirche | ICG fluorescence-guided sentinel node biopsy for axillary nodal staging in breast cancer[END_REF][START_REF] Kitai | Fluorescence navigation with indocyanine green for detecting sentinel lymph nodes in breast cancer[END_REF] It is expected that several tumor-targeting NIR fluorescent probes (like ProSense) will receive regulatory approval within the next few years.

A therapeutic challenge of any new breast cancer imaging technology is the detection of occult tumor deposits in the breast. These tumor deposits could influence surgical decision making, but do not necessarily have a prognostic relevance, because in the vast majority of cases postoperative radiation will eradicate these microscopic deposits. For instance, in preoperative MR imaging, the identification of additional tumor deposits is two to three times higher than the incidence of local recurrence, resulting in mastectomies that may not be beneficial to the patient. [START_REF] Bloom | A clinical oncologic perspective on breast magnetic resonance imaging[END_REF] Consequently, detection of tumor at the margins would be beneficial, detection of tumor deposits beyond the margins, below the cut edge of the lumpectomy cavity, could have the potential to increase surgical resection volume (or even mastectomy rates) without a benefit in survival. Since the maximum penetration depth for NIR fluorescence imaging is currently around 1 cm, [47] it is unlikely that intraoperative NIR fluorescence imaging will detect occult lesions at several centimeters distance from the primary tumor.

  mice 18 h, VisEn Medical website) and may provide flexible operation planning in future clinical applications. Histological analysis demonstrated that NIR fluorescence intensity of tumors was highest at the invasive tumor border. Resection of tumors under real-time NIR fluorescence guidance showed excellent accuracy of the technique in the intraoperative detection of tumor margins. These results suggest that clinical introduction of intraoperative NIR fluorescence imaging using a protease-activatable probe such as ProSense has the potential to increase the number of complete tumor resections in breast cancer patients undergoing breast-conserving surgery.

  Sensitivity of NIR fluorescence is mostly dependent on photon absorption of the tissue, fluorescence excitation power of the light source and concentration of the NIR fluorophore in tissue. Required camera exposure times are inversely correlated with the amount of fluorescence signal. In the current study, camera exposure times of 2-20 ms were used. The field-of-view of the Fluobeam camera system is 7 cm of diameter. Therefore, the time needed to evaluate tumor margins and excision cavities is at most several seconds. Consequently, this real-time intraoperative technique is unlikely to prolong surgical time significantly. In conclusion, this study provides preclinical validation of an innovative technique in which NIR fluorescence light is used to visualize breast tumors and to provide real-time guidance during subsequent resection. Clinical translation of these results might be very promising because of high accuracy of the technique, flexible surgical planning, increased proteolytic activity at the tumor border and upregulation of cathepsin B in a large proportion of breast cancer patients. Therefore, this study warrants clinical validation of this technique, once NIR fluorescence probes become available for clinical testing, with the ultimate goal to increase the radical resection rate of patients undergoing breast-conserving surgery.

Fig. 2 -

 2 Fig. 2 -In vitro activation of ProSense by syngeneic breast cancer rat cell line:

Fig. 3 -

 3 Fig. 3 -In vivo activation of ProSense by syngeneic rat model of primary breast cancer:

Fig. 4 -

 4 Fig. 4 -Intraoperative NIR fluorescence-guided resection of primary breast cancer and

Fig. 5 -Online Resource 1 -

 51 Fig. 5 -Ex vivo NIR fluorescence microscopy of resected breast cancer:

  Figure 1 Mieog et al.

Table 1 -Pathological assessment of inked resection margins after NIR fluorescence guided excision of primary breast cancer

 1

	Rat	Tumor	Maximum ø	Maximum ø	Radical	Minimum	Maximum	Remnant	Random non-
		ID	Specimen	Tumor	resection?	margin	margin	fluorescence	fluorescence biopsies
			(mm)	(mm)		(mm)	(mm)	tumor-positive?	tumor-positive?
	A	1	7.4	3.8	yes	<0.1	1.3	0	0/4
		2	2.8	0.8	yes	<0.1	0.9	1/1 a	0/4
		3	8.8	4.8	yes	0.7	2.4	0	0/3
	B	4	7.5	5.1	yes	<0.1	1.4	0	0/4
		5	4.9	3.1	yes	<0.1	1.0	0	0/4
		6	5.4	4.2	yes	0.1	1.0	0	0/4
		7	9.2	8.0	yes	<0.1	1.0	0	0/4
	C	8	9.1	7.9	yes	<0.1	1.1	0	0/4
		9	8.9	5.7	yes	0.2	2.5	0	0/4
		10	9.7	7.7	yes	<0.1	1.8	0	0/4
	D	11	5.3	2.4	yes	0.4	1.6	0	0/3
		12	6.1	4.3	yes	0.2	1.0	0	0/4
		13	5.7	4.4	yes	0.2	1.5	0	0/4
	E	14	8.2	6.9	yes	<0.1	0.8	0	0/4
		15	8.3	7.3	yes	<0.1	0.6	0	0/4
		16	7.3	5.6	yes	0.3	0.7	0	0/4
		17	6.6	3.0	yes	<0.1	1.9	0/1 b	0/4
	Mean ± SD	7.1 ± 1.9	5.0 ± 2.1		0.2 ± 0.2	1.3 ± 0.6	1/2	0/64
	a Macrometastasis in lymph node
	b Reactive lymph node with abundant macrophage influx

Description of a syngeneic rat model of hormone-dependent breast cancer:

	a. EMR86 breast tumors originate after transplantation of 0.5 mm 3 fresh tumor fragments
	at the mammary fat path of female Wag/Rij rats. Shown is a tumor four weeks after
	transplantation.
	b. EMR86 tumors are only induced and maintained in rats carrying estrogen pellets.
	Removal of the estrogen pellet induces apoptosis and tumor regression (N = 16
	tumors, 4 rats).
	c. EMR86 tumors are histologically classified as high-grade invasive ductal carcinomas,
	with both a cribiform and a solid growth pattern. Tumors have a stromal compartment
	of approximately 30% depending on tumor size.
	d-f. EMR86 tumor cells show strong nuclear expression of the estrogen and progesterone
	receptor in more than 90% of cells, but stain negative for HER2/neu receptor.
	Therefore, EMR86 tumors closely resemble the luminal A molecular subtype.

Acknowledgements

We want to thank Rob Keyzer and Anita Sajet for technical assistance and Fluoptics (Grenoble, France) for use of the Fluobeam® system. J.S.D. Mieog is a MD-medical research trainee funded by The Netherlands Organisation for Health Research and Development (grant nr. 92003526). Financial Disclosure: J.S.D. Mieog is a MD-medical research trainee funded by The Netherlands Organisation for Health Research and Development (grant nr. 92003526). Presented in part at the 2009 San Antonio Breast Cancer Symposium, San Antonio, TX and the 2009 World Molecular Imaging Conference, Montreal, Canada.


Eliza Wiercinska

Hildegonda P H Naber

Evangelia Pardali

Gabri Pluijm

Hans Dam

Peter Dijke

· Hildegonda

P H Naber

· Gabri

Hans Van Dam

Peter Ten Dijke
email: p.ten_dijke@lumc.nl

· H Wiercinska

· E Naber

· H Pardali

· P Van Dam

Ten Dijke

G Van Der Pluijm

The TGF-/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system

Keywords:

The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system

Introduction

Breast cancer is one of the leading causes of death in women. Morbidity and mortality are predominantly caused by metastasis, but not by the primary tumor. A critical event in the metastatic cascade is tumor cell invasion. However, the mechanisms by which primary tumor cells acquire an invasive phenotype are not well known. One of the inducers of invasion is transforming growth factor-(TGF-β). TGF-β is thought to play a dual role in cancer progression [START_REF] Massagué | TGF in Cancer[END_REF][START_REF] Akhurst | TGF-signaling in cancer--a double-edged sword[END_REF]. It can act as a tumor suppressor in the early stages, but promote cancer progression in later stages of the disease. Moreover, TGF-β is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis [START_REF] Ghellal | Prognostic significance of TGF 1 and TGF 3 in human breast carcinoma[END_REF][START_REF] Sheen-Chen | Serum Levels of Transforming Growth Factor 1 in Patients With Breast Cancer[END_REF][START_REF] Ivanovic | Elevated plasma levels of transforming growth factor-1 (TGF-1) in patients with advanced breast cancer: association with disease progression[END_REF][START_REF] Desruisseau | Determination of TGF 1 protein level in human primary breast cancers and its relationship with survival[END_REF].

TGF-β elicits its cellular responses via TGF-type II (TβRII) and type I (T RI) receptors. Upon TGF--induced heteromeric complex formation, T RII phosphorylates T RI. The activated T RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3.

These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes [START_REF] Ten Dijke | New insights into TGF--Smad signalling[END_REF][START_REF] Moustakas | The regulation of TGF signal transduction[END_REF]. Tumor suppressor activities of TGF-β/Smad signaling include amongst others repression of c-myc [START_REF] Chen | Defective repression of c-myc in breast cancer cells: A loss at the core of the transforming growth factor growth arrest program[END_REF] and induction of cyclin-dependent kinase inhibitors [START_REF] Hannon | p15INK4B is a potential effector of TGF-induced cell cycle arrest[END_REF][START_REF] Datto | Transforming growth factor induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism[END_REF].

During cancer progression accumulation of genetic mutations as well as epigenetic changes can render cells insensitive to the cytostatic effects of TGF-, and components of the TGF-pathway are frequently inactivated in a subset of cancer specimens [START_REF] Levy | Alterations in components of the TGF-superfamily signaling pathways in human cancer[END_REF]. However, breast and other cancers often only carry defects in downstream cytostatic TGF-targets, show normal TGF-signaling from receptors to Smads, and retain or gain other TGF-responsive properties [START_REF] Gomis | C/EBP at the core of the TGF cytostatic response and its evasion in metastatic breast cancer cells[END_REF][START_REF] Wakefield | TGF-beta signaling in mammary gland development and tumorigenesis[END_REF][START_REF] Dumont | Transforming growth factor-and breast cancer: Tumor promoting effects of transforming growth factor[END_REF][START_REF] Ten Dijke | Regulation of cell proliferation by Smad proteins[END_REF]. One of the TGF--induced processes contributing to tumor migration and invasion is epithelialto-mesenchymal transition (EMT). Both Smad and non-Smad signaling pathways have been implicated in TGF--induced EMT of breast cancer cells [START_REF] Deckers | The tumor suppressor Smad4 is required for transforming growth factor -induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells[END_REF][START_REF] Viloria-Petit | A role for the TGF -Par6 polarity pathway in breast cancer progression[END_REF][START_REF] Xu | TGF--induced epithelial to mesenchymal transition[END_REF]. In addition, matrix metalloproteinases (MMPs) appear to play a critical role in TGF-induced migration and invasion [START_REF] Liotta | Metastatic potential correlates with enzymatic degradation of basement membrane collagen[END_REF][START_REF] Weigelt | Breast cancer metastasis: markers and models[END_REF][START_REF] Overall | Tumour microenvironment -opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy[END_REF].

To study the role of TGF-in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of immortalized MCF10A1 (M1) normal breast epithelial cells (derived from human fibrocystic mammary tissue [START_REF] Soule | Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10[END_REF]), the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung [START_REF] Strickland | Progression of premalignant MCF10AT generates heterogeneous malignant variants with characteristic histologic types and immunohistochemical markers[END_REF][START_REF] Santner | Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells[END_REF]. Since RAS and TGF-have been reported to collaborate in promoting malignant progression of breast cancer [START_REF] Kim | TGF--induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling in MCF10A human breast epithelial cells[END_REF][START_REF] Miyazono | Transforming growth factor-signaling in epithelialmesenchymal transition and progression of cancer[END_REF], this MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.

For the analysis of TGF--induced invasion we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro. Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid [START_REF] Smalley | Life isn't flat: taking cancer biology to the next dimension[END_REF]29]. Spheroid based assays have also been shown to better predict drug resistance than monolayer cultures [START_REF] Ohmori | Blockade of tumor cell transforming growth factor-s enhances cell cycle progression and sensitizes human breast carcinoma cells to cytotoxic chemotherapy[END_REF][START_REF] Graham | Rapid acquisition of multicellular drug resistance after a single exposure of mammary tumor cells to antitumor alkylating agents[END_REF]. This MCF10 3D model system allowed us to investigate the impact of TGFsignaling on the invasive properties of breast cells in different stages of malignancy.

Our results show that both Smads and MMP2 and in particular MMP9 are critical for TGF--induced invasion of the malignant breast cancer cells.

Materials and methods

Cell culture

MCF10A series of cell lines, i.e. MCF10A (M1), transformed MCF10AneoT (M2) and its derivative MCF10CA1a (M4) were obtained from Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, USA). All MCF10A cell lines were maintained in DMEM/F12 (Gibco) supplemented with 5% horse serum (Gibco), 20ng/ml epidermal growth factor (EGF) (Upstate), 100ng/ml cholera toxin (Calbiochem), 0.5 g/ml hydrocortisone (Sigma), 10 g/ml insulin (Sigma), 100U/ml penicillin and 50 g/ml streptomycin (Gibco). All cell lines were grown in a humidified incubator at 37°C and 5% CO 2 .

3D spheroid invasion assays

Semi-confluent MFC10A cells were trypsinized, counted and re-suspended in medium containing 2.4mg/ml methylcellulose (Sigma) at the concentration of 10 4 cells/ml. 100μl of suspension was added into each well of U-bottom 96-well-plate allowing the formation of one spheroid per well. All spheroids consisted of 10 3 cells. Two days after plating spheroids were harvested and embedded into collagen. Flatbottom 96-well-plate was coated with neutralized bovine collagen-I (PureCol, Advanced BioMatrix) according to manufacturer's protocol. Single spheroids were embedded in a 1:1 mix of neutralized collagen and medium supplemented with 12mg/ml of methylcellulose. TGF-β3 (generous gift of Dr. K. Iwata, OSI Pharmaceuticals, Inc, New York, USA) and/or SB-431542 (Sigma) were directly added to the embedding solution. GM6001 and MMP2/9-inhibitor II (Calbiochem) were added in medium on top of the collagen. Invasion was monitored during the next two days and quantified by measuring the area occupied by cells using ImageJ software. Pictures were taken at day 0 and day 2 after embedding.

Lentiviral transduction

Lentiviral constructs expressing short hairpin RNAs targeting Smad4 (sh-Smad4) or a non-targeting control were obtained from the Sigma Mission shRNA library. The targeting sequence for Smad4 was 5'-GTACTTCATACCATGCCGATT-3'. The nontargeting sequence was 5'-AGAACGTCAACACCAAGTGCA-3'. Smad3 knockdown was performed using miR-Smad3 lentivirus using BLOCK-iT Pol II micro RNA interference technology (Invitrogen) as described previously [START_REF] Petersen | Smad2 and Smad3 have opposing roles in breast cancer bone metastasis by differentially affecting tumor angiogenesis[END_REF]. Cells were infected at multiplicity of infection (MOI) 2, selected with appropriate antibiotics (sh-Smad4 and the non-targeting control cells with puromycine; miR-Smad3 and miR-control cells with blasticidine), and used for experiments one week later.

Gelatin zymography

Conditioned medium was harvested from cells after 48 hr of stimulation and resuspended in 4x Laemli buffer (250mM Tris-HCl pH6.8, 8% SDS, 40% glycerol, 0.004% bromophenol blue). Samples were ran on a 10% acrylamide gel containing 0.8mg/ml gelatin, washed with 2.5% Triton and incubated at 37 C with Brij solution (50mM Tris-HCl pH7.4, 0.05% Brij-35, 10mM CaCl 2 ) overnight. Gels were stained with Coomassie Brilliant Blue (25% methanol, 15% acetic acid, 0.1% Coomassie Brilliant Blue R).

Statistical analysis

For statistical comparison of two groups, a two-tailed Student's t-test was used where applicable. Statistical analysis of three or more groups was done by one-way ANOVA followed by Bonferroni's multiple comparison test. P<0.05 was considered as statistically significant.

Other assays

Assays for cell proliferation, transcriptional reporter activity, Western blot analysis, RNA isolation, cDNA synthesis and quantitative PCR are found in supplementary methods

Results

Establishment of a spheroid cell culture model to study TGF--induced invasion of breast cancer cells

To analyze the role of TGF--signaling in 3D cultures, spheroids of

MCF10A1 (M1) normal breast epithelial cells, H-RAS transformed MCF10AneoT

(M2) cells, or M2-derived MCF10CA1a (M4) cells were embedded into collagen matrix and stimulated with TGF-(Fig. 1a). M1 cells showed only weak invasion without stimulation, but invaded significantly better upon TGF-stimulation. In contrast, the RAS-transformed M1-derivative M2 invaded already efficiently without any stimulus, which was further increased 4-5 fold upon TGF-addition. M4 cells showed the strongest invasion, both with and without TGF-addition (Fig. 1a,b), in line with their metastatic capacity. Other TGF-family ligands, such as Activin-A, BMP-6, BMP-7 or BMP-9 failed to induce invasion in any of the cell lines (data not shown). Thus, the invasiveness in our spheroid model in vitro correlates with the grade of aggressiveness of the M1, M2 and M4 cells in vivo [START_REF] Strickland | Progression of premalignant MCF10AT generates heterogeneous malignant variants with characteristic histologic types and immunohistochemical markers[END_REF][START_REF] Santner | Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells[END_REF].

Next, we examined whether the differences in invasion between M1, M2 and M4 cells can be explained by differences in TGF-responsiveness. All three cell lines were found to express type I, II and III receptors as shown by TGF-affinity crosslinking assays (Suppl. Fig. 1b), which is in line with a previous study [START_REF] Tang | TGF-switches from tumor suppressor to prometastatic factor in a model of breast cancer progression[END_REF]. To investigate if all cells activate TGF-/Smad transcriptional activity to the same extent, we made use of the CAGA 12 -luciferase reporter, which contains 12 repeats of the Smad binding element CAGA. All cells showed TGF-/Smad transcriptional activity in response to TGF-. However, induction in M2 was markedly lower than in M1 and M4 (Suppl. Fig. 1a). Furthermore, all cells showed Smad2 phosphorylation upon TGF-stimulation (data not shown). Thus, the differences in receptor binding and transcriptional activity did not correlate with the levels of spheroid invasiveness of the three cell lines. TGF-is a potent inhibitor of proliferation of epithelial cells and this inhibitory effect is often lost during cancer progression. To test whether the differences in basal and TGF--induced invasion between M1, M2 and M4 cells is associated with differences in growth rate, we performed proliferation assays.

Interestingly, the growth of all three cell lines was slightly inhibited by TGF-, but no major differences were detected between the cell lines (Fig. 1c).

Taken together, our results demonstrate that the described spheroid invasion assay closely reproduces the differences in the aggressive properties of the three cell lines [START_REF] Strickland | Progression of premalignant MCF10AT generates heterogeneous malignant variants with characteristic histologic types and immunohistochemical markers[END_REF][START_REF] Santner | Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells[END_REF][START_REF] Tang | TGF-switches from tumor suppressor to prometastatic factor in a model of breast cancer progression[END_REF], whereas the observed differences are not due to differences in proliferative capacity or responsiveness to TGF-.

SB-431542 inhibits basal and TGF--induced invasion

Next, we examined whether the basal and TGF--induced invasion in this spheroid model is dependent on the T RI kinase. For this purpose we used SB-431542, an ATP analogue and selective inhibitor of the kinase activity of T RI, activin type IB receptor (ActRIB) and activin receptor-like kinase (ALK)7 [START_REF] Inman | SB-431542 is a potent and specific inhibitor of transforming growth factor-superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7[END_REF]. As expected, SB-431542 potently inhibited TGF--induced invasion of M1, M2 and M4 cells (Fig 2a,b), indicating that TGF--induced invasion is T RI-kinase dependent. Surprisingly, the basal invasion of the spheroids was also strongly inhibited, suggesting autocrine TGF-signaling, since we found these cells to express all TGF-isoforms. The basal invasion of M1, M2 and/or M4 cells might also be mediated by ligands other than TGF-, e.g. ligands that signal via ActRIB and ALK7. However, Activin-A failed to induce invasion (data not shown) and only marginally induced CAGA 12 -luciferase transcriptional activity, possibly caused by low expression of activin receptors in these cells (Suppl. Fig. 1a).

TGF--induced invasion is Smad3/4 dependent

As TGF-β signaling potently induced invasion of the aggressive and metastatic M4 cells, we used these cells to further investigate the role of the TGF-β pathway in metastasis-associated invasion. Recent studies by our group and others have demonstrated that Smad3 and Smad4 are key players in inducing metastasis of MDA-MB231 cells [START_REF] Deckers | The tumor suppressor Smad4 is required for transforming growth factor -induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells[END_REF][START_REF] Petersen | Smad2 and Smad3 have opposing roles in breast cancer bone metastasis by differentially affecting tumor angiogenesis[END_REF][START_REF] Kang | Breast cancer bone metastasis mediated by the Smad tumor suppressor pathway[END_REF]. To investigate the role of Smad3 and Smad4 in M4, we knocked-down Smad3 and Smad4 through lentiviral vectors. The Smad4 shRNA construct used strongly reduced Smad4 protein, but did not affect the TGF--induced phosphorylation of Smad2 and Smad3 (Fig. 3a). The Smad4 knockdown vector also strongly reduced TGF-/Smad-induced transcription as measured by a CAGA 12luciferase reporter (Fig. 3b). Moreover, M4 spheroids embedded in collagen showed a severe defect in TGF--induced invasion upon Smad4 knockdown (Fig. 3c andd), indicating that TGF--induced 3D invasion is Smad4-dependent.

Although the knockdown of Smad3 was less efficient than the knockdown of Smad4, it significantly reduced the levels of phospho-Smad3, while leaving TGF-induced phosphorylation of Smad2 unaffected (Fig. 3a). Moreover, this level of Smad3-knockdown, like knockdown of Smad4, severely inhibited TGF-/Smadinduced transcription (Fig. 3b). Knockdown of Smad3 also reduced the levels of TGF--induced invasion of M4 cells in collagen (Fig. 3c andd).

To further investigate the role of Smads in TGF-β-induced invasion we performed knockdown experiments in the premalignant cell line M2 (Suppl. Fig. 2a).

Also in these cells Smad3 or Smad4 knockdown caused inhibition of TGF-β-induced invasion (Suppl. Fig. 2b andc). Taken together, these results emphasize the importance of the Smad3/4 pathway in TGF-β-induced invasion.

Critical role of MMP2 and 9 in TGF--induced invasion

Next, we asked ourselves which downstream target genes are necessary for TGF-induced invasion in this model system. MMPs play important roles in tumor cell migration and invasion, both through degradation of components of the extracellular matrix, and by activation of growth factors and cleavage of adhesion molecules [START_REF] Egeblad | New functions for the matrix metalloproteinases in cancer progression[END_REF].

Analysis of TGF--induced mRNA expression identified MMP1, MMP2 and MMP9

among the strongest TGF--responsive genes in M4 spheroids (Suppl. Fig. 3a and Fig. 4a, b, respectively). Since the spheroid assay is performed in collagen I matrix, MMP1, a known collagen I protease, was expected to be a critical mediator of TGF-βinduced invasion. However, knockdown experiments showed that MMP1 is not necessary for TGF--induced invasion in this system (Suppl. Fig. 3b-d). We next examined the involvement of the gelatinases MMP2 and MMP9, as these proteins have been correlated with poor prognosis and lung metastasis in breast cancer [START_REF] Van 't Veer | Gene expression profiling predicts clinical outcome of breast cancer[END_REF][START_REF] Minn | Genes that mediate breast cancer metastasis to lung[END_REF].

TGF-was found to strongly induce MMP2 and MMP9 mRNA levels both in spheroids (3D) and in monolayer (2D): MMP2 expression was induced approximately 10-fold upon TGF-stimulation in both 2D and 3D (Fig. 4a and4b); MMP9 approximately 100-fold and 40-fold in spheroids and monolayer respectively (Fig. 4a andb). To confirm that this induction was seen also at the protein level, we analyzed conditioned medium by gelatin zymography. As shown in Fig. 4c, TGF-induces pro-MMP2 and both pro-and active MMP9 in M4 cells.

To further investigate the role of MMPs in 3D invasion, spheroids of M4 were analyzed in the presence or absence of the general, broad-spectrum, MMP-inhibitor GM6001. Interestingly, GM6001 strongly inhibited TGF--induced invasion (Fig. 4d,e), but had no significant effect on the basal invasion. We subsequently analyzed the effect of a selective MMP2/9 inhibitor [START_REF] Tamura | Highly selective and orally active inhibitors of type IV collagenase (MMP-9 and MMP-2): N-sulfonylamino acid derivatives[END_REF]. Importantly, treatment of M4 spheroids with selective MMP2/9 inhibitor like the broad-spectrum inhibitor reduced TGF-induced invasion without an effect on basal invasion (Fig 4f,g).

As the TGF--induced invasion of M4 spheroids was dependent on Smad3/Smad4 (Fig. 3), we next examined the role of Smad3 and 4 on TGF--induced MMP2 and 9 expression. Both knockdown of Smad3 and Smad4 resulted in strongly reduced TGF--induction of MMP2 and 9 in M4 cells (Fig. 4h). Taken together, TGF--induced MMP2 and 9 expression is dependent on both Smad3 and Smad4, and is critical for TGF-β-induced invasion of M4 cells in this spheroid model system

Discussion

To increase our understanding of breast cancer cell invasion and the role of TGFtherein, we established a spheroid model in which MCF10A1 cells and its malignant derivatives invade into a collagen matrix in a TGF--dependent manner. When comparing the invasive properties of the different MCF10A1 cell lines we found that basal invasion as well as TGF--induced invasion increased from the relatively benign M1 cells, to higher levels in the RAS-transformed M2 derivative, to even higher levels in the metastatic M4 variant, and thus nicely correlated with the relative state of aggressiveness of these three cell lines [START_REF] Strickland | Progression of premalignant MCF10AT generates heterogeneous malignant variants with characteristic histologic types and immunohistochemical markers[END_REF][START_REF] Santner | Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells[END_REF][START_REF] Tang | TGF-switches from tumor suppressor to prometastatic factor in a model of breast cancer progression[END_REF]. In contrast, there was no correlation between invasion and cell proliferation, and the growth inhibitory effect of TGF-β on the cell lines was only weak. The fact that the relatively benign M1 cells showed only a minor growth-inhibitory effect upon TGF-treatment might be due to chromosomal rearrangements during immortalization, leading to loss of the CDKN2A locus and amplification of MYC [START_REF] Cowell | Molecular characterization of the t(3;9) associated with immortalization in the MCF10A cell line[END_REF][START_REF] Kadota | Delineating genetic alterations for tumor progression in the MCF10A series of breast cancer cell lines[END_REF].

Our spheroid invasion assay resembles the in vivo process more closely than 2D models, because cells in collagen I-embedded spheroids are amongst others in different metabolic states and interact in more natural fashion with their surroundings [29]. Moreover, the composition of extracellular matrix in breast cancer is often altered, resulting in fibrotic stiff foci with a high collagen I content. It has been demonstrated that increased collagen I content promotes breast cancer formation and invasion [START_REF] Provenzano | Collagen density promotes mammary tumor initiation and progression[END_REF] and is associated with greater incidence of metastasis [START_REF] Ramaswamy | A molecular signature of metastasis in primary solid tumors[END_REF]. In a previously developed 3D model by Bissell and co-workers cells are grown in a reconstituted basement membrane (rBM) matrix. This model provides a rapid assay to distinguish between normal and malignant mammary epithelium but focuses mainly on cell morphology [START_REF] Petersen | Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells[END_REF]. In this assay morphogenesis and organization of the distinct MCF10A cell lines inversely correlates with malignancy [START_REF] Debnath | Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures[END_REF][START_REF] Li | p21-Activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a threedimensional culture model for premalignant progression of human breast cancer[END_REF]. In addition, 3D cultures of MCF10A cell lines have been used to assess sensitivity to kinase inhibitors [START_REF] Li | Three-dimensional overlay culture models of human breast cancer reveal a critical sensitivity to mitogen-activated protein kinase kinase inhibitors[END_REF]. Our spheroid model complements these assays by specifically focusing on invasion. Moreover, as it is easy to conduct and very quantifiable it can also be used for screening.

In our assays the basal 3D invasion was strongly inhibited by treatment with SB-431542, an inhibitor of type I receptors for TGF-, Activin and Nodal. This suggests that cells produce TGF--like activity and stimulate invasion in an autocrine manner. This is in agreement with a recent report in which it is shown that the ECM can induce TGF-signaling in the absence of TGF- [START_REF] Garamszegi | Extracellular matrix-induced transforming growth factor-beta receptor signaling dynamics[END_REF]. We cannot exclude that Activins or Nodal also contribute to basal invasion. In fact, Activin and Nodal have also been implicated in breast cancer progression [START_REF] Incorvaia | MMP-2, MMP-9 and activin A blood levels in patients with breast cancer or prostate cancer metastatic to the bone[END_REF][START_REF] Strizzi | Emerging roles of nodal and Cripto-1: from embryogenesis to breast cancer progression[END_REF][START_REF] Adkins | Antibody blockade of the Cripto CFC domain suppresses tumor cell growth in vivo[END_REF], However, Activin-A only very weakly induced transcriptional activity and failed to induce invasion in our spheroid model.

Our results reveal a critical role for Smad3 and Smad4 in TGF--induced invasion of M4 cells. This is consistent with our previous findings that had demonstrated a key role for Smad3 and Smad4 in TGF--induced EMT and breast cancer metastasis [START_REF] Deckers | The tumor suppressor Smad4 is required for transforming growth factor -induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells[END_REF][START_REF] Petersen | Smad2 and Smad3 have opposing roles in breast cancer bone metastasis by differentially affecting tumor angiogenesis[END_REF][START_REF] Dzwonek | Smad3 is a key nonredundant mediator of transforming growth factor signaling in Nme mouse mammary epithelial cells[END_REF]. Abrogation of the Smad pathway in M4 cells by overexpression of a Smad-binding defective mutant of TβRI or a dominant negative Smad3 mutant was also reported to suppress metastasis [START_REF] Tian | Smad-binding defective mutant of transforming growth factor type I receptor enhances tumorigenesis but suppresses metastasis of breast cancer cell lines[END_REF][START_REF] Tian | Reduction in Smad2/3 signaling enhances tumorigenesis but suppresses metastasis of breast cancer cell lines[END_REF]. However, our results do not exclude a role for non-Smad intracellular signaling pathways downstream of TGF-receptors I. Various studies have shown that RAS and TGF-collaborate in promoting malignant progression of breast cancer [START_REF] Kim | TGF--induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling in MCF10A human breast epithelial cells[END_REF][START_REF] Miyazono | Transforming growth factor-signaling in epithelialmesenchymal transition and progression of cancer[END_REF]. Both TGF-and RAS can activate the MAP kinases ERK, JNK and p38 and their down-stream transcription factors, such as AP-1 family members. Interestingly, in the MCF10A cell lines Smads, MAPKs as well as AP-1 proteins have been reported to contribute to TGF--induced migration and MMP induction [START_REF] Kim | TGF--induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling in MCF10A human breast epithelial cells[END_REF][START_REF] Tian | Smad-binding defective mutant of transforming growth factor type I receptor enhances tumorigenesis but suppresses metastasis of breast cancer cell lines[END_REF]. However, it remains to be established whether and which MAPK and AP-1 members collaborate with Smad proteins to mediate basal and/or TGF--induced invasion of the MCF10A cell lines in our spheroid model system.

Our studies reveal a key role for MMP2 and MMP9 in TGF--induced invasion of the highly aggressive M4 spheroids. MMP2 and 9 were initially known for their ability to degrade native type IV collagen [START_REF] Stetler-Stevenson | Progelatinase A activation during tumor cell invasion[END_REF], in line with the idea that the main function of MMPs in invasion was degradation of the extracellular matrix, in particular the basal lamina [START_REF] Liotta | Metastatic potential correlates with enzymatic degradation of basement membrane collagen[END_REF]. Various matrix components that can be degraded by MMP2 and MMP9 are in fact produced by the epithelial cells themselves. However, besides degradation of extracellular matrix MMPs can also activate growth factors, including TGF-, and cleave adhesion molecules, such as E-cadherin [START_REF] Egeblad | New functions for the matrix metalloproteinases in cancer progression[END_REF]. It is likely that these functions of MMP2 and 9 also contribute to TGF--induced invasion.

MMP2 and 9 appear attractive targets for therapeutic intervention of breast cancer invasion. However, MMPs are multifunctional proteins and may also have anti-tumor actions. The latter appears in particular an issue for targeting of MMP9 [START_REF] Overall | Tumour microenvironment -opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy[END_REF]. Tumors of K14-HPV16 mice in a null MMP9 background were found to be more aggressive, indicating that MMP9 inhibits certain aspects of tumor progression [START_REF] Coussens | MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis[END_REF]. On the other hand, knockdown of MMP9 reduces metastasis of T RI-expressing cells [START_REF] Safina | ALK5 promotes tumor angiogenesis by upregulating matrix metalloproteinase-9 in tumor cells[END_REF]. This does not to be the case for MMP2, as overexpression in MDA-MB-231 cells augments orthotopic tumor growth and metastasis in nude mice [START_REF] Tester | Pro-matrix metalloproteinase-2 transfection increases orthotopic primary growth and[END_REF]. In human cancer cells, MMP2 is one of the predictors for lung metastasis [START_REF] Minn | Genes that mediate breast cancer metastasis to lung[END_REF]. The development of specific MMP inhibitors remains a challenge, as the enzymatic pocket of these proteins does not differ from one another.

One striking finding is that the basal invasion of M4 spheroids is not affected by the MMP inhibitors. This is in contrast to the results obtained with the T RI inhibitor, which blocks both basal and TGF--induced invasion. One could speculate that basal invasion depends on other factors induced by TGF-, whereas MMPs are necessary for overt invasion.

In conclusion, we have established a 3D model for TGF--induced invasion.

Using this model, we demonstrate that Smad3 and Smad4 are crucial for TGF-induced invasion by inducing MMP2 and MMP9. This supports the idea that targeting these molecules might aid in the prevention of metastasis in patients. The 3D assay also offers a valuable screening model system to further study TGF-/Smad-and MMP2-and MMP9-dependent breast cancer invasion. experimental metastasis of MDA-MB-231 human breast cancer cells in nude mice. Cancer Res 64:652-658

Figure 1

 1 Figure 1 TGF-β-induced invasion of spheroids of normal MCF10A1 (M1), RAS-

Figure 2

 2 Figure 2Basal and TGF-β-induced spheroid invasion is inhibited by SB-431542. M1,

Figure 3

 3 Figure 3 TGF-β-induced spheroid invasion is Smad3/4 dependent. MCF10CA1a

Figure 4

 4 Figure 4 TGF--induced spheroid invasion is dependent on matrix metalloproteinase

Acknowledgements

We thank our colleagues, in particular Lukas Hawinkels, for valuable discussion and help with experiments. We are grateful to Niek Henriquez, Petra van Overveld and Geertje van der Horst for help with our experiments to set up the invasion assay. We thank Ed Leof (Mayo Clinic, Minnesota, USA) and Ken Iwata (OSI Pharmaceuticals, New York, USA), for reagents, Fred Miller (Barbara Ann Karmanos Cancer Intitute, Detroit, USA) for the cell lines. This work was supported by the Dutch Cancer Society (RUL 2005-3371), European Union Framework 6 grant BRECOSM (5032240) and Tumor Host Genomics (518198), Centre for Biomedical Genetics, Swedish Cancerfonden (09 0773) and Ludwig Institute for Cancer Research.

Conflict of interest

The authors declare no conflict of interest.