List of Figures

List of Tables and Sheets

Data Sheets

List of Abbreviations

INTRODUCTION

L'utilisation des médicaments chimio-thérapeutiques pour le traitement du cancer est entravée par les toxicités limitant la dose avant qu'il n'ait une efficacité anti-tumorale suffisante. Les cellules souches mésenchymateuses (MSC) ont la capacité de migrer vers les cellules cancéreuses, comme le montrent les études pré-cliniques précédentes (de la Torre et al., 2020). Dans le cas des cellules souches, le rôle de la mécanique et l'efficacité des systèmes de libération des cellules peuvent être mis en évidence sous l'hypothèse motivante que des types spécifiques de cellules souches sont plus adaptés à certaines applications en fonction de leurs propriétés.

La présence de différents types de populations de MSC dans les dents a été décrite. Selon le site de prélèvement, il s'agit de la pulpe dentaire (DPSC), du ligament parodontal (PDLSC), de la papille apicale (SCAP), du follicule dentaire (DFSC) et du tissu gingival (GSC), bien qu'ils soient appelés génériquement "cellules souches dentaires". Cet ensemble de cellules souches est particulièrement intéressant car les dents, malgré leur petite taille, sont une source de cellules abondantes pour les procédures thérapeutiques, et leur préparation peut être liée à l'extraction dentaire de routine, ce qui n'impose pas de charge supplémentaire au patient. En général, celle des cellules imite la mécanique des tissus d'origine et, par conséquent, la mécanique inhérente à certains DPSC peut être avantageuse pour les techniques de besoin de transplantation et de migration des cellules souches; Normalement, les DPSC libérées de la pulpe dentaire migrent en réponse à des lésions tissulaires ou à un stimulus inflammatoire. Sur la base de leurs fonctions inhérentes dans la structure de la dent, les DPSC ont déjà prouvé des potentiels pré-cliniques prometteurs dans le domaine de la médecine régénératrice.

Les cellules souches mésenchymateuses de la pulpe dentaire (DPSC) ont été isolées et caractérisées pour la première fois il y a plus de dix ans comme des cellules hautement clonogènes capables de générer des colonies densément calcifiées. L'identité des DPSC en tant que MSC a été confirmée par leur capacité à se différencier en cellules neurales ectodermiques et en adipocytes, odontoblastes, ostéoblastes, chondrocytes et cellules myoblastes d'origine mésodermique, confirmant ainsi leur plasticité (Hollands et al., 2018). Les DPSC sont actuellement reconnues comme l'une des MSC multipotentes les plus accessibles et les plus attrayantes, avec des taux de croissance élevés, pour une utilisation en ingénierie tissulaire et en médecine régénérative. La thérapie basée sur les DPSC est prometteuse, mais comme la plupart des preuves disponibles ont été obtenues à l'aide de xénogreffes non humaines, il ne s'agit pas d'une technologie mature (Ledesma-Martínez et al., 2016).

« Les DPSC, au-delà de la médecine régénérative »

Les propriétés régénératrices, immune-modulatrices, et anti-tumorales des MSC ont été exploitées pour réparer les lésions tissulaires, interférer avec le cancer ainsi qu'avec le développement de troubles immunitaires et de maladies neurodégénératives. Cette large utilisation a également été soutenue par la facilité relative de récolte et de traitement à partir de différentes sources, et par l'abondante disponibilité après expansion in-vitro. Au-delà de la médecine régénératrice, le tropisme des MSC aux tumeurs et la non-immunogénicité ont jeté les bases de leur application dans la recherche en oncologie.

En particulier, les MSC libèrent des facteurs solubles ou des extra-vésicules (EV) qui, agissant par des mécanismes autocrines et paracrines, sont capables de moduler la survie et la prolifération des cellules cancéreuses, les voies migratoires, et de plus d'induire une immune-modulation de l'hôte.

Les thérapies à base de cellules souches constituent une approche prometteuse pour le traitement de plusieurs maladies chez l'humain. Ces dernières années, ces cellules ont suscité un intérêt particulier en raison de leur potentiel de régénération tissulaire mais, récemment, cet intérêt s'est tourné vers la compréhension du rôle que les cellules souches mésenchymateuses jouent au-delà de cela : leur rôle dans le développement et la progression du cancer.

Malgré l'enthousiasme initial, la littérature actuelle met en évidence les incohérences et les divergences croissantes des données sur la question de savoir comment les DPSC peuvent favoriser ou inhiber la croissance des tumeurs dans divers cancers, y compris notre étude sur le cancer du sein [START_REF] Dogan | Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions[END_REF], Hanyu et al., 2019, Brrunetti et al., 2018, Salehi et al., 2018). Plusieurs facteurs peuvent contribuer aux observations pré-cliniques contrastées, notamment les tissus d'origine des MSC (pulpe dentaire), l'isolement, l'expansion ex-vivo, et les protocoles de culture. En outre, d'autres complications peuvent découler du cadre clinique, du type de tumeur, du moment et de la quantité, et de la voie d'administration remettant souvent en question le nombre effectif de cellules se dirigeant vers le site de la tumeur. Toutes ces questions cruciales, qui entravent la fiabilité de la recherche et l'amélioration clinique des thérapies basées sur la DPSC, doivent être traitées par des études approfondies sur les propriétés biologiques et moléculaires de ces cellules.

« La bio-ingénierie de la DPSC pour la thérapie cellulaire » Dès le début du projet de thèse, une définition claire de la cible de l'application a été délimitée comme thérapie anti-cancéreuses et combinée (avec médicament) à base de cellules DPSC. En réfléchissant davantage à la biologie complèxe du cancer, on constate que les niveaux de base atteints par les MSC sont généralement sous-optimaux pour entraver cet objectif. Cela a contribué à fournir une justification solide pour concevoir les DPSC d'une manière plus efficace.

La nouveauté de l'oeuvre de notre projet repose sur deux aspects principaux : il est le premier travail représentant un modèle de DPSC, en tant que cellule souche adulte amorcée in-vitro, pour l'administration de médicaments et la thérapie combinée du cancer. L'autre preuve de la nouveauté est Les résultats sont divisés en six chapitres principaux, sous la forme de six articles représentant l'ensemble des travaux.

RÉSUMÉ DES RESULTATS

Le projet de thèse sert à l'application des cellules souches de la pulpe dentaire (DPSC) :

(1) Comme transporteurs médicaments anti-cancéreux (chimiothérapie classique) : par la libération de vésicules issues de la DPSC contenant des médicaments. Étant appliquée comme transporteur, celui vise à maximiser la dose de médicament.

(

« Adoption de caractéristiques fibroblastiques »

Ainsi, quelques observations ont démontré que les DPSC peuvent modifier leur phénotype après l'exposition au paclitaxel (chapitres 4 et 5) :

1-un changement de morphologie lors de l'exposition au PTX, devenant plus proche de celle des fibroblastes, mis en évidence par les images de microscopie optique ;

2-un changement vers un phénotype plus fibroblastique, mis en évidence par une augmentation de la MMP1 intracellulaire, mis en évidence par la méthode Raman ;

3-une diminution de la prolifération, mis en évidence par les tests MTT.

La diminution en prolifération est supposée être induite par la quiescence du cycle cellulaire avant l'action du PTX (Bosco et al., 2015). Certaines études de la littérature ont examiné l'effet d'autres médicaments cytotoxiques sur les DPSC. Une sénescence prématurée des DPSC a été signalée à de faibles concentrations du médicament Mitoxantrone MTX, tandis qu'une apoptose à médiation de caspases a été observée pour une dose conventionnelle élevée, de manière similaire aux fibroblastes comparés (Seifrtova et al., 2013)

GENERAL ABSTRACT

The presence of different types of mesenchymal stem cell (MSC) populations in teeth have been described, which depending on the harvest site being the dental pulp (DPSCs), periodontal ligament (PDLSCs), apical papilla (SCAPs), dental follicle (DFSCs), and gingival tissue (GSCs), although they are generically referred to as "dental stem cell". This set of stem cells is particularly interesting because teeth, despite their small size, are a source of abundant cells for therapeutic procedures, and their preparation can be linked to routine tooth extraction, which does not place an additional burden on the patient. periosteum (PSC) stem cells. Reproduced from [START_REF] Abbas | Combination of mesenchymal stem cells, cartilage pellet and bioscaffold supported cartilage regeneration of a full thickness articular surface defect in rabbits[END_REF], highlighted for the dental MSCs.

The mesenchymal stem cells of the dental pulp (DPSCs) were isolated and characterized for the first time more than a decade ago as highly clonogenic cells that were able to generate densely calcified colonies. The identity of the DPSCs as MSCs has been confirmed by their ability to differentiate into neural ectodermal cells and adipocytes, odontoblasts, osteoblasts, chondrocytes, and myoblast cells of mesodermal origin, confirming their plasticity (Hollands et al., 2018). The DPSCs are currently being recognized as one of the most accessible and attractive multipotent MSCs, with high rates of growth, for use in tissue engineering and regenerative medicine [START_REF] Tatullo | Dental pulp stem cells: function, isolation and applications in regenerative medicine[END_REF]. DPSC-based therapy is promising but ecause most of the available evidence was obtained using nonhuman xeno-transplants, it is not a mature technology (Ledesma-Martínez et al., 2016).

« DPSCs beyond regenerative medicine »

Regenerative

« DPSC bioengineering for cell-based therapy »

Stem cell-based therapies (CBT) provide a promising approach to the treatment of several diseases in humans. Over the past years, these cells gained particular interest due to their tissue regeneration potential. Recently, this interest turned toward understanding the role that mesenchymal stem cells (MSCs) play beyond regenerative medicine: their role in cancer development and progression.

From the beginning of the thesis project, a clear definition of the target of the DPSC application was demarcated as a cancer cell-based therapy. By more reflection about cancer biology, the baseline levels achieved by MSCs are typically sub-optimal to hinder this target. This has helped provide a solid rationale to engineer DPSCs to more efficiently act on our target tissue by secreting relevant molecules and by interacting with the cancer cells through cell-cell contact and ultimately improve the therapeutic outcome.

Continued exploration of engineering approaches that address challenges for DPSC application should improve the therapeutic efficacy for the cancer clinical indications. In particular, boosting the potency of DPSCs through engineering strategies, such as small-molecule (drug) priming, nanoparticle (NP) engineering, or genetic modification. Non-genetic approaches (Fig. 2) should be emphasized and examined, throughout measurable efficacy assays, therapeutic biomarkers, and mechanistic unraveling in preclinical developmental and clinical translational stages. to the use of free small molecules. Reproduced from (Levy et al., 2020).

General Overview

Introduction about cancer

Cancer is considered from non-communicable diseases (NCD), and is expected to rank as the leading cause of death and the single most important barrier to increasing life expectancy in every country of the world in the 21st century (WHO) [START_REF] Organization | Global Health Observatory[END_REF]. The GLOBOCAN 2018 estimates indicated 18.1 million new cases of cancer and 9.6 million deaths from cancer in 2018 [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF]. Cancer is an important cause of morbidity and mortality worldwide, in every world region, in both economically developed countries as well as in developing countries. A study has estimated about 1.4 million cancer deaths in the European Union (EU) for 2019 [START_REF] Malvezzi | European cancer mortality predictions for the year 2019 with focus on breast cancer[END_REF]. [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF].

Takeaway awareness notice

Understanding the statistics, as well as knowing the personal risk factors, can help with early diagnosis and treatment of breast cancer, alongside routine breast cancer screening. Hereditary and genetic factors, including a personal or family history, and inherited mutations (susceptibility genes), account for only 5% to 10% of breast cancer cases. Nonhereditary factors are the major drivers of the observed international incidence. The main recognized risk factors are related to menstruation (early menarche or later menopause), reproduction (no over-20-weeks pregnancy, postponement of childbearing, and fewer children), exogenous hormone intake (oral contraceptive use and hormone replacement therapy), nutrition (alcohol intake), and greater levels of obesity; whereas breastfeeding and physical activity protect from the disease. The primary risk factors for breast cancer are not easily modifiable because they stem from prolonged, endogenous reproductive and hormonal factors, although prevention may be beneficial through the promotion of the protective factors [START_REF] Brinton | Cancer Epidemiology and Prevention[END_REF].

At present, various therapeutic strategies for cancer management are available and there are three main types: chemotherapy, surgery, and radiotherapy. However, chemotherapy plays a significant role in cancer treatment. Conversely, several advances in the field of surgery and radiotherapy have been recorded over time.

2. Cancer chemotherapy

Chemotherapy (possibly along with targeted therapy) enter the bloodstream and circulate throughout the body to reach and destroy cancer cells in almost all parts of the body, so chemotherapy is considered a type of systemic therapy. It treats both the main tumor as well as any cancer cells that have broken off and spread to other parts of the body. Chemotherapy is still the primary mainstay of cancer treatment, and is frequently used for advanced cancers, elderly patients, and adjuvant (postoperative) therapy cases. Treatment for metastatic cancer is usually a systemic therapy. However, the survival of cancer patients is still poor, with high mortality and high morbidity rates, despite all the therapeutic advances. This is due to numerous issues in conventional chemotherapy.

(a) Normal tissue cytotoxicity

The morbidity comes in the form of serious side effects resulting notably from the use of nonspecific anti-cancerous drugs. The clinical chemotherapy drugs in their current form are delivered in untargeted variable doses, resulting in unpredictable and unwanted damage to non-diseased tissues, and sometimes life-threatening clinical side effects. Some severe side effects include immediate signs of toxicity and late signs of chronic toxicity [START_REF] Koeppen | Berne and levy physiology e-book[END_REF]Stanton, 2017, Schirrmacher, 2019). Those are usually caused by the anti-cancer drugs' adverse effect on the bone marrow, hair follicles, and cells in the digestive tract and reproductive system. Hence, currently, there is an increasing interest in the novel combination/conjugation of chemotherapy. This can especially give hope for the breast, colorectal, and pancreatic cancer.

In regards to chemotherapy effect on unborn babies, studies have shown that certain drugs (such as doxorubicin, cyclophosphamide, and the taxanes) used during the second and third pregnancy trimesters do not raise the risk of birth defects, stillbirths, or health problems shortly after birth. Though they may increase the risk of early delivery, researchers still do not know if these children will have any long-term effects.

Furthermore, in many cases, the poor survival of patients greatly relates to the inability to deliver drugs to the metastatic sites, away from the main tumor mass [START_REF] Menon | Mesenchymal stromal cells as a drug delivery system[END_REF]. More toxicity to the nontargeted tissues is related also to less efficacity of the anti-tumor effect. It is, therefore, crucial to develop new delivery strategies for chemotherapy drugs in the clinical use that are specific and do not affect healthy tissues.

(b) Chemotherapy resistance

There is resistance against chemotherapeutic drugs and chemotherapeutic resistance reports are increasing day by day. It has been suggested that chemo-resistant cancer cells that continue to be active after the chemotherapy are responsible for tumor recurrence [START_REF] Liang | Circumventing tumor resistance to chemotherapy by nanotechnology[END_REF]. However, we need to understand the mechanisms by which cancer cells develop chemo-resistance. Some key mechanisms have been suggested by which the tumor cells are more likely to develop chemo/drug resistance; these include efflux pump-mediated resistance in chemotherapy and non-efflux pump-mediated resistance in chemotherapy.

(c) Advancements in chemotherapy

Chemotherapy is the first line of treatment against cancer, both curable and advanced cancers, and it improves the general survival and quality of life for cancer patients. However, toxic effects and chemoresistance are major hurdles in chemotherapy and to avoid these problems caused by traditional chemotherapeutic regimens, new regimens are being suggested:

I) Combination modality: complementary agents (drug targeting, immune therapy), inclusion of protective agents (carriers), critical timing of administration.

II) Dosing schema: dose-dense, dose intensification, and dose escalation.

III) Individualized dosing: drug pharmaco-kinetics/ genomics.

Nanoparticle carriers have been developed like dendrimers, polymers, liposomes, micelles, inorganic, organic nanoparticles etc. Platin agents, 5-fluorouracil, and paclitaxel can be used for preparation nano drugs. Shortcomings with these nanoparticle-based drugs that often come across include: poor drugs' solubility, rapid degradation, need of large-scale output procedures, a fast release of the pharmaceutical from its carrier scheme, stability difficulties, the toxicity from the polymer, and numerous to say [START_REF] Selvamuthukumar | Nanostructured lipid carriers: a potential drug carrier for cancer chemotherapy[END_REF]. Delivery systems for chemotherapy drugs in the clinical use have been investigated extensively; yet still, further exploration into the best means of delivery is anticipated to overcome these problems associated with conventional chemotherapy.

Also, increasing efforts have been placed on optimizing drug dosing with the main goals of increasing antitumor efficacy while reducing associated toxicity. Especially the past decade has seen a marked rise in clinical trials of metronomic chemotherapy and combined treatments with novel therapeutic strategies, such as drug repositioning and targeted therapies to generate more potent non-toxic regimens [START_REF] André | Metronomics: towards personalized chemotherapy?[END_REF]. "Metronomic chemotherapy" has emerged as a modality of drug administration which involves the frequent administration of low dose conventional chemotherapy to target activated cells in tumors, having advantages over conventional dosing [START_REF] Maiti | Metronomic chemotherapy[END_REF]. This strategy is well-suited for global oncology and, if combined with tumor molecular analysis, it is poised to move towards personalized chemotherapy [START_REF] André | Metronomics: towards personalized chemotherapy?[END_REF]. The main characteristics of metronomic chemotherapy are [START_REF] Mross | Metronomic anti-cancer therapy-an ongoing treatment option for advanced cancer patients[END_REF]: Clinical-wise, many patients benefit from frequent infusion treatment cycles. However, it can lead to more problems with low blood cell counts, so it is not an option for all patients. Taxol is a chemotherapeutic sometimes given in this way for some breast cancer patients. Those therapeutic approaches aim to lower the chance of cancer recurrence and improve survival for some patients [START_REF] Maiti | Metronomic chemotherapy[END_REF]. On the other hand, distant frequency cycles are opted to allow rest periods and body recovery from the adverse effects of chemotherapy. It is assumed that the sustained long-term release of the drug can lead to improved treatment. It is also crucial to develop new delivery strategies for drugs in the clinical use that are specific and do not affect healthy tissues. We conclude that sustained high chemotherapy dose (dose-dense) with the least side effects (collateral toxicity) is the goal of cancer chemotherapy.

 Frequent

3. Development of biological therapy

Breast medical oncology utilizes both standard chemotherapies and new 'biological' or 'targeted' therapies to attack cancer specifically while minimizing harm to normal tissues.

Chemotherapeutic cytostatic drugs and small molecule inhibitors (SMIs) are considered chemical therapies, where the latter is targeted therapy interfering with transduction of oncogenic signal.

Biological therapies include [START_REF] Schirrmacher | From chemotherapy to biological therapy: A review of novel concepts to reduce the side effects of systemic cancer treatment[END_REF]: The distinction between chemical or biological types of cancer therapy is not sufficient to explain why therapy is well-tolerated or not. Side effects may be useful to define physiological from nonphysiological therapies. However, the novel drugs were approved based on reasonably certain estimates of benefit but less certain estimates of harm [START_REF] Niraula | Risk of incremental toxicities and associated costs of new anticancer drugs: a meta-analysis[END_REF][START_REF] Barnes | Efficacy, safety, tolerability and price of newly approved drugs in solid tumors[END_REF].

I)
Notably in 2017, meta-analyses (of 74 studies, >48000 patients) including the toxicity of novel drugs approved for up to 2015 was performed. Novel anticancer agents (after year 2000) were classified into four groups: agents targeting specific oncogenes, less-specific targeted agents where no biomarker to select patients, immunotherapy, and cytotoxic chemotherapy. Targeted therapy appears more efficacious than new forms of cytotoxic therapy. No apparent difference in efficacy between different types of target. Immunotherapy appears to have a better safety and tolerability compared to other cancer therapies [START_REF] Barnes | Efficacy, safety, tolerability and price of newly approved drugs in solid tumors[END_REF]. Still, ideal combination cancer therapies based on tumor immunology have to find an optimal position between maximal antitumor immunity and minimal autoimmunity [START_REF] Schirrmacher | From chemotherapy to biological therapy: A review of novel concepts to reduce the side effects of systemic cancer treatment[END_REF].

4. Stem cell therapy/ transplantation (SCT) for cancer

The ability of MSCs to react tissue injury and inflammation along with the prompted immune tolerance present them as an attractive platform for transplantation in human diseases. Moreover, enormous challenges remain for MSC therapies-from their diverse origins, the highly variable culture and cryopreservation conditions, the challenges associated with administration of MSCs, and the challenges of the host environment-that can also lead to unpredictable therapeutic outcomes. The goals of stem cell therapy for cancer patients are to:

I) Prevent

(a) MSC complementary therapy

MSCs have been utilized in clinical trials since the mid-1990s when autologous MSCs were first grown ex-vivo and administered to patients with breast cancer undergoing autologous stem cell transplantation, with the principal goal of augmenting hematopoietic engraftment. The earliest studies suggested that there was some improvement in engraftment, and these naturally led to studies of the application of MSCs to the allogeneic transplant setting [START_REF] Lazarus | Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use[END_REF][START_REF] Koç | Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy[END_REF].

(c) MSC reducing or inhibition therapy

Cells composing the tumor microenvironment (including the MSCs) can interact directly and indirectly with cancer cells by mutually altering cells' properties and functions. This has led to the proposal of inhibition cell therapy. New ongoing research is currently directed towards the use of MSCs inherent inhibitory effect towards the cancer. However, MSCs and tumor cells interact in numerous ways being in other cases supportive of cancer growth. The exact process responsible for the effect of MSCs on cancer growth is still not clear. While some studies refer this mostly to the secretome of MSCs [START_REF] Zhu | Human mesenchymal stem cells inhibit cancer cell proliferation by secreting DKK-1[END_REF], others demonstrated that MSC-cancer contact is determining [START_REF] Bajetto | Different effects of human umbilical cord mesenchymal stem cells on glioblastoma stem cells by direct cell interaction or via released soluble factors[END_REF][START_REF] Takigawa | Mesenchymal Stem Cells Induce Epithelial to Mesenchymal Transition in Colon Cancer Cells through Direct Cell-to-Cell Contact[END_REF][START_REF] Kostadinova | Mesenchymal stem cells inhibit the growth of prostate carcinoma cells in a long-term cultivation[END_REF].

A synergistic effect involving different mechanisms was described in the literature. Several direct and/or indirect mechanisms of interaction contribute to MSC-mediated stimulation of cancer cell growth including notch signaling, nanotube formation, gap junctional intercellular communication, and/or the exchange of cytokines/chemokines, extracellular vesicles and exosomes (Yang et al., 2015[START_REF] Melzer | Interaction of MSC with tumor cells[END_REF]. Moreover, one study has shown that interactions observed between selected umbilical cord MSCs and cancer cells, which caused breast cancer cell death, may include (Chao et al., 2012):

I)

2. Specific Overview

2. Novel 'combination therapy' with anti-cancer agents

Novel combination of chemotherapy with biological therapy can especially give hope for high risk breast, colorectal, and pancreatic cancers. Finally, combination/ conjugation chemotherapies aim to decrease drug doses [START_REF] Pinto | Current Cancer Treatment-Novel Beyond Conventional Approaches[END_REF] by focusing on maximizing efficacy and minimizing systemic

toxicity. An example of combination therapy of MSC with an anti-cancer drug is suggested by [START_REF] Seyhoun | Combination therapy of sorafenib with mesenchymal stem cells as a novel cancer treatment regimen in xenograft models of hepatocellular carcinoma[END_REF]. Below is a schematic diagram depicting the outcomes of various case studies related to the treatment of chemotherapeutic agent alone or combined with DPSCs (Fig. 3), demonstrating therapy goals for the combined approach. Though it has many advantages of maximizing the therapeutic effect, anti-cancer drugs in this way still contribute to adverse effects on collateral tissues. Therefore, drug targeting strategies are needed to be incorporated to achieve the best overall outcome, as by drug nanocarriers or biological carriers. [START_REF] Chakraborty | The novel strategies for next-generation cancer treatment: miRNA combined with chemotherapeutic agents for the treatment of cancer[END_REF].

2. 2. Targeted combination therapy

MSCs can be isolated, cultured and expanded, and as well are known to be inherently tumor-homing and immunosuppressive. In addition to their potential anti-cancer effect, the use of stem cells as cellular vehicles of drug-loaded nanoparticles seems to be a very promising strategy for targeting tumor tissues and maximizing drug loading. Different types of stem cells could be used as cellular vehicles, such as embryonic stem cells, adult stem cells or induced pluripotent stem cells. Within the adult stem cell group, the mesenchymal stem/stromal cells (MSCs) are the most common cell type used, given their several advantages, such as their availability, easy preparation, non-immunogenicity and immunomodulatory properties. From a clinical point of view, MSCs isolated from many sources (bone marrow, adipose tissue, umbilical cord tissue, amniotic fluid, placenta, and dental sources) are the first choice of stem cells for use in cancer therapies. It is well known that MSCs specifically migrate, home and survive in tumor sites without being incorporated into normal tissue [START_REF] Droujinine | To grab the stroma by the horns: from biology to cancer therapy with mesenchymal stem cells[END_REF]. A tumor has been seen as a "wound that never heals" which enrolls MSCs in its microenvironment [START_REF] Dvorak | Tumors: wounds that do not heal[END_REF]. MSCs can migrate towards a tumor, since these cells respond to tissue damage, hypoxia and inflammation, as found in tumor microenvironments [START_REF] Dvorak | Tumors: wounds that do not heal[END_REF], Spaeth et al., 2008). This tropism property makes MSCs a promising strategy for gene therapy, drugs or nanoparticles delivery systems in cancer therapy.

2. 2. Rationale for MSC-based chemo-combination therapy

The results of Pessina et al. were the first demonstration that, through a simple in vitro procedure, human MSCs can be loaded with anti-cancer drugs and used in vivo to release them into a tumor microenvironment (Pessina et al., 2011b). The experiments started with the accidental exposure of mouse stromal cells (BM-derived) to the anticancer drug (Pessina et al., 2011a). This caused bone marrow MSCs to acquire anti-proliferative potential towards co-cultured cells. So, they hypothesized that these stromal cells may act as a reservoir for the drug that, subsequently, may release some of its metabolites or even the drug in its original form, thus acquire the capacity to kill tumor cells in their proximity. MSCs were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time-dependent manner. PTX primed MSCs possess a potent anti-tumor and antiangiogenic activity in vitro that was dose-dependent, and demonstrable by using their conditioned medium or by co-culture assay.

"MSC+ drug" therapy goals

Similar results can be obtained using MSCs for anti-tumor agent delivery, as the recently-developed nanovector formulations of anticancer compounds (such as nanoparticle albumin-bound PTX). These mechanisms have several potential advantages over the free-form drugs: protecting drugs from being degraded in the body before they reach their target, enhancing tumor drug uptake, allowing for better control over the timing and distribution of drugs to tumor tissue, and preventing drugs from interacting with normal cells thereby decreasing toxicity. Cell-targeted therapy has a higher advantage, mainly considering that MSCs themselves produce anti-tumor factors which can kill cancer cells both in vitro and in vivo.

In another setting, PTX efficacy in cancer cells could be decreased by intrinsic/ acquired drug resistance mediated by different mechanisms, including the overexpression of the drug efflux transporter P-gp [START_REF] Yusuf | Paclitaxel resistance: molecular mechanisms and pharmacologic manipulation[END_REF]. PTX released from PTX-primed MSCs is secreted as free PTX

and as PTX stored in extra-vesicles EVs (Pascucci et al., 2014[START_REF] Melzer | Taxol-loaded MSCderived exosomes provide a therapeutic vehicle to target metastatic breast cancer and other carcinoma cells[END_REF]. In a new study [START_REF] Borghese | Adipose-Derived Stem Cells Primed with Paclitaxel Inhibit Ovarian Cancer Spheroid Growth and Overcome Paclitaxel Resistance[END_REF], authors suggest that PTX-MSCs might provide PTX stored in EVs that, similarly to liposomal drugs, may cross plasma membranes more efficiently, and by increasing PTX accumulation, counteract P-gp mediated efflux (Coccè et al., 2019[START_REF] Melling | The challenges and possibilities of extracellular vesicles as therapeutic vehicles[END_REF].

So in summary, the advantages of MSC system of drug delivery can be:

1-Maximized therapeutic loading: human MSCs primed with paclitaxel acquire a potent antitumor anti-angiogenic activity without any genetic cell manipulation. [START_REF] Chulpanova | Application of mesenchymal stem cells for therapeutic agent delivery in anti-tumor treatment[END_REF].

2. 2. 2. Rationale for MSC-based nanoparticle-combination therapy

In the last two decades, the use of drugs contained in nanoparticles (NPs) has emerged as a solution in cancer therapy to direct drug delivery to the tumor and to reduce systemic damage. NPs size range is between 1 to 100 nm and they allow the absorption of high quantities of drug due to a large surface area-to-volume ratio. Nanomedicine appears as a very promising field in cancer therapy, as demonstrated by the large successful in vitro and preclinical proofs of concept [START_REF] Patra | Nano based drug delivery systems: recent developments and future prospects[END_REF].

Small molecules (including drugs), peptides, proteins, DNA or miRNA have been loaded into NPs to be delivered to tumors. For biomedical applications, NPs should have a series of characteristics such as biocompatibility, degradability, stability, delivery efficiency and sustained release. Different types of NPs, such as organic (liposomes and polymers), inorganic (metallic, metal oxide, ceramic, and quantum dots) and carbon-based NPs (nanotubes, fullerenes), have been used in the field of medicine, especially in the treatment and imaging of tumors [START_REF] Wang | Functional nanoparticles and their interactions with mesenchymal stem cells[END_REF]. Nanoparticles have advantages over conventional anti-tumor drugs because they can be multi-functionally designed to pinpoint several targets in the tumor microenvironment. Although the use of these encapsulated formulas is in initial stages, they appear as a potential way to reach the difficultly penetrable core of solid tumors (de la Torre et al., 2020).

"MSCs-drug-NP" therapy goals

The strategy of MSC transporting of anti-tumor agents has some downsides such as the low loading capacity and the rapid diffusional clearance of the drugs when released out of the MSC cells.

Additionally, anti-cancer drugs may have some cytotoxic effects on MSCs and result in their death before arrival to tumor sites. The effects of chemotherapeutics on MSCs have been quite controversial, from a reduction in proliferation and apoptosis, to resistance while retaining proliferation and differentiation potential, and this may be due to the culture conditions of MSCs and duration of treatments (de la Torre et al., 2020). Although paclitaxel exposure does not up-regulate the expression of the trans-membrane pump P-glycoprotein 1 in MSCs, a mechanism by which cancer cells resist paclitaxel treatment [START_REF] Pires | Inhibition of P-glycoproteinmediated paclitaxel resistance by reversibly linked quinine homodimers[END_REF], Bosco et al., 2015), some other drugs may be least uptaken by MSC due to drug efflux effect [START_REF] Sadhukha | Nano-engineered mesenchymal stem cells as targeted therapeutic carriers[END_REF][START_REF] Cheng | Engineered mesenchymal stem cells for targeting solid tumors: therapeutic potential beyond regenerative therapy[END_REF]. Hence, long-term in vitro and in vivo studies are necessary to understand the mechanisms behind the influence of chemotherapy on MSCs.

The encapsulation of chemotherapy drugs into NPs increases the drug-loading capacity of MSCs while reducing potential toxic effects on MSCs (de la Torre et al., 2020), and also limit drugs' diffusional clearance [START_REF] Yao | Maximized nanodrug-loaded mesenchymal stem cells by a dual drug-loaded mode for the systemic treatment of metastatic lung cancer[END_REF][START_REF] Layek | Nano-engineered mesenchymal stem cells increase therapeutic efficacy of anticancer drug through true active tumor targeting[END_REF].To ensure targeting of the therapeutic anti-cancer dose to the tumor site, stimuli-response strategies are being developed. Drug-NPs transported by MSCs is one method to significantly reduce undesired side effects of anticancer treatments, on the patient and on transporter MSCs, but this combined system MSC/NP is still in its initial stages. It has been reported

that the type of NP and its size, as well as its concentration, and incubation time could interact with and alter the physical and phenotypical properties of MSCs [START_REF] Wang | Functional nanoparticles and their interactions with mesenchymal stem cells[END_REF]. However, other studies suggested that MSCs loaded with NPs preserved their morphology, proliferation, migration and homing capacity [START_REF] Paris | Vectorization of ultrasound-responsive nanoparticles in placental mesenchymal stem cells for cancer therapy[END_REF][START_REF] Labusca | Stem cells as delivery vehicles for regenerative medicine-challenges and perspectives[END_REF]. This "non-genetic" engineering approach, provided by the nanoparticle loading, has unique advantages in cancer therapy and is complementary to existing genetic cell manipulation (Wang et al., 2015[START_REF] Layek | Nano-engineered mesenchymal stem cells increase therapeutic efficacy of anticancer drug through true active tumor targeting[END_REF]. A number of new studies has revealed promising results for the application of MSCs as cellular carriers NP-drugs towards cancer tissue (Zhao et al., 2017[START_REF] Timin | Safe and effective delivery of antitumor drug using mesenchymal stem cells impregnated with submicron carriers[END_REF], Wang et al., 2019).

2. 3. Clinical trials of MSCs in cancer therapy

A new frontier for MSC application is the achievement of genetic engineering based methodology to convert MSCs into therapeutic vehicles to graft into the tumor and produce or release engineered EVs and nanoparticles, or cytotoxic agents. These strategies inspired by successful preclinical studies, were confirmed in few human cancer studies. The first clinical trial of gastrointestinal tumors worldwide utilizing genetically-engineered MSCs in humans has been reported (TREATME1) [START_REF] Niess | Treatment of advanced gastrointestinal tumors with genetically modified autologous mesenchymal stromal cells (TREAT-ME1): study protocol of a phase I/II clinical trial[END_REF]. This is a successful phase I/II clinical trial.

Another two registered clinical trials with MSCs have primarily focused on ovarian cancer. One of them is a phase I clinical trial in which human MSCs transfected with interferon beta (MSCs-IFNβ).

Similarly, a phase I/II trial was initiated to find the side effects and best dose of MSCs infected with oncolytic measles virus and to observe its effect on patients with ovarian cancer. Also, in a clinical approach for the treatment of lung cancer, allogeneic MSCs expressing a full-length version of TRAIL have been used. MSCs as gene-therapeutic vehicles aim to deliver the TRAIL [START_REF] Lin | Mesenchymal stem cells and cancer: clinical challenges and opportunities[END_REF].

Also, one clinical trial for treating liver cancer with MSCs is on the registry and is recruiting subjects.

The purpose of this trial was to study whether MSCs may influence the outcome of graft versus host response in liver transplantation of liver cancer patients. In a phase I clinical test, allogeneic bone marrow-derived MSCs were infused in men with localized prostate cancer [START_REF] Schweizer | A phase I study to assess the safety and cancer homing ability of allogeneic bone marrow derived mesenchymal stem cells in men with localized prostate cancer[END_REF].

2. 4. Challenges with stem cell treatments

The primary objective of studies is to assess the safety and cancer-homing ability of MSCs. MSCs may home primary tumors by insufficient levels to kill cancer cells or inhibit tumor growth. More showed that 48h-serum free DPSC-CM does not affect tumor growth or drug resistance after 24 hours in vitro, neither the tumor proliferation rate after 21 days in vivo (Hanyu et al., 2019). Another study, DPSC and DPSC differentiated into osteoblasts was primed with pro-apoptic chemokine (TRAIL),

where direct cell-cell interaction was fundemental for human myeloma cell inhibition [START_REF] Brunetti | High expression of TRAIL by osteoblastic differentiated dental pulp stem cells affects myeloma cell viability[END_REF]. One other study, they used DPSC alone for post-cancer reconstruction of acute esophageal injury (an in-vivo study). The transplanted DPSCs showed homing, proliferation, and transdifferentiation into mature esophageal epithelial cells in vivo and repaired the damaged esophageal tissue in the DPSC group. Most patients with this disease originally had a malignant tumor, but the researchers have not determined the effect of DPSC transplantation on cancer recurrence (Zhang et al., 2018). On the other hand, another source of the dental MSCs has been investigated for PTX delivery: the gingival papilla stem cells (GinPa MSCs) which have shown promising in-vitro cancer cell inhibition capacity [START_REF] Brini | Cell-mediated drug delivery by gingival interdental papilla mesenchymal stromal cells (GinPa-MSCs) loaded with paclitaxel[END_REF][START_REF] Coccè | Drug loaded gingival mesenchymal stromal cells (GinPa-MSCs) inhibit in vitro proliferation of oral squamous cell carcinoma[END_REF], Coccè et al., 2019).

Some studies in the literature have discussed the effect of other cytotoxic drugs on DPSCs (Seifrtova et al., 2012, Seifrtova et al., 2013, Garg et al., 2015, Kang et al., 2018). The DPSC cells were more sensitive to the cisplatin than the dermal fibroblasts they were compared with, inducing MAPK along with caspases activation and apoptosis in DPSCs (Seifrtova et al., 2012). Increased pulp sensitivity to cold testing was also observed in cisplatin-treated with radiotherapy patients (Garg et al., 2015).

Mitoxantrone (MTX), a topoisomerase-II inhibitor of antibiotic anti-cancer class, caused DPSC premature senescence at low concentrations while caspase-mediated apoptosis for high conventional dose, similarly to compared fibroblasts (Seifrtova et al., 2013). All genotoxic effect with proliferation blocking, most noticeable after 72h, was drug dose-dependent (Seifrtova et al., 2012, Seifrtova et al., 2013).

3. 2. Chemotherapy drugs: clinical background

Breast cancer is the most common type of cancer in women and accounts for one-third of all cancers in women. Chemotherapy drugs used for breast cancer therapy can be given after surgery (adjuvant)

or before surgery (neo-adjuvant). For the (neo)adjuvant drugs and also drugs against advanced (metastatic) breast cancer, chemotherapy includes: taxanes most commonly paclitaxel (Taxol), platinum agents (cisplatin, carboplatin), pyrimidine antagonists (5-fluorouracil, capecitabine, gemcitabine), and other drug classes.

Although drug combinations are often used to treat early breast cancer, advanced breast cancer more often is treated with a single drug (monotherapy). Chemotherapy is given in cycles, followed by a rest period to give you time to recover from the effects of the drugs. Cycles are most often 2 or 3 weeks long. The schedule varies depending on the drugs used. Chemotherapies are often given for a total of 3 to 6 months, depending on the drugs used.

Focus on paclitaxel drug

Paclitaxel (PTX) is a novel anticancer agent that have been proved effective for different cancer types, with different stages including metastatic cancers, with known or unknown primary sites [START_REF] Yasui | Carcinoma of unknown primary site treated with carboplatin+ paclitaxel+ bevacizumab+ erlotinib and its maintenance chemotherapy[END_REF]. The brand name is Taxol, which is given as intravenous therapy. Paclitaxel binds to βtubulin in assembled tubulin, thereby reducing depolymerization. Taxanes stabilize microtubules and dampen microtubule dynamics to prevent the normal formation of mitotic spindles [START_REF] Jordan | Microtubules as a target for anticancer drugs[END_REF]. This leads to chronic activation of the spindle assembly checkpoint, leading to mitotic arrest [START_REF] Gascoigne | How do anti-mitotic drugs kill cancer cells[END_REF]. Extended mitotic arrest eventually leads to cell death [START_REF] Kavallaris | Microtubules and resistance to tubulin-binding agents[END_REF]. It was also shown that the taxanes strongly induce tumor necrosis factor (TNF) alpha production, which causes toxic immune cell activation, and may also promote apoptosis [START_REF] Sprowl | Alterations in tumor necrosis factor signaling pathways are associated with cytotoxicity and resistance to taxanes: a study in isogenic resistant tumor cells[END_REF].

Paclitaxel can be used for the treatment of all stages of breast cancer, primary and metastatic. It is also used for the treatment of inflammatory breast cancer (IBC), an uncommon type of invasive breast cancer. Most women with IBC will receive a taxane such as paclitaxel (Taxol) as the second type of chemotherapy drug (together with doxuribicin). Paclitaxel has been studied at variable doses (15-825 mg/m 2 ) and infusion times (1h to 4 days). It can be included in both weekly and every 3-weeks dosing cycles, as recently reviewed by [START_REF] Stage | Clinical pharmacokinetics of paclitaxel monotherapy: an updated literature review[END_REF]). Paclitaxel's mechanistic, clinical, and side effects on breast cancer are recently reviewed by [START_REF] Abu Samaan | Paclitaxel's Mechanistic and Clinical Effects on Breast Cancer[END_REF].

3. 3. Raman microscopy

The application of Raman spectroscopy to analyze biomolecules and even tissues was first demonstrated as early as the 1960s, and by the mid-1970s biomedical applications were explored.

Raman spectroscopy can be performed on live cells (Salehi et al., 2013b), and also provides high spatial resolution, being an optically scatter-based microscope technique, commonly employed in a confocal mode, enabling subcellular analysis of biological processes at an organelle level. Raman spectral maps of whole live cells can be performed over minutes to hours, but in cases where spectral quality is favored over speed, for detailed analysis, measurement protocols often entail fixing the cells at fixed time points after exposure to exogenous agents, having optimized protocols for cell fixation [START_REF] Meade | Studies of chemical fixation effects in human cell lines using Raman microspectroscopy[END_REF].

"The Spectroscopy system"

The Raman spectroscopy is a non-destructive method of analysis based on the photon-matter interaction, allowing to identify the molecules' vibrations. Each molecule has its unique vibrational modes, it's characteristic signature. Thus, this technique provides chemical information from the vibrations of the molecules after their excitation by a monochromatic laser. Thereafter, the spectra are obtained to allow different chemical mapping of the analyzed sample area. for measurement of samples [START_REF] Wen | Application of Raman spectroscopy in biopharmaceutical manufacturing[END_REF].

Advances in

"The Confocal system"

Confocal Raman microscope is one of the best techniques to probe pharmaceutical compounds. This is because the laser beam allows direct sharp focusing on very small visible and sub-visible particles down to low micron size using the high magnification provided by the Raman microscope. Detailed structural information can be obtained from the Raman spectrum of the drug. This includes the secondary chemical structure, the state of the hydrogen bonding, the structural conformation, and intermolecular interactions. For drugs, the Raman spectrum not only provides information about the active ingredient but also about the excipients "secondary ingredients of the drug". Hence we can obtain information about the distribution, crystal habit and polymorphism of the excipients when performing a Raman analysis [START_REF] Wen | Application of Raman spectroscopy in biopharmaceutical manufacturing[END_REF]. Confocal microscope allows depth scan with a high level of depth resolution. It will suppress unwanted fluorescence background. The Raman microscope (Fig. 6a) focuses a laser beam down to a small spot on the order of 1µm 3 . By placing an aperture at the back of focal plane of the microscope a confocal mode is achieved. In our system a multimode fiber optic plays the role of confocal aperture (Fig. 6b).

3. 4. Spectral data analysis

In each measurement, every single Raman spectrum carries a bunch of information at the position where the spectrum is recorded. The relevant information can be extracted to allow further analysis.

To obtain all the information, the spectra need to be prepared using appropriate filters and algorithms, therefore the data can be visualized as a reconstructed (false) image processed and exported. Although cellular components are very similar in terms of Raman vibrations, however, with appropriate data treatment, it is possible to increase the spectral contrasts between them and reveal the very small differences (Salehi et al., 2013a).

First: Spectral pre-processing in Raman spectroscopy

In the case of Raman spectroscopy, sole recorded spectra can suffer from contaminations, and therefore, background, substrate and instrumental contributions are routinely subtracted. A wide underlying spectral background is often observed in Raman spectra of biological samples, while the relatively high amount of spectral information in Raman spectra means that the derivative spectra are difficult to interpret [START_REF] Marshall | Choice of spectroscopic lineshape model affects metabolite peak areas and area ratios[END_REF].

Multiple points can be summarized for spectral processing :

-An important route towards minimizing pre-processing is appropriate sample presentation. When measured in immersion, the water acts as an index matching fluid, visually reducing the scattering of the laser spot by the sample and significantly enhancing the spectra. Measurement in water immersion also significantly reduces any damage in tissue samples due to the laser, the water providing a heat sink to protect from photothermal effects [START_REF] Bonnier | In vitro analysis of immersed human tissues by Raman microspectroscopy[END_REF].

-In addition to background from the sample, in optically thin single cells grown samples, the substrate itself can contribute significantly. Choice of substrate can minimize this contribution, and UV-grade CaF2 is widely accepted as an optimal choice for 532 nm wavelength laser [START_REF] Kerr | Optimal choice of sample substrate and laser wavelength for Raman spectroscopic analysis of biological specimen[END_REF].

-Confocal operation, available in some Raman spectrometers, can reduce the spectral contribution of the substrate, as previously described by Puppels et al. for single cells [START_REF] Puppels | Studying single living cells and chromosomes by confocal Raman microspectroscopy[END_REF][START_REF] Puppels | Description and performance of a highly sensitive confocal Raman microspectrometer[END_REF].

-In cases where the spectral background cannot be completely eradicated experimentally, background removal is commonly performed, by one of numerous algorithms (Bulmer et al., 1975, Haight and[START_REF] Haight | Automated image background removal in line-imaging Raman spectroscopy[END_REF]. However, it should be noted that such subtraction of arbitrary backgrounds does not guarantee a reproduction of the true spectral features, as a spectral minimum between many overlapping Gaussian features may not be the true minimum of the signal. Prior knowledge of the contributing factors should be used, wherever possible. Accordingly, it is common to record and subtract "dark" and substrate spectra from the sample spectra before removing any arbitrary background.

-Further artifacts in Raman spectroscopy include nonlinearity of axes and dark noise. In dispersive multichannel Raman spectrometers employing a CCD sensor, the data point spacing during acquisition can be irregular not only due to the use of different gratings. It may change from day to day due to different calibration settings and even drift over the course of a day due to variations in temperature and/or humidity. A number of commercial instruments now incorporate a post recording calibration procedure, although it is not clear as yet whether a standard procedure has been adopted by the biospectroscopy community.

-As Raman spectroscopy measures scattered intensity (as opposed to a transmission ratio), in addition to calibration of the spectral axis, intensity calibration is necessary in cases that the results from different instruments and laser sources are to be compared. To this end, the use of standard reference materials (SRM), subsequent mathematical treatment, and employing computational methods using a correction curve.

-Electronic noise (flicker, shot, and thermal noise) is an unpredictable and constant occurrence primarily in the spectral intensity, and can have a huge impact on the quality of any signal [START_REF] Mccreery | Raman spectroscopy for chemical analysis[END_REF] and Raman spectra are commonly additionally subjected to a noise reduction protocol to "smooth" the spectra, increasing the signal to noise and accentuating the true spectral features.

Second: Spectral post-processing in Raman spectroscopy

Analysis of the subtle changes in spectral profiles associated with, for example, disease, biological processes or the influence of external agents require the use of multivariate analytical techniques. Such multivariate methods have become invaluable to a wide range of fields, including pharmaceutical sciences. Sophisticated spectral analysis is required to extract specific information related to an external variable such as exogenous exposure dose or time, requiring supervised methods which reduces the dimensionality of the data and correlates information, here represented by the spectra data set, for example, to time evolution or drug concentrations or to a gold standard assay, to track the dependent evolution of the spectral signatures in, for example, subcellular regions. The regression coefficients obtained can be analyzed and provide information about the contribution of spectral variations. As a function of frequency, the coefficients illustrate the spectral features which are influenced by the external factor selected [START_REF] Farhane | Monitoring doxorubicin cellular uptake and trafficking using in vitro Raman microspectroscopy: short and long time exposure effects on lung cancer cell lines[END_REF]. Using multivariate data analysis, Raman microspectroscopy was shown to be capable of tracking the kinetics of the uptake and accumulation of drugs at a subcellular level in vitro.

Specifically considering vibrational spectroscopic datasets, multivariate methods allow analysis of multiple spectra simultaneous and interdependently. This then allows for comparisons to be made between spectra and groups of spectra within a dataset and to identify trends these may contain e.g. spectral pathologic markers in control and non-control samples, identification of nanoparticle containing spectra, response to external agents etc.

An important consideration in the application of multivariate methods to spectral data analysis is the requisite size of dataset. Statistical significance is a critical consideration, and when patient diagnosis is the outcome, misclassification has serious consequences. In the construction of multivariate classifiers, robustness of the model should also be rigorously tested.

.3. 5. Our project: Raman-based studies

This thesis is composed of four main parts of Raman study, as steps of four main investigations:

Firstly, investigation of the DPSCs' drug uptake and release by Raman: three different classes of anticancer drugs (chapters 2, 3 and 5) ;

Secondly, investigation of paclitaxel's pro-apoptotic effect on DPSCs and our therapy's effect on MCF7 cells measured by cytochrome-C translocation (chapter 3) ;

Thirdly, investigation of paclitaxel-induced pathology and resistance in DPSCs and cancer cells by intracellular matrix metalloproteinase MMP1 detection: a comparative study (chapter 4) ;

Fourthly and lastly, a preliminary study of metal nanoparticles (MNPs) uptake by DPSCs and cancer cells (chapter 7).

3. 6. Our project: Therapy-based studies

This thesis is composed of four main chapters, as steps of novel therapy development of nongenetically primed dental pulp stem cells (DPSC). The chapters will answer four inquiries:

Firstly, the Proof-of-Principle for the anti-cancer drug uptake and delivery by the DPSCs:

POP studies (containing the two first articles, chapters 2 and 3) ;

Secondly, the Proof-of-Mechanism of the application of DPSCs as drug delivery vector: three distinct drugs to three cancer cell types: POM studies (containing the two following articles, chapters 4 and 5);

Thirdly, the Proof of-Concept of the therapeutic effect of paclitaxel-primed DPSCs on breast cancer:

POC study (containing one article, chapter 6) ;

Fourthly and lastly, the promising application for the nanoparticle-primed DPSCs for cancer application: preliminary study (one article, chapter 7).

France). Tooth surfaces are cleaned using 2% chlorhexidine and cut around the cementum-enamel junction using sterilized discs. Teeth are then broken into 2 pieces to reveal the pulp chamber. The pulp tissue is gently separated from the crown and root and then digested in a solution of 3 mg/mL collagenase type I and 4 mg/mL dispase for 1 hour at 37°C. The solution is then filtered through 70 μm falcon strainers and added to αMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin with the addition of 1 ng/mL bFGF (basic fibroblast growth factor) and placed in 75 mL flasks. The detailed protocol is represented in the figure 7.

4. 2. Fluorescent transfection of DPSC cells (A) Lentiviral transduction

Viral transduction of cells with a fluorochrome label was chosen for co-culture studies assuring no interference or modification with drug activity, and also to be maintained and not to fade through generations, allowing accurate quantification. This method generated cells for co-culture that stably express RFP and fluorescence did not diminish during the whole culture period. Prior to lentiviral infection, the transduction conditions were optimized for each cell line and each plasmid (Fig. 11). In addition, a puromycin titration was performed to identify the minimum concentration of puromycin that caused complete cell death after 3-5 days. The lentiviral transduction of DPSC was based on manufacturer's protocol (Qiagen).

Briefly, DPSCs were plated in 75 cm 2 culture flasks until 90% confluency in complete medium, as described above. Then, as transduction reagent, SureENTRY (Qiagen) was added at a concentration of 8 μg/mL together with the RFP-containing viral particles at a concentration of 1.4×10 7 transducing units (TU)/ mL (or using a multiplicity of infection MOI of 50) with an antibiotic-free medium.

Following overnight incubation, medium containing viral particles was removed and replaced with fresh medium containing 2 μg/mL (active concentration) of puromycin (Sigma-Aldrich) in order to isolate the puromycin-resistant colonies. The primary stem cells were infected with one spin infection, and transduced cells underwent fluorescence-activated cell sorting (FACS) analysis after the last infection cycle, for the presence of fluorescence proteins. Sorted and puromycin-selected polyclones were subsequently used for the assays.

Figure 8: Lentiviral transduction of RFP-DPSC (transient study). Cignal Lenti Positive Control (RFP) with

varying multiplicity of infection MOI from 12.5 (lowest) to 50 (highest expression), transduced around 20,000 DPSC cells (24 hours before transduction 10 4 cells were plated per well of 96-well plate) and after 72 hours RFP fluorescence was visualized using fluorescent microscopy.

(B) Sorting of transduced living cells

A fast way of purifying transduced cells could be by using FACS, where an almost pure population can be obtained in a short time. For the preparation of sorting, the cells were trypsinized and resuspended in PBS, and then kept at 4 °C until sorting. Propidium iodide PI (Sigma-Aldrich) was added to the staining tube in the last step, with a final concentration of 0.1 µg/mL for live cells, to facilitate the elimination of dead cells. Incubation with PI was 15 minutes in the dark followed by washing. All the samples were filtered through test tubes with 40μm nylon mesh cell strainers (Corning Falcon) to minimize cell aggregation within the samples and the analysis is performed within an hour. For sorting live cells, we used the BD FACS Melody cell sorter coupled with FACS-chorus software (BD Biosciences, CA, USA). Following the sorting of both cell types, RFP expression was analyzed. The purity of subpopulations after sorting was confirmed by analyzing post-sort samples in the sorter again, reaching a purity of above 95%. For the establishment of a stable culture, cells were maintained in growth medium containing the selective dose of puromycin until the start of experiments.

4. 2. Confocal Raman system 4. 2. Raman data acquisition

Raman spectra were collected using a WITec Confocal Raman Microscope System alpha 300R (WITec Inc.). Excitation in confocal Raman microscopy is generated by a frequency-doubled Nd:YAG laser (Newport) at a wavelength of 532nm, with 50mW maximum laser output power in a single longitudinal mode. The incident laser beam is focused onto the sample through a 60× NIKON water immersion objective having a numerical aperture of 1.0 with a working distance of 2.8 mm (Nikon).

The laser power after the objective is 15mW but the power absorbed by cells in PBS is lower.

The spatial and depth resolutions are 300nm and 1μm, respectively. The mixed scattered radiation passes through an edge filter to block the Rayleigh radiation from the Raman signal, which is then directed to the electron multiplying charge coupled device camera EMCCD (DU 970N-BV353). The electron multiplying charge coupled device chip size was 1600 × 200 pixels, the camera controller was a 16-bit A/D converter operating at 2.5 MHz. The acquisition time of a single spectrum was set to 0.5

sec. An area of 150 × 150 points per image was recorded leading to the acquisition of 22500 spectra for one image. The advantage of our analysis method using EMCCD is the high speed of scan. Each pixel can be recorded with a very good signal to noise ratio at low integration times. For the high spatial resolution of the system (300 nm), a complete image of a whole cell is recorded for a better analysis. Data acquisition and processing were performed using Image Plus 2.08 (or the updated version 5.2) software from WITec.

4-K-means cluster analysis (KMCA)

K-Means clustering analysis (KMCA) is a statistical method which aims to partition data into clusters based on similarity. A good example of KMCA in Raman spectroscopy is shown in our previous study to identify regions in the Raman dataset, corresponding to anti-cancer drug or metal nanoparticles, and differentiate them from neighboring cytoplasm (as well as the mitochondria, nucleus and nucleolus) (Salehi et al., 2013). The figure below (Fig. 9) shows a diagram illustrating the main steps in the K-means clustering algorithm.

Figure 10 : Schematic outlining the key steps in the K-means clustering analysis algorithm.

From a spectroscopic imaging perspective, an initial number of seed locations is chosen. The spectra are then assigned to one of the seed locations. Once all spectra have been assigned, the mean spectrum or centroid is calculated and the distance between each spectrum and centroid is calculated. The spectra are then reassigned if necessary and the process is repeated until no spectra change groups. Reproduced from (Byrne et al., 2016).

5-Pearson correlation coefficient

Beside the K-mean cluster, a spectral correlation matrix was calculated (Miljković et al., 2010) to find the most similar spectrum to the reference spectrum of paclitaxel and cytochrome c. To quantify the similarity, as a "distance", the Pearson's correlation coefficient (r) was calculated for each pair of spectra. The value of (r) can vary between -1 and 1, thus it can be expressed as a percentage ranging from -100%, no correlation, to 100%, the perfect match. From these values, a pseudo color map can be constructed, reflecting the quantified similarities. All calculations were performed with a homemade code written in MatLab (R2008a, Math Works Inc.). A flowchart of the basics of this method is presented in figure below (Fig. 10).

4. 3. Studies of cell apoptosis

The term apoptosis refers to programmed cell death or cell suicide program as a process of normal cell development or in response to pro-apoptotic agents. In a 3 h-induction with apoptotic stimuli, the cells displayed morphological landmarks of apoptosis-associated mitochondrial fragmentation, perinuclear redistribution of mitochondria, nuclear condensation, and cytoplasmic shrinkage [START_REF] Tang | Reversibility of apoptosis in cancer cells[END_REF].

The initiation of apoptosis was further confirmed by the biochemical landmarks of apoptosis [START_REF] Wang | The expanding role of mitochondria in apoptosis. Genes & development, 15, 2922-2933[END_REF], the activation of apoptotic protease caspases, the cleavage of pro-caspase-3, and also the dysfunction of mitochondria. Apoptosis is commonly evaluated by examining multiple cellular events such as loss of mitochondrial membrane symmetry (tested by annexin-V), translocation of cytochrome-C from mitochondria to the cytosol, and activity of apoptosis-related proteases (like caspases).

The extrinsic pathway of apoptosis involves the recruitment and activation of procaspase-8 and caspase-8 then directly activates the effector caspases such as caspase-3 to initiate the execution process. Upon activation of the apoptotic events, mitochondrial outer membrane permeabilization is induced and the release of proapoptotic mitochondrial contents into the cytoplasm, such as cytochrome c and other soluble mitochondrial intermembrane space proteins [START_REF] Pradelli | Mitochondrial control of caspase-dependent andindependent cell death[END_REF].

Released cytochrome c leads the formation of the apoptosome, leading to induced cleavage and activation of other effector caspases [START_REF] Jiang | Cytochrome C-mediated apoptosis[END_REF]. A common execution of extrinsic and intrinsic pathways is initiated by the cleavage of effector caspases, caspase -3/-6/-7 and results in DNA fragmentation, cytoskeletal reorganization, cytoplasmic condensation, and formation of apoptotic bodies [START_REF] Elmore | Apoptosis: a review of programmed cell death[END_REF].

The following table summarizes the tests used throughout the thesis to investigate for apoptosis :

4. 4. Assays for cell migration

Five tests measuring the cell migration were performed throughout the thesis, that are summarized in the table below in terms of test category, cell, analytical method, and variable tested (table 3). The homing into spheroids, O-ring, and transwell assays are fully explained in the chapter 5. The morphological and spheroid dissemination assays are found in the chapter 6.

Introduction

Previous work have demonstrated that paclitaxel (PTX) arrests the cell cycle in the G0/G1 and G2/M phases [1]. Bosco et al. (2015) suggested that mesenchymal stem cells (MSCs) are resistant to the cytotoxic effects of paclitaxel observing the inhibition of their proliferation, inhibiting apoptosis, and inducing quiescence [2]. The quiescence ability of stem cells is critical for the maintenance of stem cell potency and of their function during tissue homeostasis and tissue repair after an injury [3].

Moreover, authors have concluded that paclitaxel causes induction of the fibroblastic phenotype in MSCs based on the changes in expression of distinguishing markers, like the matrix metalloproteinase MMP1, and changes in morphology and in differentiation potential [2]. In this study, we intend to quantify intracellular MMP1 in the objective of examining the "fibroblastic" phenotype state of human dental-pulp stem cells (DPSCs) after their exposure to PTX. Based on a previous finding in literature [2], the present study hypothesizes that PTX incorporation increases the fibroblastic collagenase MMP1 level in the DPSC cell, as a resistance mechanism to the drug.

In Raman confocal microscopy, intracellular organelles can be resolved due to the subcellular resolution achieved with the optical microscopy. Added to that, recording the Raman spectra at higher spectral resolution, compared to other spectroscopy techniques, valuable biochemical information can be gained [4], with more potential for elucidating molecular 'modes of action' by analysis of the spectral changes [5]. In the context of the action of chemotherapeutic drugs in-vitro, it is possible to monitor both the first molecular events initiated by the drug chemistry and the subsequent cascade of events determining the efficacy of a drug [5]. While understanding the chemistry can be key to establishing quantitative structure-activity relationships, understanding of the subsequent pathways may help to understand the mechanisms of drug resistance and sensitivity in different cell types and ultimately in patients [5]. Therefore, to have a global idea on the effects of external agents, including drugs or nanoparticles on cells, there is a need for advanced label-free methods enabling to monitor the cascade of cellular events which occurs after drug exposure.

In our earlier studies, the paclitaxel drug has been traced within DPSC stem cells and MCF7 breast cancer cells, by using single-cell Raman imaging, without altering the drug effect or the cell structure neither [6,7]. We have also shown that paclitaxel can have different modes of action with different Raman spectroscopic signatures depending on its dose [8], and this was confirmed also for other anticancer agents by using Raman spectroscopy [5]. For instance, by using the drug dose with viability assays as independent experiments, Salehi et al. showed that it was possible to experimentally quantify intracellular PTX concentration in the cancer cells [8]. These results were confirmed with simulated data, by using mathematical equations [8], further emphasizing the power of the Raman technique and potential to high throughput analysis for in-vitro drug testing. In another context, Salehi et al. also showed the potential of Raman spectroscopy for label free real-time apoptosis detection (at 1-hour interval over up to 4h) of live MCF7 after exposure to high dose paclitaxel through the time-dependent release of cytochrome C from mitochondria [9]. Cellular responses to pro-apoptotic treatment have been also revealed in DPSC and MCF7 cells [6].

Therefore, Raman spectroscopy can shade the light on the subcellular uptake and accumulation pathways of chemotherapeutic agents, characterize and fingerprint their mode of action and interaction mechanisms, and potentially identify cell resistance traits [5]. The potential of vibrational spectroscopy in conjunction with multivariate analysis techniques as a diagnostic tool has thus been well demonstrated, and the concept of 'spectral cytopathology' has been established [10]. The Raman technique can identify biochemical changes in tissue allowing detection of pathological change before histological features are present. A further potential application which is currently attracting increasing attention is in the understanding and screening of cellular resistance to therapeutic treatments to guide strategies for personalized therapies generally [11], and specifically in breast cancer [12]. We hereby show that confocal Raman microscopy can detect metabolic modifications, such as the MMP level change in the cells, after exposure to paclitaxel.

Previous works addressed the chemical effects of drugs on cancer cells by measuring specific changes in DNA, lipid, and protein features after drug exposure [13][14][15]. In one study, the effect of docetaxel drug on MCF7 was studied, comparing the treated versus untreated cells and investigating the druginduced morphological changes in combination to chemometric analysis [16]. Lin et al. also used Raman spectroscopy to assess the paclitaxel cytotoxicity on human lymphoma cells [17]. For large and complex data sets, post-processing or data mining is necessary in order to reduce the number of variables though retaining the most important variations within the dataset. More sophisticated spectral analysis is required to extract specific information related to an external variable such as exposure dose or timing, requiring supervised methods to reduce the dimensionality of data and to correlate complex information.

In the present study, we have standardized a method based on supervised Pearson correlation analysis of the cell spectra for intracellular MMP1 quantification. Cancer and stem cells fall in the category of "differentially-uncommitted cells": cells with the ability of originating different cell types by undergoing a cascade of changes in gene expression [18]. Cancer cells in culture commonly present a variety of organization of cell structures as well as abnormal rates of cell division. The same occurs

Priming with paclitaxel

Paclitaxel 6 mg/mL (Taxol, Teva pharmaceutical Ind.) was used for all the experiments. DPSCs were cultivated for 24h on polished and disinfected CaF2 (Crystran Ltd, Dorset, UK) substrates. Paclitaxel is then added to the cell culture medium at increasing concentrations of 1-10-100 µM followed by 1h incubation. In another setting, the DPSC, MCF7, and MCF7 TAX19 were seeded in the same way and incubated with 10 µM PTX for 6h. The CaF2 substrates with adhered cells are fixed with 2% PFA followed by thorough rinsing with 5x PBS and reserved in petri dishes containing 5 mL PBS.

Viability assay of DPSC incubated with PTX

DPSCs (3x 10 4 cells/cm 2 ) were seeded on 96-well plates (VWR). After 24h, PTX was added in increasing concentrations (1-10-100 µM) and incubated for 1h incubation. In another setting, 10 µM PTX was added to DPSC, MCF7, and MCF7 TAX19 for incubation periods from 3h to 72h, without medium changing. Non-treated cells (in drug-free medium) were always considered as controls. Cell number was quantified by measuring absorbance at 540 nm by Thiazolyl Blue Tetrazolium Bromide (MTT) assay (Euromedex) as previously described [6].

Raman data acquisition

Raman spectra were collected using a WITec Confocal Raman Microscope System alpha 300R (WITec Inc.). Excitation in the confocal Raman microscopy is generated by a frequency-doubled Nd:YAG laser (Newport) at a wavelength of 532nm, with 50mW maximum laser output power in a single longitudinal mode. The incident laser beam is focused onto the sample through a 60× NIKON water immersion objective having a numerical aperture of 1.0 with a working distance of 2.8 mm. The laser power after the objective is 15mW but the power absorbed by cells in PBS is lower. The spatial and depth resolutions are 300nm and 1μm, respectively. The mixed scattered radiation passes through an edge filter to block the Rayleigh radiation from the Raman signal, which is then directed to the electron multiplying charge coupled device camera EMCCD (DU 970N-BV353). The acquisition time of a single spectrum was set to 0.5 sec. An area of 150 × 150 points per image was recorded leading to the acquisition of 22500 spectra for one image. The advantage of our analysis method using EMCCD is the high speed of scan. Each pixel can be recorded with a very good signal to noise ratio at low integration times. Data acquisition and processing were performed using Image Plus 2.08 (or the updated version 5.2) software from WITec.

Raman data analysis

Human recombinant MMP1 was purchased from Sigma-Aldrich (SRP3117) in powder form. Two forms of MMP8 were purchased: powder form ThermoFisher (10254-H08-H2), and in solution from

Novus biologicals biotechne (NBP2-51559). For our study, the Raman data analysis of MMP1 protein, based on the spectral correlation matrix, was performed [25] to find the most similar spectrum to the reference spectrum of MMP1. Calculating the correlation matrix is a well-established method by our team, frequently done beside the KMCA method for the spectral analysis [6,8,9].

First, the Raman spectral references of the MMP1 and MMP8 were calculated. Then, to quantify (as distance) the whole cell spectra similarity with the references spectra, the Pearson's correlation coefficient (r) was calculated for each pair of spectra, using a homemade code written in MatLab (Math Works, Inc, Massachusetts, USA). The value of r can vary between -1 and 1, thus it can be expressed as a percentage ranging from -100%, no correlation, to 100%, the perfect match. From these values, a pseudo color map can be constructed, reflecting the quantified similarities. Quantification of MMP1 pixel count and cell area measurements was performed by ImageJ (version 1.51a, NIH) to obtain intracellular quantification of MMP1. The relative ratio of MMP1 was derived by normalizing the selected MMP1 pixel count to the respective cell area (pixel/µm 2 ) in each image.

Statistical analysis

The results of cell viability MTT tests were analyzed with student's t-test. The MMP1 intracellular ratios were compared within each condition (with PTX compared to control without PTX) using the "Mann-Whitney Rank Sum" test. For all experiments, statistically significant values were defined as p<0.05.

Results

Intracellular localization of MMP1

Integrated Raman intensities in the specific spectral region (2800-3000 cm-1) corresponding to the C-H stretching mode are used to provide a chemical map of lipids and proteins in the DPSCs (Fig. 1A).

Then the Pearson correlation pseudo-map is created (Fig. 1B), where the colors designate the highest correlation (red) to the no correlation (blue). This indicates that the MMP1 based on its spectrum (Fig. 1C) was found located in the cytoplasm (Fig. 1B; yellow arrows) and nucleus (Fig. 1B; red arrows), observed in both stem and also in cancer cells (not shown). No further clustering was performed to determine the exact compartment of MMP1 in the cytosol.

MMP1 intracellular level calculation

In this experiment, we investigated how Paclitaxel treatment modulated the expression of the fibroblast marker MMP1. In a first experiment, DPSCs were treated with paclitaxel different concentrations (1, 10, or 100 µM), while in a second study cancer cells were treated with only 10 µM PTX for comparison. Correlation of the MMP1 was measured after short incubation with paclitaxel (1h and 6h respectively for first and second experiments), then the ratio of MMP per DPSC or cancer cell area was calculated.

MMP1 intracellular level increases, in line with viability decrease, in

DPSCsPTX

The results of MMP1 intracellular protein in DPSC by 30% correlation of the maximum value showed a low ratio of MMP1 in normal DPSCs. A dose-dependent fold increase was observed in the ratio of MMP1 in DPSC incubation with paclitaxel (compared to non-treated control) even with the relatively low treatment doses after 1h. Even at 1µM PTX, cells showed high increase of MMP to cell ratio by 2.5 folds (n=9). Treatment with high-PTX concentration showed 2.9 folds (n=10) more MMP compared to control non-treated cells (n=8) by our method (Fig. 3A). These data are consistent with a significant DPSCs loss in viability for the highest PTX dose (Fig. 3B).

Cell viability after exposure to PTX

All three cell types are treated with 10 µM PTX for up to 72 h. Viability of DPSC and PTX-resistant MCF7 TAX19 (MCF7-r) cells was not affected while that of MCF7-s was significantly reduced, as

shown in figure 4.

Low MMP1 intracellular level in DPSC while high levels in breast cancer cells

We also performed MMP1 spectral analysis by confocal Raman spectroscopy in DPSCs in comparison with MCF7 breast cancer cell line (n=25) and its PTX-resistant subline (n=26), as shown in Fig 5A.

By only 10% correlation measurement (Fig. 5B), a significant higher MMP1 level was observed in cancer cells, both sensitive and resistant, compared to the stem cells (p<0.001). After 6 hours incubation with 10 µM PTX, less MMP1 level was observed in the sensitive (n=26) and no effect in the PTX-resistant (n=64) cancer cells when exposed to treatment, as shown in Fig 5A . For DPSCs, and considering a higher correlation measurement (30%), very much lower MMP1 level is found when compared to the cancer cells. To note that this concentration and incubation time with PTX could induce increase in MMP1 intracellular ratio but not significantly.

Figure 5: MMP1 intracellular ratio comparison between cells of three resistance traits to paclitaxel: sensitive cancer (MCF7-s), PTX-resistant cancer (MCF7-r), and highly resistant stem cells (DPSC). (A)

Histogram shows decreased MMP1 level in treated cancer cells, while an increase in the DPSC cells, with

Comparison with other quantimetric approaches in the literature

For the detection of intracellular MMP, conventional methods such as immunohistochemistry (IHC)

staining for localization and quantification, zymatography, Western blotting (WB), qRT-PCR, and microarrays have been widely used. For definitive proof, multiple antibodies and methods should be used that have no background binding and staining and confirmed using orthogonal techniques of extracted intracellular proteins. Hence, usually two or ideally three techniques are used for result validation. There are two types of intracellular MMP1 test analysis: the differential gene expression (latent or pro-form) and the intracellular protein expression (active-form). We have collected beneath the literature evidencing our non-invasive spectral method of analysis.

I) Qualitative results

In one study, MMP-1 staining was not shown to co-localize in Golgi apparatus or lysosomes but was clearly co-localized with mitochondria and nucleus, in all cell types including fibroblasts and epithelial cells [29]. By IHC staining, one cohort study detected positive MMP1 nuclear and cytosolic staining in the breast cancer cells, analyzed at protein level [30], and also by semiquantitative RT-PCR, IHC and western blot (WB) [31]. Although nuclear localization is confirmed, the nuclear/ chromosomal MMP1 activity and function intracellularly is not yet evidenced [32].

I) Quantitative results

In one study, MMP1 and MMP2, had hundred times higher expression in human adult fibroblast compared to MSCs [33]. Also, high expression of MMP1 is confirmed in MCF7 [34] and breast cancer tissues [35][36][37]. Generally, MMP1 is at the top of the list of the MMP family that were found to be dysregulated in human cancers [38]. Also compatible with our results, no significant difference in the MMP1 was found for paclitaxel-resistant MCF7 compared to the sensitive breast cancer cells [39,40]. The methods used are summarized in the table below (Table 1).

Although in the non-injured somatic cells, PTX induces an upregulation, it is expected that treatment of cancer cells may cause MMP1 reduction [34,41]. In recent studies, paclitaxel suppressed growth and proliferation through down-regulation of MMP-mediated signaling pathway in breast cancer cells [42], as well as in glioblastoma cells [43] and ovarian cancer cells [44], notably the MMP9 isoform.

Although not tackled by other literature, treatment with PTX caused a reduction of MMP1 level according to our findings.

Table 1: Review of other literature validating Raman results using invasive metric methods.

Condition (its finding) Cell type Technique Reference MSC (low MMP1)

BM-MSC Microarray RT-PCR [33] BM-MSC (normal) RT-PCR WB shRNA [45] MSCsPTX (increased MMP1) BM-MSC RT-PCR WB [2] Breast cancer (high MMP1) MCF-7, MDA-MB231 IHC WB [34] Patient-isolated cells Genetic arrays [35][36][37] PTX-resistant breast cancer (high MMP1) MCF-7/Pac DNA microarray [39] MCF-7/Pac RNA Genechip [40] *Index; RT-PCR, real-time polymerase chain reaction; WB, western blot test; shRNA, small heparin RNA vector; IHC, immune histochemical staining.

Validation of the method

Discussion

By their nature, label-free techniques register all information within the sampling area, and identification of specific responses requires more sophisticated techniques to data-mine the differential responses due to cell injury or change. Regression and correlation approaches can be employed to extract the specific spectroscopic signatures of cellular changes which are caused by external stimuli, like drugs. These analyses can elucidate the spectroscopic signatures of the direct (chemical) effects of the drug as well as the subsequent cellular metabolic responses [46]. Raman spectroscopy opens the perspective in label-free fingerprinting and monitoring of biological processes in-vitro, with potential applications in fundamental cytological research, pre-clinical pharmacological development, and ultimately improved individualized clinical therapeutics [5]. Our results revealed that DPSCs have very low MMP1, which is not the case of cancer cells. We have observed an increase of MMP1 protein level after exposure of the DPSCs to increasing high concentrations of PTX during a short time-period. Transformation of cells towards malignant or high metastatic behavior involves a sequence of mutations that affects the expression of genes which are biologically responsible for tumorigenic properties of cancer cells, and used as markers to identify the cancer cells [18]. However, the increase we see in DPSCsPTX is still extremely low as compared to the cancer cells, even when very high drug dose is tested. Further experiments and timely comparison are needed to propose that the intracellular MMP1 induces any cancerous transformation of the DPSCsPTX.

We observed an increase of MMP1 protein level only in the case of PTX incorporation to DPSCs.

Mesenchymal stem cells are normally long-lived cells that can readily enter and exit quiescence depending on external signals [3]. In the literature, a quiescent (reversible G0 cell cycle) state induction in human MSCs caused by paclitaxel incorporation, is proposed as a defense mechanism and suppression of apoptosis-induction in many anticancer drugs [2]. In nearly all instances, a significant correlation between expression of MMP1 and cell survival has been documented. An increased MMP1 level can be associated with injury to the respective cells.

In the same context, it was suggested that the increase in intracellular fibroblastic collagenase in a way confers a resistance to apoptosis [2,29]. MSCs resist to paclitaxel by adopting a non-proliferating fibroblast phenotype (of which an increase in MMP1 and 3). On induction of apoptosis, intracellular MMP1 co-localized within mitochondria and nuclei, result in resistance to apoptosis and thus may explain the well-known association of this enzyme with tumor cell survival and spreading [29].

Elucidation of the intracellular pathways that MMP1 utilizes to confer resistance to apoptosis may further the understanding of molecular mechanisms by which DPSCs resist to chemotherapeutic drug, and of the intracellular functions of this enzyme.

This effect of PTX refers to DPSC's inherent ability of increased stress resistance and escape from cell death due to exogenous agents. DPSC may escape apoptosis by change in their MSC phenotype. To the best of our knowledge, no comparison was made between stem cells and cancer cells in terms of MMP-1 intracellular level. The effect on MMP1 breast cancer after paclitaxel treatment was not noted in the literature, but the intracellular level is expected to decrease because of the lower grade of proliferation of MCF7 after PTX therapy, although it's not a mechanism of action for the drug. In addition, MMP1 level may correlate with the status of physiology or disease [47,48] rather than the drug's mechanism of action. It is not evident that this increased MMP1 in DPSC is enough to induce tumorigenesis transformation. Further tests should be done to exclude this risk.

Moreover, a study of Zhao et al. (2017) has confirmed the association of the level of therapeutic efficacy of MSC to their MMP1 expression level, measured by mRNA profiling, and also the downregulation of MMP1 in exogenous MSCs, put in comparison to MSCs derived from cancer stroma [45]. It was revealed that in MSCs, the efficacy of inhibiting cancer cell proliferation was partially mediated by their MMP1 expression, as a key factor in regulating the cancer growth, via the mitogenactivated protein kinase (MAPK) pathway [45]. It was confirmed that downregulation of MMP1 in MSCs leads to reduced cancer cell apoptosis while increased proliferation [45]. In another recent study (2018), the gene expression levels of MMP1 and were highly upregulated in fibroblasts associated to cancer (CAFs) after their exposure to taxane [49].

The mesenchymal stromal cells and fibroblasts being a part of cancer stroma have a tumor supportive role, while exogenous MSCs are normally applied for their inherent tumor-inhibitive capabilities.

From the controversies in the literature, we can deduce that the inherent role (whether inhibitive or supportive) of cells when co-cultured with cancer cells can be enhanced by the MMP1 level. In our case, the DPSC by itself or pre-loaded are confirmed to cause the inhibition of breast cancer cells, and thus, we agree with the study of Zhao et al [45] stating that the mediated effect of MMP1 in the MSCs inhibitory function against cancer. MMP1 in MSCs has been identified as anti-cancer factor, playing an important role in suppressing cancer cell proliferation in the co-culture of MSCs and cancer cells [45] and inducing apoptosis of cancer cells [45,50]. These findings provide a theoretical basis for the mechanism in the boosted inhibition capacity of DPSCsPTX against breast cancer cells (compared to unloaded or naïve DPSC), that needs to be confirmed by further studies.

We conclude that MSC priming with PTX increases the intracellular MMP1 level, which has been suggested to mediate inhibition of malignant cells proliferation [45]. This can explain the very potent anti-tumor effect of the DPSCsPTX in compared to the unloaded cells. Importantly, apart from paclitaxel effect on MSC proliferation and phenotype, in-vitro analyses of the chemotherapy influence of the treatment on the stem cells functional characteristics in the studies do not take into account the potentially-relevant influences of the in-vivo microenvironment and the stem cells' interaction with other cell types. Although our non-labeling technique allows the study of proteins inside the cell, it cannot exactly define if MMP1 is released extracellularly, similarly as other intracellular detection techniques. The level of MMP1 and other MMPs can be strongly enhanced in cells by intracellular accumulation only and not secretion [36].

In looking to various literature, experimental care is required to definitively reveal MMPs function inside the cell. Immunohistochemical and immunocytochemistry localization needs to be performed using high resolution imaging and complimented with confocal microscopy analysis which includes 3D slices throughout the cell where possible to avoid overlap artifacts being interpreted as evidence for cytosolic and nuclear MMP protein localization and activity [32]. Indeed, many initial reports of intracellular MMPs relied upon histology or Western blots only, often with just one or more antibodies, subcellular fractionation of nuclear and cytosolic compartments or subcellular organelles need to be performed for characterization with convincing, rigorous multiple positive and negative controls [32].

In this regard, it is thought that advanced non-labelling techniques with standardized analyses for multiple conditions are therefore encouraged. The ability of vibrational spectroscopy to characterize and differentiate responses of pathological from non-pathological cell types to drugs opens up potential clinical applications as a companion diagnostics (CD) tool, and ultimately in development of personalized medicine approaches [51].

To extract information regarding the biochemical changes behind the spectral differences, more sophisticated data processing is required [52]. In such case, correlation with accepted or "gold standard" assays can be used to guide and validate the interpretation of the vibrational spectroscopic results. In addition to standard cytotoxic assays, the spectroscopic results can be correlated with more precise biomarkers [52]. Resistance spectral pathways have been specifically targeted by some research groups [53][54][55]. In monitoring the response to exogenous agents in-vitro, we have demonstrated that spectral methods like Raman spectroscopy can be potentially employed to test cellular resistance pathway in stem cells, by integration with other tests or studies. Our method has been validated for use by comparing to different studies' chemometric methods, comparing different commercial references (in different forms), and comparing to another isoform in the same MMP family. Nevertheless, these differential spectral responses remain multivariate in nature, and therefore, identification of differential spectroscopic "signature", with all the biomolecule "bands", may be more appropriate. Furthermore, for realistic applications potential, it is important that combinatorial signatures which are identified as characteristic of the cellular interaction and/ or response pathway, are translatable across cell lines and ultimately patient samples [5].

Conclusions

In view of the high expectations of MSCs use as drug carriers, our work suggests a novel possibility to perform extensive and high-quality confocal Raman analysis to better understand the cellular mechanisms involved in drug resistance, such as drug uptake and release and cell fate. Our study significantly improves the knowledge on DPSC mechanisms, opening interesting perspectives for further investigation on primary dental pulp derived stem cells. By taking into account the above data and the previous data collected, it appears evident that Raman spectroscope provides an interesting method useful for studying the plasticity of MSCs addressed for targeted delivery. Our study provides data investigations for the metabolic functions of resistance (or perhaps recovery from damage) related with the MSCs role in cell therapy, and also proposes a mechanism related to Taxol resistance. In our study using confocal Raman, it was not possible to distinguish between the pro-MMP1 from the active form of MMP1. To date, there is limited knowledge of the mechanisms and localization of MMPs to intracellular compartments. Perhaps advances in the application of spectral methods in correlation with other preclinical tests can help accentuate understanding of the MSCs still-unresolved mechanisms.

Introduction

Approaches that involve non-genetic modification of cells for therapeutic purposes can be used for targeted anti-cancer therapy. In each category, multiple types of cells have been explored because of their unique properties, such as a long circulation time, ability to home to specific tissues, and immune response. Different types of cells are suitable for different applications based on their properties, including the mesenchymal stem cells (MSCs). These cells have been studied for their capability to deliver various cytotoxic agents for chemotherapy (drugs, prodrugs, or related factors), as well as for immunotherapy, and transporting nanoparticles, anti-angiogenic agents, or oncolytic viruses or genes [1,2]. MSCs derived from the dental pulp are an easy source of stem cells for application in cellular therapy. For this aim, high numbers of cells are needed, added to the necessity of low patient morbidity, especially in autologous therapy applications. Cancer patients generally are sensible for surgery procedures, unless already deliberated in treatment planning. Extraction, isolation, and manipulation of dental pulp stem cells (DPSCs) are generally of minimal invasive nature requiring minor procedures.

On the other hand, for heterologous therapy, those cells are considered as medical waste from healthy persons, so they can be overseen for potential use when validated.

In this study, we examine the feasibility of the DPSC as anti-cancer drug "vectors" by investigating the subtle differences that occur in these cells upon exposure to lethal (10 µM) concentrations of Application of this high concentration of drugs leads to cell death in the tumor counterpart. Taxanes represent the most applied chemotherapy approaches and are the first line of treatment for patients with metastatic as well as early-stage breast cancer [3], thus are chosen for intense study on the DPSC in our study. Importantly, certain negative mutations associated with mitosis regulation are responsible for the development of resistance in cancer cells, and thus the effectiveness of taxane therapy is decreased [4].

As DPSC is a mesenchymal stromal cell that is used for different in-vitro biotechnology applications (principally tissue regeneration), we thought it is very important to have information also on their sensitivity to anti-cancer drugs with different cytotoxic mechanisms. In normal physiology, the dental pulp cells maintain and repair the periodontal tissue and respond to damage, resulting in the migration to the damaged area and the creation of odontoblasts and dentine in an attempt to repair the damaged tooth [5]. These observations led to the rising preclinical studies concerning DPSCs use in regenerative medicine. Consequently, DPSCs can be also investigated in chemotherapy-exposed cancer patients, as they have vital roles in repair and tooth support in damage and stress situations. Studies like ours can be envisaged for the mere effect on the patients stem cells inside teeth and their functional responsiveness to chemotherapy exposure.

Furthermore, the presence of drug release can be confirmed by using single-cell imaging method that is non-invasive and label-free, thus does not alter drug effect or neither the cell structure. Different drugs delivered by the loaded DPSCs, through their conditioned medium, were traced inside the cancer cells using two different analysis methods, after short incubation time to confirm the fast drug release by the primed DPSCs.

Materials and Methods

Primary mesenchymal stem cells

Human wisdom teeth extracted for orthodontic reasons were recovered from healthy patients (15-18 years old). Written informed consent was obtained from the parents of the patients. This protocol was approved by the local ethical committee (Comité de Protection des Personnes, Montpellier hospital, France). DPSCs were isolated and characterized as previously described [6,7,8]. DPSCs were cultured in αMEM (Modified Eagle's Medium, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 100 μg/mL penicillin and streptomycin (PS, ThermoFisher), with the addition of 1 ng/mL bFGF (basic fibroblast growth factor, R&D system) at 37°C and 5% CO2. DPSCs used for all experiments were between passages 2 to 8.

Primary MSCs derived from human bone marrow and adipose tissue, BMSC and ADSC were offered from (Dr. Daniel Noel, IRMB, Montpellier). These cells were cultured in αMEM supplemented with 10% FBS, 100 µg/mL PS, 1 ng/mL bFGF, and experiments were done in early time passages.

Cancer cell lines

The MCF7 (ATCC-HTB22), derived from a metastatic breast cancer patient, is a standard cell line model to use for cancer research [9]. Paclitaxel-resistant subline MCF7 TAX19 (offered from Dr. Peter Coopman, IRCM, Montpellier) was previously established [10] after culturing the parental MCF7 cells in increasing paclitaxel concentrations, and then maintained in paclitaxel-free medium. The colorectal cancer cell line, HCT116 (ATCC-CCL-247), was also a gift (from Dr. Celine Gongora, IRCM Montpellier) for use with our experiments. All the fore-mentioned cell lines were grown in DMEM media (Dulbecco's MEM, Gibco), containing 10% FBS and 1% PS. Accordingly, in direct co-culture experiments, the medium was changed gradually in two steps that, subsequently, the cells were cultivated in αMEM.

Priming with anti-cancer drugs

Paclitaxel 6 mg/mL (Taxol, Teva pharmaceutical Ind.) was used for all the study experiments. 5-Fluorouracil was a gift from IRCM in the concentration of 380 mM. Oxaliplatin was a gift from (Dr.

Celine Gongora, IRCM, Montpellier) and was diluted in PBS to the final concentration of 12 mM.

Both stock solutions were aliquoted and stored at -20°C. For cytotoxicity studies, sub-confluent cultures (1×10 5 cells/mL or 6×10 5 cells/ 25 cm 2 flask) were exposed to 10µM of drugs diluted in the complete medium of each cell type.

Proliferation assay of DPSC and cancer cells

Proliferation assay of DPSC with anti-cancer drugs

In vitro anti-proliferation assays of direct PTX, 5FU, and OXP on DPSCs cultures with correspondent cancer cells. Proliferation was assessed by constructing growth curves over a 7-day period. Briefly, 4000 cells/cm 2 DPSCs or correspondent cancer cells were seeded in 96-well plates (VWR). After 24h, cells were treated with chemotherapeutic drugs by adding drug-diluted or drug-free medium in the wells. Viability (3h-48h) and proliferation (3-5-7 days) were assessed by MTT assay, as previously described [11]. Medium was replenished on half period (between days 3,4) by same dilution of drug concentrations. Cell number was quantified by measuring absorbance at 540 nm by Thiazolyl Blue Tetrazolium Bromide (MTT) assay (Euromedex) as previously described [11]. Viability of DPSC, BMSC, and ADSC cells after 24h priming with PTX was also measured by MTT assay. Cells without drug were considered as control groups.

Proliferation recovery of PTX-primed DPSCs

DPSC were treated with the chemotherapeutic drugs paclitaxel (10 μM) for 12 h. The media containing the drug was removed and rinsed two times in PBS, then detached by trypsinization. The cells (4000 cells/well) were seeded in 96-well plates with complete media without PTX and allowed to proliferate for a period of 21 days with replenishment of medium every 3-4 days, and cell numbers were determined at different time points each 3 days (day 3 to 21). On 7-day time point, cells were trypsindetached and viable cell percentage was determined by trypan blue exclusion. Trypan blue 0.4% stain (Invitrogen) was mixed at 50% with cell suspension (5 mg/mL concentration). Cells were then counted using a hemocytometer. % Viability = 100 -(blue cells count/ total cells count× 100).

Effect of drug-primed MSCs conditioned medium

For subsequent experiments with PTX, after 24h of drug priming the cells were washed twice with phosphate buffered saline (PBS 1x) to remove non-internalized compound, then trypsinized for subsequent experiments or imaging. Conditioned media were collected from cells after fresh aMEM with 10% FBS had been incubated with the cells for the selected period, stored at 4°C for use on the following day, or freezed at -20°C for later use.

Conditioned media of MSCsPTX-CM were obtained from the seeding of 2x 10 4 MSCs in 24-well plate. After 48h (sub-confluent culture), medium was changed by fresh complete medium for another 48h. The 48-h CM (MSCs ±PTX CM) was diluted a first time to 10% v/v CM, and a second time dilution to 1% v/v CM. Last dilution was added to MCF7, and cell viability was evaluated by MTT assay after another 48h days for all conditions.

For experiments with resistant cancer cells, DPSCs were incubated for 12h with 10 µM PTX.

DPSCsPTX-CM (4h-CM) were tested for their in-vitro anti-proliferative activity against MCF7 (MCF7-s) or MCF7 TAX19 (MCF7-r) for efficiency comparison. The effect of conditioned medium from PTX-treated DPSCs was studied on both MCF7 subtypes for up to 7 days. For all tests, conditioned or control media were added to 1x10 4 cells cancer cells (MCF7, MCF7 TAX19 , or HCT116) in 96 multiwell plates (VWR), 24h after their initial seeding. Medium without drug served as the control for normalization.

Drug tracing after delivery to cancer cells

In another setting, the cell-conditioned media (DPSCs5FU-CM and DPSCsOXP-CM) were collected after 4h of adding fresh complete medium, then was added to MCF7 and HCT116 cells cultivated for 24h onto polished and disinfected calcium fluoride CaF2 (Crystran Ltd) substrates. Cancer cells were incubated for only 3 hours with CM from drug-primed DPSC cells. The CaF2 substrates with adhered cells are fixed with 2% PFA with thorough rinsing with 5x PBS and reserved in petri dishes containing 5 mL PBS, for transfer under confocal Raman spectroscope.

For details about acquisition system, check the Methods chapter (II.2.1 Raman data acquisition). The reference spectra of drugs (5FU and OXP) was calculated by Raman excitation. Then, we tried to seek out the drugs inside the cancer cells using their reference spectra. A first analysis method used is to check the drug map by K-mean cluster analysis. A second analysis method to detect drug inside the cells is using Pearson's correlation map with reference spectrum of drug, as described above. For all the represented images, C-H cell image and the particle map image were merged using Adobe Photoshop software.

Lentiviral transduction of DPSCs and MCF7

Lentiviral transduction of RFP-DPSCs and GFP-MCF7 was done based on manufacturer's protocol (Qiagen), and followed by FACS sorting (BD Biosciences). The purity of subpopulations after sorting was confirmed by analyzing post-sort samples, reaching a purity of above 95%. For the establishment of stable cultures, cells were maintained in growth medium containing the selective dose of puromycin until the start of experiments.

Migration assays of DPSCs ±PTX toward MCF7

The impact of PTX loading on DPSC's endogenous intrinsic tropism towards MCF7 cells was tested.

Ring migration assay, considered as gap closure method, is used to determine the average DPSCsPTX

Ring migration test for 1 day

Cell cloning 4.7-mm rings (Bel-Art, SP scienceware) are preserved in 70% alcohol solution, and are rinsed in sterile 1x PBS immediately before use. The rings were placed vertically in each well of 24well plates (Corning), with one ring in each well. Cell suspensions were added carefully to both sides of each ring. 5 x10 3 MCF7 cells were seeded inside the ring, and 85 x10 3 cells outside the ring, reaching about 80% confluency when cells were attached [12]. Outside numbers of DPSC was calculated with double the numbers of DPSC to that of cancer cells (according to their respective surface seeding areas). Twelve hours after cells attached firmly, the rings were carefully removed straight up using tweezers. The cells were washed gently with PBS to remove cell debris then were replenished with fresh aMEM containing 10% FBS and 100 μg/ml of puromycin.

At 24h, the gap of ring placement and inside of cell rings are evaluated by Celigo plate-based imaging, using the "cell migration: wound healing" application. Full images for 45% of the well (central zone)

are acquired where the gap area is defined by the software. The automated method detected migrating cells into the central entire gap depending on the RFP-fluorescence signal. The percent gap closure is defined as the area of well occupied by cells divided by the total selected well area.

Transwell migration test for 7 days

In order to quantify the ability of DPSCs to migrate on the long term in presence of MCF7, transwell migration assay was performed. The test was assessed by a 24-well transwell system insert of 8 μmpore size filter membrane (ThinCert, greiner bio-one). 2000 RFP-DPSC cells loaded or not with PTX, in 10% FBS 1% puromycin medium of 100 μL volume cells were added to the upper chamber and cultured in the same conditions as previously described. To the bottom chamber was added 6000 GFP-MCF7 cells in 600 μL medium containing 10% FBS 1% puromycin or only medium as negative control. After 7-days incubation period, the inserts were fixed with 4% PFA, then the cells on the upper surface of the filter membrane were removed by cotton swab and manually scraping (using small blunt excavator) the non-migrated DPSCs from top of the inserts. Cells migrated to the undersurface of the porous membrane were imaged using a LED coupled to fluorescence microscope (Celigo). Migrated cells on the whole membrane surface were counted by automated method for all control and test wells, to determine the number of migrated cells that already passed or still within the pores. Differential migration % is defined as the ratio of test migration towards "MCF7+FBS" to the migration of control group towards "FBS only".

Test of DPSCs ±PTX penetration into 3D spheroids

Round-bottomed, 96-well ultra-low attachment spheroid microplates (Corning) were used for spheroid formation. MCF7 or MCF7 TAX19 cells were seeded at a density of 5×10 In-vitro recovery assay was also used to test the impaired proliferation effect following PTX priming, as well as the proliferation recovery on long term after drug withdrawal. DPSCs were followed for up to 21 days (day 21 not shown), until the proliferative ability was partially, but not fully, recovered (Fig. 1D). On day 7 following culture in drug-free medium, DPSCs viability was assessed by trypan blue test. With the fact that no significant difference in viability was observed up to several days (>95% viability on day 7, data not shown), we can conclude that the observed reduction was due to decreases in cell proliferation and not due to decreased viability.

We observe that the trait of proliferation with DPSCs constant incubation with PTX (Fig. 1C) for 3 and 5 days seems to strongly correlate with that following short-term DPSC's drug priming (Fig. 1D) for 3 and 6 days too. This suggests that the severe proliferation impairment in the latter case is due to the presence of high drug released in the environment, rather than the drug internalization process in the first place. Moreover, proliferation recovery after PTX withdrawal was found directly related to the number of DPSCs directly exposed to the drug (see Annex Fig. A2-a).

DPSC±PTX migration ability in 2D model

On short-term migration (24h), a ring stopper was used so as when removed it creates an exclusion zone into where the cells migrate. The green shaded area identifies area occupied by cells (Fig. 2A), whereas the green line is the selected gap area for analysis. The DPSC±PTX cells were allowed to migrate for 24h, and Figure 39B shows the relative cell migration for the two conditions. Our findings show that drug-loaded DPSCs tend to better migrate into the center of well where MCF7 is present (Fig. 2B) with no statistical difference in the median values compared to unloaded DPSC.

To evaluate the tumor tendency migration of DPSCsPTX, the migration of unloaded and loaded DPSCs through the membrane pore toward MCF7 cells in the bottom chamber was tested after 7 days (Fig. 2C). Negative control without MCF7 cells was added to compare the differential migration of

DPSC±PTX homing ability in 3D model

Red

DPSCs can deliver different drugs and overcome drug resistance

We investigated the effect of 1% (v/v) conditioned media from several types of drug-primed DPSCs.

Results in figure 4A, showed a significant decrease in viability for all conditions. However, HCT116 cells viability was less affected than that of MCF7 cells. Quantitative methods for loss of cancer cell viability indicate the uptake of drug, however, we wanted to further ascertain the presence of drug by using single-cell imaging. Figure 4B, shows the efficiency of DPSCsPTX -CM on PTX-resistant MCF7, starting early days incubation. These findings indicate that exposure to DPSCsPTX factors can overcome the time-delayed trait of resistance to paclitaxel. In the same setting, a comparison between DPSCsPTX -CM with that from bone marrow MSCs and adipose derived MSCs, under the same conditions on MCF7 cells, with the viabilities after the 24h PTX priming period (Fig. 4C), has shown comparable effect with no significant difference (Fig. 4D). This indicates that DPSCs can be ulterior source for the therapeutic approach but does not indicate necessarily their superiority at these studied conditions. Regression analysis and comparison with PTX alone confirmed the high potency of DPSCsPTX-CM treatment on both cell types (see Annex Fig. A3). The comparative results

showed a higher inhibitive ability of the DPSCsPTX-CM compared to high drug dose alone (see Annex Fig. A3). CM from DPSCsPTX was the only treatment capable of producing potent effect on resistant cells, on day 2, compared to DPSCs alone or PTX alone (see Annex Fig. A4).

Raman spectroscopic method confirmed the delivery of 5FU and OXP drugs in cancer cells incubated with CM, shown as a big cluster in MCF7 cells (Fig. 5A) and a small cluster in HCT116 (Fig. 5B), respectively. DPSC mitochondria as initiation of apoptosis, directly following 12h-priming, and after the release of PTX [11]. This can confirm too our suggestion that the drug uptake or release does not affect the cell viability, but rather cause their proliferation impairment. We suggest that the 12h priming period is not high enough to induce an effect on viability after 7 days, meaning that the clinical observation of DPSC reduction in the number after priming was only a result of suppression of growth. Our results also suggest that paclitaxel may cause reversible damage to some DPSCs and are in accordance with other published research [7].

The migration tests performed in our study are aimed to measure the intrinsic migration property of the DPSCs when loaded with paclitaxel, to check any change in their motility when exposed to PTX.

In a first test, we calculated the gap closure per surface area, driven by the co-culture with conditioned medium through horizontal expansion of adherent cells. In previous reports, it was found that cell confluency when the cells are attached did not affect cell migration rates [12]. An error margin could be produced when fixing the ring by fleeing of the different cell types outside/ inside the ring. To suppress that in our experiment, we did not do quantification at time-point (T0), but we opted to quantify the whole ring gap plus within the ring (interior), to perform standardized comparison. We used the data analysis to determine the amount of cell growth and/or migration within the gap or empty area. The error margin was considered equal in all wells (generated by same individual), and especially as the analysis was not limited to selected positions. In contrast to the previous method [12], analysis was made for the full gap by an automated method, and also two different types of cells were used as a variation proposed by the previous protocol.

In transwell migration test for 7 days, results from separate cell counts of DPSC and MCF7 in transwell suggest that equal or higher migration may be contributed to DPSCsPTX, providing the same number of cancer cells. We here point out that our earlier results of MMP1 upregulation may result in an increased cell migration activity. A study comparing the expressions of several genes in the MSC showed that the increase in MMP1 expression was associated with highly-migrating MSCs, highlighting the important role of MMP1 in addition to MMP3 in MSC migration [34]. Furthermore in the literature, the direct interaction between cancer cells and MSCs were examined, showing that MSC migration increased in the culture with cancer cells [35]. Cytokines activated by the cancer cells are responsible for many of the trophic effects reported for MSCs. For instance, the co-culture of MSC with cancer cells was found to increase MSC migration in a dose-dependent manner by significantly increased MSC secretion of MMP1 [35][36][37][38].

In addition, our results indicated the unchanged tumor tropism of DPSC when loaded with PTX and their suitability for drug delivery. However, our study focused on the impact of PTX loading on DPSC migration. Further investigations are also needed to understand the nature of DPSC migration towards cancer. The study was not inclusive in terms of DPSC differential motility to MCF7. DPSCs in both experiments were not starved before experiment (FBS was always maintained), and the starting cellcell ratio was relatively low (1 DPSC: 3 MCF7). An enhanced protocol for assessment will be by using MCF7-CM without supplement for migration testing. Those are two factors that may be addressed to seek a significant change in DPSCs (and DPSCsPTX) migration toward cancer. The homing and penetration of DPSC cells into 3D spheroids was not impaired. It is not weird because the mesenchymal stem cells and their exosomes have tropism related to tumor hypoxia as a potent mediator directing MSCs' migration [39]. MSCs can migrate towards a tumor, by responding to tissue damage, hypoxia and inflammation, as found in tumor microenvironments [40]. They are attracted to several growth factors, cytokines and proteases existing in the tumors [41]. The cytokine production differs in 2D versus 3D cancer cultures [42]. This may indicate the higher prominence of cell-based cancer therapy in direct 3D co-culture models (and in-vivo tissue similarly), as compared to monolayer, indirect culture, or cell-free therapy types. The resistant cancer cells that we use demonstrate a model for time-delayed resistance [10], thus may suggest a lower dynamic of cytokine secretion (more resistance) and therefore result in slow cytokine-mediated penetration of DPSC±PTX cells until day 9. In a unique study (2018), DPSC homing was confirmed following radiation cancer therapy onto an injured tissue in rat model, adding that most patients with acute esophageal injury originally had a malignant tumor [43]. This study suggested that the cells may release a malignant signal or chemical substance that stimulates DPSC homing and engraftment through the blood circulation [43]. However, the experimental duration of this study was too short (28 days) to identify long-term complications of DPSC transplantation, including any carcinogenic changes [43].

Finally, the effect of CM from pre-primed DPSC was very potent, even at 1% CM concentration. it was noted earlier that the kinetics of incorporation and release of PTX by bone marrow MSCs has a higher trend of drug release during the first 24h of incubation [15], hence our choice for drug priming was between 12h (mainly for DPSCsPTX) or 24h (for other cells/ drugs comparison) in the different experiments. However, some hydrophilic small molecules or microvesicles have shown inefficient bioavailability due to poor cell membrane permeability [44]. Namely, the cellular uptake of small molecule drugs should be regarded [44], and this could account to the lower effect on HCT116 cells, for all CM+drug types. HCT116 cells may require a more prolonged exposure with drug (more concentration or time) to elicit appreciable responses. A comparable effect of CM from DPSCsPTX compared to other MSC sources is considered promising outcome for future application of these cells, considering their ease of isolation. Further in-depth studies are needed for comparatively determining whether special MSC sources are putatively more potent in cancer treatment in terms of their efficacy and safety.

As a conclusion, our evidence agrees with literature data which demonstrated that MSCs exposure to PTX reduce the cells' proliferation activity, 2D migration ability, and maybe differentiation potential, without significantly affecting their viability. Some studies in literature have discussed the effect of other cytotoxic drugs on DPSCs [45][46][47][48]. Our study is the first one -to the best of our knowledge-that addresses the DPSC's migration toward cancer cells, although the differential ability is not clearly evident. The migration of drug-exposed DPSCs was never tackled in the literature as well. Importantly, apart from paclitaxel effect on MSC proliferation and phenotype, in-vitro analyses of the chemotherapy influence of the treatment on the stem cells functional characteristics like cell motility do not take into account the potentially-relevant influences of the in-vivo microenvironment and the stem cells' interaction with other cell types.

To sum up, these functional properties should be investigated as key requirements for the stem cells' potential use in regeneration of chemotherapy-induced tissue damage, such as cell adhesion and multipotent differentiation. Generally, no alteration to stem cell surface markers (stemness) or to cytoskeleton structure had been highlighted by other studies, but an association of functional impairment of the cytoskeleton was indicated [7]. MSCs were proven to retain their stem cell characteristics after their recovery from chemotherapeutic treatment [7,[49][50][51][52][53]. As PTX is widely used in clinical practice (for treating breast, colorectal, ovarian, lung, gastric, bladder, and head and neck cancers), attention should be paid to the dental pulp stromal damage caused by this and other similar agents. The DPSCs used in this study are extracted from young-aged patients' teeth. Although the rate of doxorubicin absorption by all aged MSCs followed the same trend, lower MSC age is found to be associated with higher doxuribicin resistance, as referred to in one study [54]. Allogenic transplantation of stem cells derived from young donors are considered to potentially overcome agerelated limitations [55], thus emphasizing on the allogenic banking of DPSCs for future cell-based applications in cancer therapy [START_REF] Chaubron | Allogenic banking of dental pulp stem cells for innovative therapeutics[END_REF].

Conclusions

Our study significantly improves the knowledge on DPSC mechanisms, opening interesting perspectives for further investigation on primary dental pulp derived stem cells for cancer therapy. Our study provides a proof-of-mechanism and important investigations for the metabolic functions of recovery (and detoxification) related with the MSCs role in cell therapy, and also the mechanism related to taxol resistance.

In summary, there are numerous chemotherapeutics with different modes of action. The choice of appropriate therapy priming of DPSCs should be customized for patients, according to the precise determination of the tumor type and stage of disease, to allow decreasing cancer growth, resistance, or metastasis. On the other hand, the drugs that we tested for DPSC priming are, commonly administered as multidrug therapy for oral cancers. Taxanes, platins, and fluorouracil (TPF), in standard combination or monotherapy are used for the head and neck tumors [START_REF] Rapidis | Docetaxel in the treatment of squamous cell carcinoma of the head and neck[END_REF][START_REF] Lowe | Taxane, platinum and 5-FU prior to chemoradiotherapy benefits patients with stage IV neck node-positive head and neck cancer and a good performance status[END_REF]. DPSC priming and delivery efficacy for these drugs were tested for a perspective application in future trials.

[ PRELIMINARY STUDY : THERAPEUTIC NANOPARTICLES ]

In this thesis, we evaluated the combination of DPSCs and chemotherapeutic agents for treating cancer.

The approach of DPSC priming with therapeutics is a new promising approach, utilizing the unique properties of these cells for obtaining the targeted therapy goal for cancer treatment. The thesis discussed the role of DPSCs themselves in the delivery application and cancer therapy, provided incorporation with physic-technical aiding methods for the resolving of the newly developed therapies, suggested an understanding of the DPSC-based chemoresistance and interaction with both cytotoxic agents and cancer cells, and detailed the use of combination therapy by DPSCsPTX for breast cancer treatment.

The thesis was set up to " evaluate results by considering cells, secretome, and 3-dimensions as an integrated whole." This added complexity to our in-vitro studies, as careful attention should be paid for the setting-up of the experiments according to the specific aim, and during the interpretation of data.

1. General Conclusions

The main general conclusions that can be derived from the research work are :

-The use of paclitaxel in cancer therapy is hampered by the dose-limiting toxicities before having sufficient anti-tumor efficacy. DPSCs have the ability to nurse to cancer cells as shown for the first time through our results. It is hypothesized that the penetration of tight stroma and transavation by stem cells require a level of flexibility and deformability that is considerably different from that observed in healthy somatic cells. The role of mechanics and efficiency of stem cell delivery systems can be highlighted under the motivating hypothesis that specific types of stem cells are more suitable for certain applications depending on their properties.

-Generally, the mechanics of cells mimics the mechanics of tissues of origin and consequently, the inherent mechanics of certain DPSCs can be advantageous for the techniques for transplantation and migration of stem cells; DPSCs are released from the dental pulp and normally migrate in response to tissue damage or inflammatory stimulus. Based on their inherent functions in the tooth structure, DPSCs have already proved promising preclinical potentials in the field of regenerative medicine.

-The thesis has tested the differential ability of DPSCs to hamper the proliferation of cancer cells.

DPSCs have a history of very promising regeneration potential in many fields of dental and other tissues. In this thesis, we have proved that the DPSCs in distance had an intrinsic in-vitro regeneration potential while, on the other hand, they can be simply conditioned by co-culture with cancer cells "cancer-dependent DPSC conditioning" to inhibit cancer.

-The context "employment of DPSCs" emerged throughout our addressed investigations, where

DPSCs have unique tumor inhibition properties only when co-injected with cancer cells. This potential differential ability of DPSC in cancer therapy will be very interesting in their application.

-Engineering of MSCs with therapeutic drugs is necessary as it offers a safer and more efficient cancer therapy as compared to the unstable and heterogenous non-engineered cells. The significant challenge is then to efficiently deliver the desired therapeutic agent into DPSCs to generate cells with a high payload, high viability, and yet without inducing harmful phenotypic changes, all together for the aim of increasing their killing potency.

-In addition, drug-loading approach is a potential alternative to the genetic method of producing therapeutically-engineered MSCs, forwarded by the need for less preparation, less system complexity, and less cost when compared to other approaches.

-Breast cancer is addressed in the thesis as the main malignancy in women. Also, the autologous graft is possible in case of bad prognosis cancer in young age women, being the main indication for stem cell therapy application (at least in the early steps of novel therapy development).

-In contrast to the classical use of fluorescent tags for drugs or the common separation methods for detecting intracellular drugs, Raman microscopy as a non-labeling non-invasive technique of high sensitivity and resolution (300 nm), was used as a unique tool in the research for obtaining numerous detailed information.

2. Summary of Main Findings

From the limitations of the four studies, the following main findings could be drawn :

-Unlike some previous reports of reduced migratory properties of MSCs exposed to drugs, no significant change in tumor tropism was observed through our experiments. This property is particularly relevant to the use of primed DPSCs for targeting cancer. MSCs showed endogenous tropism to cancer cells' conditioned medium.

-Our tests collectively have shown big evidence that the drug bioavailability is improved by the DPSC incorporation (most likely DPSC secretome) in direct contact with cancer. The synergistic effect revealed both superior and faster efficacy.

-It is not enough to study only the "drug-primed DPSCs" to prove their effectiveness.

Unloaded DPSCs have also shown inhibitory functions when in direct contact with cancer cells. The unloaded cells were investigated in the aim of recovered DPSC functions after the release of the therapeutic drug (after 3 weeks), even if partial in-complete recovery toward normal function was obtained. Also, it is not guaranteed that 100% of the stem cells will upload the drug (a percentage of unprimed DPSCs will be present).

-The thesis has pointed out to the context of "naïve DPSCs" and "committed DPSCs " which appeared clear through our investigations. The direct interaction with cancer cells contributed to modulated cell functions and activity. The thesis has also tackled the issue regarding the means of DPSCs delivery : by injection (systemic) or by transplantation (co-injection) directly to cancer. In our study, we emphasize the DPSC-cancer cell contact and interaction (maximized crosstalk) as the main known route of DPSC inhibitory efficacy against breast cancer.

-DPSC factors have contributed to overcoming chemotherapy resistance, a huge obstacle in the way of cancer treatment. Under very low chemotherapy doses, an important synergistic effect has resulted in high efficacity against resistant cells.

-The potential clinical application of Raman spectroscopy as a companion diagnostics (CD) tool for distinguishing the cancer and stem cells, and also for detecting mechanisms of cellular response to drugs (or any exogenous agent) through the search for specific "biomarkers" based on a non-labeled approach, incorporated with other cellular assays.

3. Perspectives and Recommendations

The following perspectives and recommendations from the thesis can be forwarded :

- -The risk of the malignant transformation of DPSC after their therapeutic payload was not decisive in the thesis, and further experiments should be addressed. The heterogeneity of the cells present in tumors and their plasticity with the epithelio-mesenchymal transformation (EMT) of cancer cells require great caution regarding the use of DPSCs with the tumors (in-vitro assays on cell lines in the presence of FBS (non-GMP condition)).

-The ability to differentiate and maintain regeneration abilities during cancer treatment should be also further investigated. DPSC stem cell markers should be checked according to the ISCT criteria (international society for cellular therapy). Further tests of the paracrine effect of DPSC in co-culture are needed to be put in evidence, for the safety of their employment.

-Even if it was not directly tackled, based on our results, the DPSC proliferative effect on the nonmalignant cells is expected and needs to be further investigated on healthy breasr cells, for instance.

In an earlier study, it was suggested that the utility of MSC to initiate apoptosis was specifically aimed at breast cancer cells, but not at the normal cells (Chao et al., 2012).

-In a promising perspective, the same stem cell transplant can be used for cncer post-construction therapy, as a diffuse procedure now for breast tissue construction. New research is directing towards the use of stem cells as both delivery vehicles for cytotoxic (or generally therapeutic) delivery as well as making use of their regenerative abilities after the delivery step [START_REF] Scioli | Adipose-derived stem cell-mediated paclitaxel delivery inhibits breast cancer growth[END_REF]. This can be regarded in anticipation for firstly post-surgical tissue reconstruction and secondly as a preventive measure from dormant cancer recurrence or activation.

-A secretome profiling of DPSCs is strongly stressed out in conditions of indirect/ direct co-culture.

Due to the changed tendancy with the interaction with cancer cells, the hypothesis of contactdependent secretome modulation should be evidenced. Therefore, before envisaging the transfer of the data obtained to the human clinic, it will be necessary to precisely determine the chronology and the relationships between the environment of the tumor cells (roles of cytokines due to chronic inflammation… etc.) and the pulp stem cells. Similarly, the question of the immunosuppressive properties of DPSC on tumor development must be asked by in-vivo testing.

-The cancer dependent MSC conditioning represents another potentially clinically-relevant variable to be considered when DPSCs are anticipated as therapeutic agents. DPSC exosomes or extravesicles (DPSC-EVs) conditioned by co-culture with cancer can be also investigated as safe vehicle tools when the cytotherapy is contraindicated. The modified EVs from DPSCs can be used as a selective drug delivery system -It was suggested by our study that the downregulation of MMP1 in DPSCs may be leading factor to reduced apoptosis and increased cancer cell proliferation. Further tests are needed to imply this hypothesis based on (Zhao et al., 2017) findings.

-The drugs tested in incorporation with DPSCs can be for breast cancer, colorectal cancer, and also can be tested for the head and neck tumors (perspective application).

4. Research Openings

Further subject openings can be generally overlooked:

-Currently, paclitaxel is effective for various human diseases, such as neurodegenerative diseases, different cancers, and chronic kidney disease [START_REF] Kitasato | Clinical outcomes of chronic kidney disease patients treated with everolimus-eluting stents (EES) and paclitaxel-eluting stents (PES)[END_REF][START_REF] Meng | Prodrug strategies for paclitaxel[END_REF][START_REF] Niwa | Safety and efficacy of carboplatin plus nab-paclitaxel for treating advanced non-small-cell lung cancer with interstitial lung disease[END_REF]. The delivery approach of DPSCsPTX can be generalized to different tissues besides the different cancer types.

-A new potential for primed MSCs for the delivery of therapeutics is tested towards treating the novel coronavirus disease [START_REF] Jeyaraman | Mesenchymal Stem Cells (MSCs) as a Novel Therapeutic Option for nCOVID-19-A Review[END_REF][START_REF] Muraca | Mesenchymal stromal cells and their secreted extracellular vesicles as therapeutic tools for COVID-19 pneumonia?[END_REF]. The COVID-19 pandemic, designated as a public health crisis, has started in November 2019 and to this day is rapidly spreading around the world. The acute respiratory distress syndrome and tissue damage resulted from SARS-CoV-2 represent the most threatening complication of COVID-19 infection. Great interest has been devoted to the possible clinical applications of MSCs, their extracellular vesicles, and nanoparticles that convey much of the biological effects. Currently, several studies are in clinical trials, including of whom one trial recruited in Jordan, and others in different countries of the world. In a promising look, two of these clinical trials employ the use of DPSCs [START_REF] Raza | Primed'Mesenchymal Stem Cells: a Potential Novel Therapeutic for COVID19 Patients[END_REF]. Mainly the immunomodulatory regenerative potential of MSCs could be a novel application to improve the responses of COVID-19

patients.

-Finally, in order to be useful for downstream translational efforts, this priming methodology must be scalable in production: improved priming efficacy, DPSC bio-banks, and scale-up production. In our approach, there is relatively no need for conditioning preparation procedures, for instance, centrifugations or other cell treatments. However, the reparative senescence for DPSCs, which is pointed out in our study and confirmed in the literature, may limit their high scalability (scale-up production). In a wider perspective, given the foreseeable needs of the application DPSC cytotherapy, it can be proposed the development of an automated cell factory manipulators (with high layer expansion vessels) and microcarriers-based bioreactor culture systems for scale-up production. New research has begun to seriously find solution routes in this regard.

-From a clinical dental clinical point-of-view, cells during the tooth eruption phase undergo higher expression of certain factors to aid the process. MMP1 is one factor that was found highly expressed in the eruptive pathway of rat molars (de Pizzol Júnior et al., 2018). The study further concluded that MMP1 enzymatic failure during the eruptive pathway may disturb tooth eruption. During periods of accelerated growth, MSC cells are instantly activated and replenished. Advancements in the understanding of this have provided novel insights into the dental stem cells niche to benefit clinicians with more specifically marked MSC subpopulation in the human tooth for successful tissue regeneration [START_REF] Shi | Pulp stem cells derived from human permanent and deciduous teeth: Biological characteristics and therapeutic applications[END_REF]. However, the majority of these markers are known from in-vitro propagation studies and they do not reflect the properties of DPSCs under physiologic condition or reparative process. Dental pulp stem cells may also be affected at the physiologically stressful conditions through some phenotypic changes. Raman spectroscopy analysis can be useful to determine level changes in incorporation with other invasive techniques.

APPENDIX B : PROOF-OF-MECHANISM (1)

Sorting of transduced living cells using FACS

For the preparation of FACS experiment, the cells were trypsinized and resuspended in PBS, and then kept at 4 °C until sorting. Propidium iodide PI (Sigma-Aldrich) was added to the staining tube in the last step, with a final concentration of 0.1 µg/mL for live cells, to facilitate the elimination of dead cells. Incubation with PI was 15 minutes in the dark followed by washing. All the samples were filtered through test tubes with 40μm nylon mesh cell strainers (Corning Falcon) to minimize cell aggregation within the samples and the analysis is performed within an hour. For sorting live cells, we used the BD FACS Melody cell sorter coupled with FACSchorus software (BD Biosciences, CA, USA). Following the sorting of both cell types, GFP/RFP expression was analyzed. The purity of subpopulations after sorting was confirmed by analyzing post-sort samples in the sorter again, reaching a purity of above 95%. For the establishment of a stable culture, cells were maintained in growth medium containing the selective dose of puromycin until the start of experiments.

(B) Figures in Annex

Figure 1 .

 1 Figure 1.1 : Main categories of stem cells ...........................................................................................

1 :

 1 Figure 1.1 : Main categories of stem cells ...........................................................................................

Figure 1 . 2 :

 12 Figure 1.2 : Non-genetic bioengineering solutions to boost the functions of DPSCs .........................

Figure 1 . 3 :

 13 Figure 1.3 : Schematic diagram describing the role of chemotherapy and biological combination cancer therapy-responsive and chemotherapy resistant types ............................................................

Figure 1 . 4 :

 14 Figure 1.4 : Overview of MSCs and tumor cells interaction as an approach for cancer therapy ........

Figure 1 . 5 :

 15 Figure 1.5 : Hypothetical mechanism of action of nanoengineered MSCs .........................................

Figure 1 . 6 :

 16 Figure 1.6 : Confocal microscope setup ..............................................................................................

Figure 1 . 7 :

 17 Figure 1.7 : Protocol for isolation and culture of DPSCs from third molars .......................................

Figure 1 . 8 :

 18 Figure 1.8 : Lentiviral transduction of RFP-DPSC (transient study) ..................................................

Figure 1 . 9 :

 19 Figure 1.9 : WITec alpha 300R confocal Raman microscope (LBN-EA4203) ..................................

Figure 1 . 10 :

 110 Figure 1.10 : Schematic outlining the key steps in the K-means clustering analysis algorithm..........

Figure 1 . 11 :

 111 Figure 1.11 : Flowchart of Pearson correlation coefficient analysis ...................................................

Figure 2 . 1 :

 21 Figure 2.1 : Schema representing the experiment (CM-Raman) steps. ...............................................

Figure 2 . 2 :

 22 Figure 2.2 : DPSC's uptake of Paclitaxel ............................................................................................

Figure 2 . 3 :

 23 Figure 2.3 : MCF7 uptake of Paclitaxel ...............................................................................................

Figure 2 . 4 :

 24 Figure 2.4 : Drug to cells ratio for long versus short paclitaxel incubation .........................................

Figure 2 . 5 :

 25 Figure 2.5 : Average of ratios (drug-to-cell pixels ratio). ....................................................................

Figure 3 . 1 :

 31 Figure 3.1 : Viability assay results on control and drug-treated cells. .................................................

Figure 3 . 2 :

 32 Figure 3.2 : Predominant bands in the Raman spectra .........................................................................

Figure 3 . 3 :

 33 Figure 3.3 : Paclitaxel uptake by DPSC (Step 1) .................................................................................

Figure 3 . 4 :

 34 Figure 3.4 : Two DPSCs after 4h-release of PTX (Step 2) ..................................................................

Figure 3 . 5 :

 35 Figure 3.5 : The viability of DPSC after releasing paclitaxel ..............................................................

Figure 3 . 6 :

 36 Figure 3.6 : MCF7 after paclitaxel (PTX) uptake from CM released by DPSCs (Step 3) ..................

Figure 3 . 7 :

 37 Figure 3.7 : MCF7 in buffer solution (Step 3) .....................................................................................

Figure 4 . 1 :

 41 Figure 4.1 : Localization of intracellular MMP1 in DPSC cell, as detected by confocal Raman spectroscopy with Pearson correlation analysis. ................................................................................

Figure 4 . 2 :

 42 Figure 4.2 : Raman spectroscopy images of MMP1 Correlation in DPSC .......................................

Figure 4 . 3 :

 43 Figure 4.3 : Correlation of MMP1 in DPSC, correlated to MTT cytotoxicity test.. ..........................

Figure 4 . 4 :

 44 Figure 4.4 : Cell different resistance patterns to PTX chemotherapy ................................................

Figure 4 . 5 :

 45 Figure 4.5 : MMP1 intracellular ratio comparison between cells of three resistance traits to paclitaxel ............................................................................................................................................................

Figure 4 . 6 :

 46 Figure 4.6 : Comparison of Raman method of spectral correlation with MMP8 isoform .................

Figure 5 . 1 :

 51 Figure 5.1 : DPSC resistance to different chemotherapeutic drugs, and recovery following exposure to PTX ................................................................................................................................................

Figure 5 . 2 :

 52 Figure 5.2 : Impact of PTX on DPSCs migration ..............................................................................

Figure 5 . 3 :

 53 Figure 5.3 : Homing ability of DPSCsPTX into 3D spheroids ..........................................................

Figure 5 . 4 :

 54 Figure 5.4 : Effect of short term DPSCsPTX-CM on MCF7 and its resistant-to-PTX subline .........

Figure 5 . 5 :

 55 Figure 5.5 : Delivery of different ant-cancer drugs by primed DPSC-CM .......................................

Figure 6 . 1 :

 61 Figure 6.1 : Effect of direct 2D co-culture of DPSCs±PTX and MCF7 ............................................

Figure 6 . 2 :

 62 Figure 6.2 : Cytometry and morphology analysis of MCF7 cancer cells co-cultured with DPSCs. .

Figure 6 . 3 :

 63 Figure 6.3 : MCF7 cancer cell spheroids proliferation after several days of treatment.....................

Figure 6 . 4 :

 64 Figure 6.4 : Effect of indirect co-culture of DPSCs±PTX and MCF7 ...............................................

Figure 6 . 5 :

 65 Figure 6.5 : Paracrine effect of DPSCs±PTX on the dissemination of MCF7 spheroids ..................

Figure 6 . 6 :

 66 Figure 6.6 : Effect of co-culture of DPSCs±PTX and MCF7 resistant-to-PTX ................................

Figure 7 . 1 :

 71 Figure 7.1: Overview of Co-metal nanoparticles of 5nm diameter ...................................................

Figure 7 . 2 :

 72 Figure 7.2 : M-H hysteresis loop measured for Co nanoparticles .....................................................

Figure 7 . 3 :

 73 Figure 7.3 : Optical images of agglomerated Co MNPs studied by Raman spectroscopy ................

Figure 7 . 4 :

 74 Figure 7.4 : Raman signature of Co MNPs based on their localization .............................................

Figure 7 . 5 :

 75 Figure 7.5 : MCF-7 cells incubated with Co nanoparticles ...............................................................

Figure 7 . 6 :

 76 Figure 7.6 : Raman reconstructed images showing Co MNPs inside MCF7, HCT116, and DPSC cells ............................................................................................................................................................

Figure 7 . 7 :Figure 8 . 1 :

 7781 Figure 7.7 : Toxicity test of cobalt metal nanoparticles on (a) HEK-293 cells, and (b) PPC-1 cells

Figure A1 :

 A1 Figure A1 : Raman spectrum of intracellular PTX in stem cell. .......................................................

Figure A2 :

 A2 Figure A2 : DPSC immediately after priming with PTX 10µM 12h.................................................

Figure A3 :

 A3 Figure A3 : MCF7 after 3h with 4h-CM (DPSC+PTX 10µM for 10h) .............................................

Figure A4 :

 A4 Figure A4 : MCF7 after 3h with 4h-CM (DPSC +PTX 10µM for 15h) ............................................

Figure A5 :

 A5 Figure A5 : Diagram representing early and later signs of apoptosis. ...............................................

Figure A6 :

 A6 Figure A6 : MCF7 after 3h with 4h-CM (DPSC +PTX 100µM for 1-2h) ........................................

Figure A8 :

 A8 Figure A8 : Methodology for PTX quantification in the above MCF7 cell scans. ............................

Figure B1 :

 B1 Figure B1 : Methodology of calculation of the MMP1/ cell ratio in DPSC cells. .............................

Figure B2 :

 B2 Figure B2 : Comparison of chemical structure of isoforms of MMP collagenase family. ................

Figure C1 :

 C1 Figure C1 : Optical images of DPSCs for morphological assessment after PTX treatment..............

Figure C2 :

 C2 Figure C2 : Proliferation of DPSC and MCF7 cells separately in transmigration chambers. ...........

Figure C3 :

 C3 Figure C3 : Linear regression analyses for high PTX dose and DPSCsPTX-CM effect ...................

Figure C4 :

 C4 Figure C4 : Inhibition effect of DPSC±PTX-CM compared to high PTX dose ................................

Figure D1 :

 D1 Figure D1 : Effect of direct 2D co-culture of DPSC±PTX and MCF7 for 5 and 7 days ...................

Figure D2 :

 D2 Figure D2 : Evolution of MCF7 spheroids as control conditions ......................................................

Figure D3 :

 D3 Figure D3 : Effect of indirect co-culture of DPSCs±PTX and MCF7 ...............................................

Figure D4 :

 D4 Figure D4 :Apoptosis detection in MCF7 cells incubated with the short-term (4-h) CM .................

Figure D5 :

 D5 Figure D5 : Effect of indirect co-culture of DPSCs±PTX and paclitaxel-resistant MCF7 ...............

Figure D6 :

 D6 Figure D6 : Effect of the inhibitive DPSC±PTX-CM on paclitaxel-resistant cancer cells ...............

Sheet 1 :

 1 Paclitaxel (PTX) drug information sheet, drug bank .....................................……..…......220 Sheet 2 : Fluorouracil (5FU) drug information sheet, drug bank .............................….………........221 Sheet 3 : Oxaliplatin (OXP) drug information sheet, drug bank ............................…...……..…......222 Sheet 4 : Cobalt nanoparticle (Co-NP) information sheet, Pro1nano ..................…………..…......223 Sheet 5 : Lentivirus (Cignal) information sheet, Qiagen.......................................…………..…......224

  l'utilisation d'une nouvelle méthode comme le microscope Raman confocal pour l'analyse multi-étape au cours du projet. La spectroscopie Raman est une méthode d'analyse non-destructive basée sur l'interaction photonmatière, permettant d'identifier les vibrations des molécules. Chaque molécule a ses modes vibratoires uniques : sa signature caractéristique. Ainsi, cette technique fournit des informations chimiques à partir des vibrations des molécules après leur excitation par un laser monochromatique. Par la suite, les spectres sont obtenus pour permettre une cartographie chimique différentielle de la zone de l'échantillon analysé. Cette thèse est composée de quatre parties principales de l'étude du Raman, comme étapes de quatre enquêtes principales : I-Étude de l'absorption et de la libération de médicaments par la méthode Raman : trois classes différentes de médicaments anticancéreux (chapitre 2, 3, et 5) ; II-Étude de l'effet pro-apoptotique du paclitaxel sur les cellules DPSC et de notre traitement sur les cellules cancéreuses, mesuré par translocation du cytochrome-C (chapitre 3) ; III-Étude de la pathologie et de la résistance induites par le paclitaxel dans les DPSC et les cellules cancéreuses par détection intracellulaire de la métalloprotéinase matricielle (MMP-1) : une étude comparative (chapitre 4) ; IV-Étude préliminaire de l'absorption des nanoparticules métalliques (MNP) par les DPSC et les cellules cancéreuses (chapitre 7). * * * * * * * * * * D'une manière générale, l'axe principal de notre étude peut se résumer en trois points principaux : (1) l'interaction entre les cellules souches de la pulpe dentaire (DPSC) et les médicaments anticancéreux, notamment le paclitaxel, (2) l'efficacité de l'approche thérapeutique combinée "DPSC+médicament" et la compatibilité pour l'application clinique, et (3) le sort des DPSC après le traitement (la transportation) avec les perspectives possibles. Cette thèse est composée de quatre parties principaux (Résultats), en tant qu'étapes du développement d'une nouvelle thérapie à base de cellules souches de la pulpe dentaire (DPSC), nongénétiquement amorcées. Les chapitres répondront à quatre questions : Premièrement, les Preuves du Principe pour l'absorption et la libération de médicaments anticancéreux par les DPSC : Études POP (contenant les deux premiers articles, chapitres 2 et 3); Deuxièmement, les Preuves des Mécanismes de l'application des DPSC comme vecteur d'administration de médicaments : résistance, migration, et délivrance de trois médicaments: Études POM (contenant les deux articles suivants, chapitres 4 et 5) ; Troisièmement, les Preuves du Concept de l'effet thérapeutique des DPSC amorcées par le paclitaxel sur le cancer du sein : Études POC (contenant un article, chapitre 6) ; Quatrièmement et finalement, l'application prometteuse des DPSC amorcées par des nanoparticules pour le traitement du cancer : Étude préliminaire (un article, chapitre 7).

  ) Comme thérapeutique cellulaire : par le sécrétome, vésicules, les facteurs cytotoxiques et apoptotiques libérés de la DPSC qui peuvent traiter des cancers et peuvent possiblement être conditionnés pour optimiser leurs fonctions inhibitrices.(3) Comme thérapie ciblée : par la migration vers le tissu cancéreux (en tant que tissus lésés non cicatrisés) basée sur les facteurs chimiotactiques libérés par les cellules de cancer. Celui-ci vise à réduire les effets secondaires de la chimiothérapie systématique.Nous effectuons donc l'amorçage des DPSC par des médicaments anticancéreux, en essayant d'améliorer la fonction de ces cellules pour la thérapie anticancéreuse. Trois types de préconditionnement de DPSC ont été montrés tout au long de notre projet : par l'amorçage de petites molécules (trois médicaments anti-cancéreux), par l'amorçage de nanoparticules (sur base du métal cobalt), et le conditionnement par co-culture avec les cellules cancéreuses cibles. Les cellules de la pulpe dentaire en tant que source non-invasive de cellules souches n'ont pas été vérifiées dans la littérature dans le cadre de l'administration ciblée de médicaments chimio-thérapeutiques. Dans ce projet, une technique unique "Microscopie Raman confocale" d'imagerie biochimique de l'absorption des médicaments aiderait à mieux comprendre le mécanisme d'action des médicaments et la localisation cellulaire par analyse spectrale sans marquage.

Figure 1 :

 1 Figure 1: Main categories of stem cells. Embryonic stem cell (ESC), induced pluripotent stem cell (iPSC), adult stem cell (ASC), hematopoietic stem cells (HSC), and mesenchymal stem cell (MSC) of dental and orofacial origin: orofacial-bone-marrow (OBMSC); dental-pulp (DPSC); exfoliated deciduous teeth stem cell (SHED); periodontal-ligament (PDLSC); dental-follicle (DFSC); adult pulp (APSC); oral-epithelium (OESC); gingival (GSC); salivary-gland (SGSC); adipose tissue (ATSC); Schneiderian-membrane (SMSC);

  , immunomodulatory, and tumor homing properties of MSCs have been exploited to repair tissue injuries, interfere with cancer, as well as immune based disorder and neurodegenerative disease development. This large use was also sustained by the relative easiness of harvest and processing from different sources, and the abundant availability after in vitro expansion. Beyond regenerative medicine, MSC tumor tropism and non-immunogenicity laid the groundwork for their application in oncology research. In particular, MSCs release soluble factors or extra-vesicles (EVs) that acting via autocrine and paracrine mechanisms can modulate cancer cell survival and proliferation, migratory pathways, and induce host immunomodulation. Despite initial enthusiasm, current literature highlights growing data inconsistencies and divergences on whether and how MSCs may promote or inhibit tumor growth in various cancers, including breast cancer. Several factors may contribute to contrasting preclinical observations, including MSC tissue of origin, isolation, ex vivo expansion, and culture protocols. Moreover, further complications may derive by the clinical setting, tumor type, administration route, timing, and quantity, often questioning the effective number of cells homing to the tumor site. All these crucial issues, hampering the research reliability and clinical improvement of MSC based therapies, should be addressed by in depth studies on the biological and molecular properties of these cells.

Figure 2 :

 2 Figure 2 : Non-genetic bioengineering solutions to boost the functions of DPSCs. (A) Priming DPSCs with small molecules is a simple and promising approach to induce the secretion of therapeutics, but the effect of small molecules (ex. drugs) is relatively short-lasting. (B) DPSCs can also be engineered with drug-loaded particles. These particles are intracellularly loaded into DPSCs to sustain their therapeutic efficacy for an extended period of time. However, particle preparation can increase the cost and complexity when compared

  Seifrtova, M., Havelek, R., Soukup, T., Filipova, A., Mokry, J. & Rezacova, M., 2013. Mitoxantrone ability to induce premature senescence in human dental pulp stem cells and human dermal fibroblasts. J Physiol Pharmacol, 64, 255-266. Tatullo, M., Marrelli, M., Shakesheff, K.M. & White, L.J., 2015. Dental pulp stem cells: function, isolation and applications in regenerative medicine. Journal of tissue engineering and regenerative medicine, 9, 1205-1216. Zhao, S., Zhao, Y., Guo, J., Fei, C., Zheng, Q., Li, X. & Chang, C., 2017. Downregulation of MMP1 in MDS-derived mesenchymal stromal cells reduces the capacity to restrict MDS cell proliferation. Scientific reports, 7, 43849.

  administration of chemotherapy without any interruptions (dose-dense)  Using a biological optimized dose  No application of any growth factors  Low incidence of treatment related side-effects  Potential for delayed development of resistance.

  Monoclonal antibodies (MAbs) that target tumor cells (e.g. expression oncogenes/ growth factor receptors), or target vascular endothelium to inhibit angiogenesis, or target inhibitory immune cell receptors to interfere with immune regulation ; II) Gene-modified immune cell therapy; these are autologous targeted (antibody-binding) cytotoxic T-lymphocytes, or therapeutic dendritic cell (DC) as antitumor vaccines for active specific immunotherapy ; III) Oncolytic viruses (OVs), which exhibit tumor selectivity and induce tumor cell death and oncolysis; these agents positively induce immunogenic cell death (ICD) by the patient's own immunity.

  Binding mechanism: breast cancer cell apoptosis from direct cell-cell contact with MSCs, at various ratios, and infusion of some substance into cancer cell by MSCs ; II) Cell-in-cell mechanism: breast cancer cell apoptosis from internalization of MSCs ; III) Indirect (cytokine) mechanism: attenuation of breast cancer cell growth from one or more cytokines secreted, predominantly, by co-cultured MSC and cancer cells or by MSC alone, without direct contact with cancer cells.

Figure 3 :

 3 Figure 3: Schematic diagram describing the role of chemotherapy and biological combination cancer therapy: responsive and chemotherapy-resistant types. Case-A: Cancer cells treated only with the chemotherapeutic agents induces less effective cancer cell death than when treated along with chemotherapeutic agents combined to DPSC, Case-B. Case-C: Chemo-resistant cancer cells treated only with the chemotherapeutic agents demonstrate no cells death due to chemo-resistance. Case-4: While chemoresistant cancer cells when treated with chemotherapeutic agents combined with targeted DPSCs causes massive cancer cells death. Idea reproduced from[START_REF] Chakraborty | The novel strategies for next-generation cancer treatment: miRNA combined with chemotherapeutic agents for the treatment of cancer[END_REF].

2 -

 2 Combination effect: MSCs and MSCsPTX release cytokine factors that induce cancer cells' apoptosis or death. They also release molecules that reduce cancer cell adhesion to endothelium, and therefore reduce cancer cell migration/ invasion, when in co-culture. 3-Possibility of overcoming chemotherapy drug resistance by the drug availability and/or MSC factors. 4-Targeted effects through the homing and migration abilities of MSCsPTX to cancer sites.

Figure 4 :

 4 Figure 4: Overview of MSCs and tumor cells interaction as an approach for cancer therapy. Upper left:MSCs intrinsic migration and homing property to cancer cells (tumor chemokine-dependent). Upper right: Non-modified MSCs pre-loaded with chemotherapeutic drugs. Lower right: Genetically-modified MSCs by prodrugs or oncogenic factors (black) or inhibitory miRNA (red). Lower left: MSC's tumor-inhibiting property, by intrinsic factors' secretion (killing cancer cells directly (cytotoxic factors) and indirectly through activation of immune effector cells and vasculature) or immune-modified MSCs by addition of immune factors(black), leading to immune cell tumor killing. Reproduced from[START_REF] Chulpanova | Application of mesenchymal stem cells for therapeutic agent delivery in anti-tumor treatment[END_REF].

Figure 5 :

 5 Figure 5: Hypothetical mechanism of action of nanoengineered MSCs. Once nanoengineered MSCs home to tumors, while free drug is effluxed out of the MSCs by efflux transporters, nanoparticles can enter the cells by endocytosis, escape into the cytoplasm, and release the drug inside the cells. The free drug inside the cells can be effluxed out into the surrounding tumor cells to elicit antitumor response. Reproduced from[START_REF] Cheng | Engineered mesenchymal stem cells for targeting solid tumors: therapeutic potential beyond regenerative therapy[END_REF].

1 .

 1 attention and patience are needed to promote the clinical translation of MSCs in tumor treatment.MSCs and their secreted exosomes have great potential for tumor therapy. Overall, it is clearly perceivable from the literature and data on the use of MSCs in oncology specifically, and beyond for other disease clinical trials, how hard is the comparison of the outcomes between different studies and striking controversies on the successful MSC delivery to target tissues. Meanwhile, to accelerate the transformation from preclinical research to clinical application, more efficacy and safety of these therapeutic approaches need to be provided by preclinical studies.Study of the art: DPSCs and cancerWhile the interactions of MSCs with tumor have been well-documented in the literature, scarce are the studies concerning the interaction between the dental pulp stem cells (DPSCs) and cancer cells[START_REF] Dogan | Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions[END_REF], Hanyu et al., 2019), and the effect of DPSC direct co-culture on cancer growth is very rarely analyzed.Dogan et al. have noted that DPSC increased cell proliferation and decreased apoptosis in prostate cancer cell cultures in direct/ indirect co-culture[START_REF] Dogan | Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions[END_REF].Hanyu et al.

  Raman spectroscopy technology in the last two decades have produced a range of Raman techniques suitable for biopharmaceutical applications. This includes Fourier Transform (FT)-Raman, dispersive visible Raman, chiral Raman, ultraviolet (UV) resonance enhanced Raman, surface enhanced Raman, confocal Raman microscopy as well as hand-held Raman devices. Each Raman technique has its unique advantages and disadvantages in terms of instrument sensitivity and suitability

Figure 6 :

 6 Figure 6: Confocal microscope setup (A) Raman microscope parts (B) Confocal aperture setup. Copyright © WITec.de

Figure 7 :

 7 Figure 7: Protocol for isolation and culture of DPSCs from third molars. (A) Pulp recovery: a stable finger support while using a diamond disc to create a 360˚ grove at 2 mm depth under the cemento-enamel junction. Notice, the molar may have open-ended apices. (B) Mechanical disruption: the crown was separated from the root (arrows) with minimum debris by wedging the chisel in the groove and applying gentle force with a hammer. (C) Chemical disruption: The exposed pulp tissue (arrow) was collected with a hemostat and Endodontic K-files and placed in 4°C culture medium with provided treatments. Selecting teeth with a large pulp chamber (arrow) ensured the removal of pulp tissue in one piece with minimal debris. (D) Expansion: Dental pulp cells formed visible colonies as viewed under an inverted light microscope (arrows). Reproduced from[START_REF] Alsulaimani | Isolation of dental pulp stem cells from a single donor and characterization of their ability to differentiate after 2 years of cryopreservation[END_REF].

Figure 9 :

 9 Figure 9 : WITec alpha 300R confocal Raman microscope (LBN-EA4203).

Figure 11 :

 11 Figure 11: Flowchart of Pearson correlation coefficient analysis. The correlation coefficient (r) is calculated through a specific formula (*) calculating the similarity distance between the spectra of each point in the cell scan(s) and the reference spectrum of the molecule in interest. Reproduced from the Ph.D. thesis of Dr. Hamideh Salehi.

Figure 1 :

 1 Figure 1: Localization of intracellular MMP1 in DPSC cell, as detected by confocal Raman spectroscopy with Pearson correlation analysis. (A) Raman reconstructed image of the C-H region, (B) Pearson correlation map of the MMP1 derived by MatLab, with high correlation MMP1 in the cytoplasm (yellow arrows) and nucleus (red arrows), and (C) the MMP1 reference spectrum.

Figure 2 :

 2 Figure 2: Raman spectroscopy images of MMP1 Correlation in DPSC with increasing PTX treatments. MMP1 reference spectrum is compared with the whole cell spectra, red pixels show the position of MMP1 correlated (30% threshold) inside the cells. For untreated DPSC (control), blue arrows show the few MMP1 pixels in the cell.

Figure 12 :

 12 Figure 12: Correlation of MMP1 in DPSC with increasing PTX treatments, correlated to MTT cytotoxicity test. (A) Ratio MMP1/cell (pixel/µm 2 ) is calculated for DPSCs treated for 1h with increasing PTX concentrations (1µM, 10µM, 100µM). Histogram shows significant increase of MMP1 ratio in cell when treated with considerable PTX concentration. Red line is logarithmic tendency curve. The results represent the mean ±SEM of triplicate cultures of multiple experiments. (B) Viability of the treated DPSC cells. The results represent the mean ± SD of triplicate cultures of one representative experiment.

Figure 413 :

 413 Figure 413: Cell different resistance patterns to PTX chemotherapy. DPSCs, MCF7 TAX19 , and MCF7 cells were incubated with 10μM PTX for up to 3 days. The results represent the mean ±SD of triplicate cultures of one representative experiment.

  DPSCsPTX having higher MMP1 in compared to their untreated group. The results represent the mean ±SEM of multiple experiments. (B) Raman scan of non-treated MCF7 cells, red pixels show the position of MMP1 correlated (10% threshold) inside the cells.

  figure 6C. Considering these investigations, the tested method is considered of relative high sensitivity and specificity to the target protein.

Figure 14 :

 14 Figure 14: Comparison of Raman method of spectral correlation with different MMP isoform from the collagenase family (MMP-8): (A) Spectrum and measured Pearson correlation of recombinant MMP8 in powder form. (B) Spectrum and measured Pearson correlation of recombinant MMP8 in solution form. (C) Pearson correlation map of the MMP1 and MMP8 in the same DPSC cell, with the MMP/cell ratio in the cell is about 3-fold higher (exactly 2.75x) for the MMP1 compared to that for MMP8.

3

  classical anticancer drugs belonging to 3 classes characterized by a unique cytotoxic activity. The first class, the anti-metabolites (5-fluorouracil), the second class includes platinum-based agents; they are DNA alkylating agents (oxaliplatin), and lastly are the anti-microtubules or antimitotic (paclitaxel).

  migrated cell count towards the cancer within the first 24 hours (short-term). Transwell migration assay is used to test DPSCsPTX migration after 7-days (long-term). Both tests are cytokine-induced indirect co-culture, driven by the horizontal (O-ring) or vertical (trans-migration test) movement of DPSC cells.

  4 cells/ mL by adding 200 µL/well. Cells aggregated and merged in 3D spheroids within 72 h, where spheroid diameter reached an average of 600 µm. On day 4 after initial seeding, 2000 RFP-labeled DPSCs±PTX were directly added to spheroids in microwells and was imaged for 1-3-6-9 days. Half volume of the culture medium was carefully changed for spheroids maintenance each two days. Two scans of wells, for spheroids and for RFP cells, were captured on each day with Celigo™ imaging cytometer (Nexcelom, Bioscience). RFP integrated intensity, measured at 0.5 mm around spheroid (50% well mask), were represented as an indication of DPSCs penetration into spheroids in three-dimensionality.

Figure 1 :

 1 Figure 1: DPSC resistance to different chemotherapeutic drugs, and recovery following exposure to PTX. DPSCs, MCF7, HCT116 cells were incubated with 10μM drugs: (a) 5FU, (b) OXP, and (c) PTX and then cultured for 7 days. (d) DPSCs were incubated with paclitaxel 10μM for 12h and then cultured for another 18 days. The y-axis shows the cell number, expressed as a percentage of (d0) control. The results represent the mean ±SD of triplicate cultures of two or three independent experiments.

  DPSCsPTX to that of unloaded DPSCs. Increased migration towards MCF7 was noted for both DPSCsPTX with no significant difference. As shown in figure2D, DPSCsPTX showed a comparable differential migration compared to unloaded DPSC group, which indicates basically an un-affected function. The analysis of differential migration had insufficient power to determine whether a particular MCF7 dose or schedule will produce statistical superior efficacy. Still, DPSCsPTX migration to MCF7 cells (and control) presented a statistically significant reduction in the number of cells (Fig.2E; 2F) compared to unloaded DPSC (p<0.05). The results (Fig.2F) are represented after standardization of total numbers of DPSCsPTX/ DPSC (equal to 55%, see Annex Fig.A2-b), to allow for more accurate quantification, in light of the defected DPSCsPTX cell proliferation. Beyond that, the significantly less migrated DPSCsPTX count after 7 days can be caused by the lower number of MCF7 caused by the cytotoxic paracrine activity of DPSCsPTX versus that of DPSCs (equal to 10%, see Annex Fig.A2-b).

Figure 2 :

 2 Figure 2: Impact of PTX on DPSCs migration. (a) Fluorescent images of ring migration wells for unloaded DPSC and DPSCsPTX towards MCF7 cells in the center of well. Cloning rings were put for 24h then removed to allow cell migration, green segmentation shows migrated cells are color in the center of wells, and (b) histogram showing average number of migrated stem cells in both conditions (48h after seeding). (c) Typical image showing transwell migration insert (8µm-pore), with the fluorescent image of migrated red DPSCs on the insert, scale bars: 2mm. (d) Histogram showing no difference in the differential migration ability towards cancer cells in the end of 7-day test for the DPSC±PTX. (e) Magnified image showing unloaded DPSC (up) and DPSCsPTX (down) after incubation on the insert for 7 days. Notice some empty pores obvious in black, and red pores indicating pores with migrated RFP-DPSCs inside, scale bars: 500µm. (f) Histogram showing reduced migration ability for DPSCsPTX towards cancer cells compared to unloaded cells on day 7. The results represent the mean ±SD. *p<0.05.

  -fluorescent DPSC cells with non-fluorescent cancer spheroids were mixed to an initial concentrations of 10,000 cancer cells and 2000 DPSC±PTX. From one starting number of cancer cells, we could obtain two morphologies of 3D spheroids (in two different experiments). By using identical microscopy settings per time-point, penetration of RFP-DPSCs ±PTX into MCF7 loose spheroids and compact spheroids (Fig.3A; 3B, respectively) was investigated. DPSC cell number was directly proportional to red-fluorescence intensity when quantified through the fluorescent plate reader (Celigo). DPSCsPTX showed slightly higher homing/ penetration ability compared to DPSCs. No significant difference between both groups was found, and data showed time-dependent increase with days. On day1 -start of penetration-, red cells are still far surrounding the spheroid with very little penetration count. On day 3 -penetration in process-, red cells are more approaching the spheroid and more cells are penetrating inside. On day 6, most red cells seem to have penetrated the spheroid. As shown by figure3C, opposite to the 2D migration findings, the intensity of penetrated RFP-DPSCs has significantly increased on day 9 for DPSCs (p<0.01) and for DPSCsPTX (p<0.001). This increase in penetrated cells was not evident toward PTX-resistant spheroids (Fig.3D).

Figure 3 :

 3 Figure 3: Homing ability of DPSCsPTX into 3D spheroids. Optical/ fluorescent superposed images of MCF7 loose spheroids (a) and compact spheroids (b) when co-cultured with RFP-DPSCsPTX at (from left to right): 1 day start of penetration, 3 days penetration in process, and 6 days where most red cells already penetrated the spheroid. Scale bar: 500 µm. (c) Histogram shows increasing RFP integrated intensity for DPSCs ±PTX measured at 0.5 mm peri-spheroid, showing significant increase of penetrated cells at day 9, for both groups (p<0.01). (d) Histogram showing the penetration into resistant MCF7 spheroids was not yet significantly increased on day 9, showing a flattened increased curve.

Figure 4 :

 4 Figure 4: Effect of short term DPSCsPTX-CM on MCF7 and its resistant-to-PTX subline. (a) CM from DPSCs primed with PTX, 5FU, and OXP drugs showed cytotoxic effect on MCF7 breast and

Figure 5 :

 5 Figure 5: Delivery of different anti-cancer drugs by primed DPSC-CM. Raman images confirming the spectral tracing of the 5-FU (a) and OXP (b) detected by two methods: clustering analysis (KMCA)on left panel, confirmed by the Pearson correlation (middle panel) calculated for each drugs' spectra as shown in right panel.

  To add more precise information regarding migration and penetration into spheroids, confocal fluorescent microscopy should be used with confocal slices taken at specific depth from the base of sphere, along with standardized controls. Penetration should be evaluated by imaging DPSC fluorescence in sections of spheroids in order to compare different fluorescent intensities across the sections.

Figure 3 :

 3 Figure 3: MCF7 after 3h with 4h-CM (DPSCsPTX 10µM for 10h). PTX intracellular spectrum in cancer cell and the PTX pixels (red).

Figure 4 :

 4 Figure 4: MCF7 after 3h with 4h-CM (DPSCsPTX 10µM for 15h). Three differential cell clusters: high cytoplasm proteins (cyan blue), nucleolus (green), and mitochondria (navy blue), and PTX (red), added to the transmembrane/ outer membrane zone and the PBS buffer. All seven image clusters performed by KMCA Raman spectroscopic analysis.

Figure 5 :

 5 Figure 5: Diagram representing cell shrinkage (early) and nuclear fragmentation (later) signs of apoptosis. Reproduced from R&D systems (Bax relocates from cytoplasm to mitochondria causing apoptosis).

Figure 1 :

 1 Figure 1: Methodology of calculation of the MMP1/cell ratio in DPSC cells. (1) Total cells' area is calculated for each image scan (using color threshold), (2) Pearson correlation map is calculated for the MMP1spectrum in each pixel of the scan (using MatLab), and (3) selection of the pixels according to the "30% of maximum" threshold selected for DPSC cells. Finally, the selected correlation map is superposed on the C-H image scan (Adobe photoshop) to give the final image of MMP1 correlation in the cells. The protocol is standardized for all the DPSC conditions: untreated DPSC (upper panel) and DPSC treated with 100µM PTX (lower panel).

  were primed with paclitaxel (10μM for 12h) and then cultured for another 15 days, to monitor cell proliferation recovery. The y-axis shows the cell number, expressed as a percentage of control. The results represent the mean ±SD of triplicate cultures from one representative experiment. **p<0.01, ***p<0.001 versus unloaded DPSC group.

Figure 3 :

 3 Figure 3: Linear regression analyses for high PTX dose and DPSCsPTX-CM effect on MCF7 cells. (a) The inhibitory effect of increasing incubation time with PTX on MCF7 and DPSC cells was analyzed by linear regression that showed less dose-dependent DPSC proliferation reduction, also confirmed by lower coefficients of determination (R 2 ) 0.85 compared to 0.98 for MCF7 cancer cells. (b) the regression analysis,for treatment of both the cell lines MCF7s and MCF7r, showed high coefficients of correlation (R 2 ) ranging from 0.93 to 0.99, comparable to that of PTX. The secretome of loaded DPSC produced a significant dosedependent inhibition of cancer cell growth, also confirmed by regression analysis (R 2 = 0.99), that is similar to the inhibition produced by direct PTX (R 2 = 0.98).

Figure 4 :

 4 Figure 4: Inhibition effect of DPSC±PTX-CM compared to high PTX dose. Short-term conditioning medium from DPSC, primed or not with PTX, is compared to high treatment dose of PTX. CM was left in incubation for 2 days with the cells, enough time to show DPSCs effect. High PTX dose was added as 10µM for 12h. Bone marrow mesenchymal stem cells (MSC) were used as primary cells of considerable resistance to treatment, as referral cell type for comparison. Those cells have proved moderate resistance compared to cancer cells, sensitive and resistant types. Resistant cancer cells demonstrate a model for time-delayed resistance, when compared to resistant MSCs. The results show a higher inhibitive ability of the DPSCsPTX-CM compared to high drug dose alone. The results represent the mean ±SD of triplicate cultures from one representative experiment. *p<0.05, **p<0.01, ***p<0.001 versus no treatment.

Figure 1 :

 1 Figure 1: Effect of direct 2D co-culture of DPSC±PTX and MCF7 for 5 and 7 days. Dose-dependent inhibition of MCF7 proliferation by DPSCsPTX (a) and DPSC (b) on days 5 and 7, showing increased cell proliferation with time. The two highest ratios (4000 and 8000 DPSCsPTX) caused maintenance of proliferation inhibition at day 7, suggesting time-dependent inhibition for these doses. Higher ratios of DPSC are needed to reveal a time-sustained effect. The results represent the mean ±SD of triplicate cultures from one representative experiment. All results in (a) are p<0.001 versus no treatment. Results in (b) are ¤: p<0.01, ¤ ¤: p<0.001 versus no treatment. *p<0.05, **p<0.01, and ***p<0.001 shows time-inhibition between day 5 and 7.

Figure 2 :

 2 Figure 2: Evolution of MCF7 spheroids as control conditions. Spheroids were grown with medium alone (negative control) or with 5µM PTX alone (positive control). *p<0.05 results show a significant reduction in

Figure 5 :

 5 Figure 5: Effect of indirect co-culture of DPSCs±PTX and MCF7 TAX19 for 5 and 7 days. The two cell types were mixed by transwell co-culture at the median ratio (1:3) with the absence of cell-cell contact. Two independent experiments for 5 days (count by imaging cytometer) and 7 days (optical densities), confirming the inhibition effect by DPSCsPTX on day 7. The results represent the mean ±SD of triplicate cultures from one representative experiment. **p<0.01, ***p<0.001 versus no treatment.

Figure 6 :

 6 Figure 6: Effect of the inhibitive DPSC±PTX-CM on paclitaxel-resistant cancer cells. Short-term (4h) medium conditioning by the DPSC±PTX supported inhibition of the resistant MCF7 TAX19 cells. Both types of conditioned media showed a potent inhibitory response, with a stronger effect by DPSCsPTX-CM (p<0.001). The inhibition effect of DPSC-CM was superior to 50% on days 5 and 7. These findings indicate an increased role of DPSC-CM, as compared to the released PTX, in the inhibition effect of DPSCsPTX-CM culture. The results represent the mean ±SD of triplicate cultures from one representative experiment. *p<0.05, **p<0.01, ***p<0.001 versus no treatment.

Table I :

 I Summary of main Raman system characteristics and the data analysis used. ......................

Table II :

 II Summary of our tests for cell apoptosis detection ...............................................................

Table III :

 III Summary table of all migration-related assays in the thesis. ..............................................

Table IV :

 IV Review of other literature validating Raman results using invasive metric methods ......

Table V :

 V Overview of all performed tests: category, description, and results ...…………..….......172

Table VI :

 VI MMP1 ratio calculation in DPSC stem cells ....................................................................

Table VII :

 VII MMP1 ratio calculation in sensitive cancer cells ............................................................

Table VIII :

 VIII MMP1 ratio calculation in resistant cancer cells ...........................................................

  Dans une étude préliminaire (chapitre 2), les DPSC ont été laissés en vue d'absorber du paclitaxel (PTX) et le milieu conditionné de 4h (CM) a été obtenu, qui représente la libération progressive de médicament et d'autres facteurs dans le milieu de culture. La microscopie confocale Raman a été utilisée pour visualiser le médicament de manière intracellulaire, à la fois pour les cellules souches et les cellules cancéreuses traitées.La détection du médicament PTX dans les cellules cancéreuses est une preuve solide que les cellules DPSC peuvent transporter et délivrer des médicaments. Une seconde étude plus approfondie sur les différentes durées d'incubation a montré l'absorption finale du médicament, en calculant la que les cellules souches issues de la moelle osseuses cultivées en présence de 10μM de PTX dans les mêmes conditions. Ensuite, le médicament anticancéreux paclitaxel a été tracé à l'intérieur de la DPSC directement après la période d'amorçage(10 µM, 12h), 4 heures après le début de la libération des extra-vésicules (DPSC-EVs), et quelques heures dans les MCF7 suite à leur culture avec le milieu conditionné.En premier lieu, l'apoptose en réaction au paclitaxel a été détectée en utilisant une méthode Raman confocale sans marquage. Le cyt-C agit comme un déclencheur de l'activation de cascade de quelques événements cellulaires et sa translocation depuis les mitochondries est considérée comme un signe nucléaire dans les cellules traitées (analyse KMCA Raman). Un autre point de repère morphologique est le changement de forme des cellules cancéreuses détecté par analyse de cytométrie en flux (SSA diffusion cytométrique). En plus de la translocation du cyt-C, des points En ce qui concerne la résistance des cellules DPSCs aux agents anticancéreux, la même méthode n'a pas montré d'apoptose des cellules DPSC incubées de 12h avec 10 µM PTX, où les cellules étaient en bon état après la longue période d'amorçage (chapitre 3). En outre, des tests de survie de DPSC avec de fortes doses (10 µM) de trois médicaments ont été effectués. Tous les effets des médicaments Outre l'imagerie des structures intracellulaires en fonction de leur composition biochimique intrinsèque, les caractéristiques spectroscopiques uniques des protéines (enzymes) peuvent permettre une détection sans marquage, d'une très haute spécificité et sensibilité, et ce sans modifier les les MCF7 sensibles au PTX voit leur viabilité altérée dès 12h de traitement. Une diminution du ratio MMP1/cellules est observée dans les MCF7 dès 6h de traitement avec 10μM de PTX. Tous nos résultats ont été confirmés par une étude de la littérature d'autres équipes utilisant des variantes d'autres méthodes invasives.Dans le même contexte, nous suggérons que l'augmentation de la collagénase fibroblastique intracellulaire confère en quelque sorte une résistance à l'apoptose.Limb et al. (2005) ont confirmé que lors de l'induction de l'apoptose, la MMP1 intracellulaire, en co-localisant dans les mitochondries et les noyaux, entraînerait une résistance à l'apoptose. Cela pourrait donc expliquer l'association bien

	de repère biochimiques tels que le clivage des caspases (test de Caspase-7), et le dysfonctionnement propriétés biochimiques des molécules d'intérêt. Dans le cas des agents chimio-thérapeutiques, il est
	mitochondrial (test MTT) ont également confirmé l'effet pro-apoptotique et cytotoxique du sur la DPSC ont été très minimes pendant les premières 24 heures d'incubation. La viabilité (jusqu'à possible de surveiller l'absorption, la distribution et l'accumulation, les interactions chimiques, et les
	DPSCsPTX. 48h) des DPSC n'a pas été fortement affectée par la cytotoxicité directe de tous les médicaments de événements cellulaires ultérieurs. Nous avons fait la distinction entre différentes caractéristiques
	De ce fait, les cellules DPSCs ont montré qu'elles sont comme un réservoir efficace capable trois classes (et modes d'action) différentes : PTX, 5FU, et OXP. spectroscopiques : interactions médicamenteuses ou réponses cellulaires.
	d'absorber le paclitaxel sans subir d'apoptose. Il a également été confirmé que les DPSC (en tant que Selon notre étude (chapitre 4), en utilisant la microspectroscope Raman confocale couplée à

quantité de PTX par cellule DPSC par deux modes d'amorçage. En utilisant une forte concentration de médicament en temps court (100 µM, 1-2 h) ou vice-versa

(10 µM, 10-15 h)

, celui ne change pas significativement l'absorption de cellules DPSC. Notre étude a suggéré qu'une incubation plus courte pourrait être préférable d'un point de vue clinique, tandis qu'en faveur de la viabilité de la cellule DPSC, une concentration plus faible (jusqu'à 10 µM) doit être choisie (chapitre 2). Le chapitre 2 est composé du premier article (publié dans Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XV en 2017) démontrant que les DPSC sont capables d'internaliser rapidement le paclitaxel (PTX) et de le libérer dans le milieu de culture, qui est nommé comme le milieu conditionné (CM). Le PTX est ensuite internalisé par les cellules issues de lignées de cancer du sein (MCF7) cultivées en présence de CM de cellules souches chargées en PTX (DPSC-PTX). Ces observations sont obtenues grâce à la microscopie Raman. Ce travail permet de poser les conditions expérimentales (10 μM de PTX, ~12h) qui seront utilisées dans les articles suivants. Le chapitre 3, (article publié dans Stem Cell research and therapy 2018) démontrent que l'internalisation du PTX n'altère pas la viabilité des DPSC, alors que les MCF7 présentent des dommages cellulaires observés en microscopie Raman. Il est aussi montré que les DPSC sont plus résistantes au PTX d'apoptose. La méthode a été établie par une étude précédente de notre équipe, Salehi et al. (JBO, 2013), qui ont démontré la capacité de la microscopie Raman confocale à détecter l'apoptose par observer la libération de cytochrome C à partir des mitochondries. La solution de cyt-C (Sigma-Aldrich) a été mesurée avec un spectre de référence pour établir une carte de corrélation de Pearson. Une comparaison entre la localisation de groupes de mitochondries et de cytochrome C dans des cellules a été fait par deux méthodes analytiques : l'analyse en classification à K-moyen (KMCA) pour la mitochondrie et la corrélation de Pearson pour la cyt-C. Ces méthodes analytiques ont été appliquées en utilisant les mêmes images de cellules sans avoir besoin de nouvelles expériences, constituant l'avantage des méthodes spectrales pour obtenir un grand nombre d'informations. Salehi et al. (JBO, 2013) a montré que le cyt-C est toujours à l'intérieur des mitochondries des cellules cancéreuses MCF7 pendant trois heures d'incubation avec du PTX 10 µM : indiquant l'absence d'apoptose. Une concentration plus élevée ou un temps plus long apparaît nécessaire pour initier l'apoptose dans ces cellules. Ce qui est intéressant, nos résultats ont montré l'apoptose dans les cellules cancéreuses MCF7 par la même méthode après la même période d'incubation (3h) avec le sécrétome issu de DPSCsPTX (chapitre 3). Celui-ci indique l'effet synergétique du sécrétome de DPSCsPTX qui est équivalente à une dose de PTX plus importante (>> 10 µM). Des autres signes d'apoptose ont été observés comme changements morphologiques apoptotiques et destruction sous-organelle, comme la fragmentation membranaire et véhicules) libèrent leur charge, le médicament et des facteurs spécifiques, à proximité des cellules cancéreuses. Dans cet article, nous montrons qu'après seulement quelques heures, les DPSC ont déjà libéré une partie, mais pas la totalité, du médicament. Par une étude de quantification : ≈ 100% des cellules DPSC étudiées ont été chargés de médicament et 86% des MCF7 ont reçu le médicament par voie de milieu conditionné. Un effet positif sur la biodisponibilité du médicament après le transport par les DPSC a été remarqué, car l'apoptose a été observée dès 3h dans le cas de CM libéré par les DPSCsPTX. La concentration de médicament dans les vésicules sécrétées après une incubation de 3 heures a été estimée ≥ 0.5 μM, en référence à l'étude précédente de Salehi et al. (APL, 2013). Le sécrétome de DPSC amorcées a été testé sur trois types de cellules cancéreuses : le cancer du sein sensible, le cancer résistant à la chiomiothérapie, et le cancer colorectal. En outre, l'absorption de médicaments a été confirmée indiquant la libération de médicaments par le sécrétome de la DPSC amorcées par les trois médicaments testés : le paclitaxel PTX, le fluorouracile 5FU, et l'oxaliplatine OXP (chapitre 5). En outre, une viabilité plus élevée des cellules MSC de la pulpe dentaire (DPSC) a été constatée par rapport à celles de la moelle osseuse (BMSC) et du tissu adipeux (ADSC), et une différence significative supérieure est constatée dans la viabilité de DPSC après 12h d'incubation (chapitre 3) ou 24h d'incubation (chapitre 5) avec du PTX 10 µM. * * * * * * * * * * l'analyse de corrélation de Pearson, nous avons pu quantifier d'une manière intracellulaire la "collagénase fibroblastique", la métalloprotéinase (MMP-1) dans des cellules non-différentielles : cellules cancéreuses et cellules souches, avec ou sans le médicament paclitaxel. Les résultats révèlent que les DPSC ont un taux MMP1 très faible, ce qui s'oppose au taux MMP1 élevé caractéristique des cellules cancéreuses. Nous avons observé une augmentation du niveau de la protéine MMP1 après exposition des DPSC à des concentrations élevées de PTX pendant une courte période. Cependant, l'augmentation que nous constatons dans les DPSCsPTX est encore extrêmement faible par rapport aux cellules cancéreuses, même lorsque des doses très élevées de médicament sont testées. D'autres expériences et une comparaison opportune sont nécessaires pour proposer que la MMP1 intracellulaire induise une transformation cancéreuse du DPSCsPTX. Le troisième article permet donc de mettre en évidence la localisation intracellulaire de MMP1 dans les DPSC par microscopie Raman avec une analyse de corrélation de Pearson. Un traitement d'1h avec une très forte dose de PTX (100 μM) provoque une augmentation significative de MMP1 dans les DPSC. Cette dose altère la viabilité des cellules. Dans cet article, les MCF7 résistantes au traitement paclitaxel sont testées. Il est montré qu'un traitement de 10 μM de PTX affecte peu leur viabilité après 72h alors que connue de cette enzyme avec la survie et la propagation des cellules tumorales. Bosco et al. (2015) ont confirmé que les MSC résistent au paclitaxel en adoptant un phénotype fibroblastique non-proliférant (dont une augmentation de la MMP1 et 3). En induisant les cellules à entrer en quiescence avant la transition de cycle cellulaire (G2/M) nécessaire à l'action du Paclitaxel, il est possible que les MSC utilisent la régulation du cycle cellulaire pour se protéger des effets cytotoxiques du Paclitaxel d'après Bosco et al. (2015). Selon nos résultats, cette propriété est unique seulement pour les DPSC comme cellules souches résistantes au PTX, mais n'est pas impliquée dans la protection des cellules cancéreuses résistantes au médicament. Cette réponse au PTX par les DPSC fait référence à la résistance inhérente au stress à ces cellules et à la fuite de la mort cellulaire causée par des agents exogènes.

Effet de synergie »

	de colonisation de l'espace créé par le retrait d'un anneau ou une culture en transwell séparant les vers les sphéroïdes cancéreux n'a pas été altéré avec le temps. Le modèle 3D, tel qu'établi par deux En parlant de l'effet thérapeutique direct sur le cancer, notre étude a fourni une approche Fait important, des diamètres sphéroïdes cancéreux (diamètres vers 500 µm) fortement réduits ont 3-Synergie de deux agents distincts de potentiels thérapeutiques limités : DPSC provoquant une
	DPSC traitées ou non au PTX et les MCF7 démontrent que le PTX n'altère pas la migration des cellules méthodes différentes tout au long de nos études, fournit des données cliniquement pertinentes, plus facilement accessible pour efficacement utiliser les DPSC comme véhicule pour l'inhibition été obtenus avec les cultures de petits ratios de 4000 et 8000 DPSCsPTX, où les deux doses testées ont légère inhibition, plus une dose faible de paclitaxel. Un des agents seul ne suffit pas à provoquer
	souches. De même, le PTX n'altère pas la capacité de tropisme et/ou pénétration des cellules dans des représentatives de la condition in-vivo et, dans de nombreux cas, comparables aux études animales. La (dose/ temps) des cellules cancéreuses en utilisant des doses faibles de médicaments classiques. L'effet provoqué une inhibition significativement plus élevée par rapport au mono-traitement au PTX de forte l'inhibition, alors que leur combinaison peut exercer un effet thérapeutique comparable à celui du
	sphéroïdes de MCF7. Enfin cet article montre l'effet du milieu de culture conditionné des DPSC ±PTX plus grande importance de la thérapie anticancéreuse basée sur la DPSC était évidente dans les modèles dépendant de la dose ou du temps fait référence aux effets du traitement par des petites molécules. Si dose. Le taux maximal d'inhibition de la croissance des sphéroïdes (30% ±0.03) a été obtenu avec paclitaxel à forte dose (chapitre 6).
	sur les lignées de cellules de cancer du sein résistantes ou non au PTX et d'une lignée de cellules de 3D de co-culture directe, par rapport aux tests en monocouche, aux effets d'interaction minimale, ou les effets changent lorsque la dose de traitement est modifiée, on dit que les effets sont « dose-l'utilisation de 8000 DPSCsPTX pendant 7 jours, ce qui était supérieur à l'inhibition obtenue avec une 4-Conditionnement par contact entre la DPSC et les cellules cancéreuses entraîne l'engagement
	cancer colorectal. Il est formellement montré que le CM de DPSCsPTX inhibe significativement la aux thérapies sans cellules. dépendants ». Lorsque l'effet d'inhibition est plutôt accru avec le temps de l'administration, on parle forte dose de PTX seul (25% ±0.03) ou avec des DPSC seules (14% ±0.06). et un effet accru d'inhibition du cancer. Celui-ci est supposé comme hypothèse depuis les effets
	prolifération des cellules cancéreuses dès J1 pour les lignées sensibles au PTX et dès J2 pour les lignées Dans une dernière étude, sous les mêmes conditions, une comparaison entre le milieu conditionné d'une « inhibition temporelle ». Combinés aux données collectives, nos résultats ont montré que les Le CM issu des DPSCsPTX peuvent, même en moindre interaction, atténuer totalement la opposés d'interaction directe/ indirecte avec les cellules MCF7 (chapitre 6).
	résistantes. obtenu après 48h à partir du même nombre de cellules de (DPSCsPTX, BMSCsPTX et ADSCsPTX) facteurs DPSC jouent un double rôle spécifique à chaque stade de la thérapie. dissémination des cellules sphéroïdes, relatif à l'effet oncolytique associé au PTX. Le moyen de
	Concernant les effets à long terme des médicaments cytotoxiques (chapitre 5), la viabilité des a montré un effet comparable sans une différence significative sur les cellules cancéreuses du sein. Ces Le chapitre 6 approfondit les résultats de l'article précédent en donnant des précisions en fonction délivrance supposé de la thérapie de DPSCsPTX pour le cancer du sein sera préférentiellement par
	DPSC n'a pas été fortement affectée mais leur capacité de prolifération et leur capacité de migration résultats n'ont pas montré une supériorité des DPSC dans les conditions étudiées, mais laissent des doses et des temps de culture. Dans le modèle en 2 et en 3D, les DPSCsPTX inhibent la voie locale (co-injection dans le tumeur solide) plutôt que par la voie systématique (veineuse), vu les
	2D (altération au 7e jour) ont néanmoins été affectées. Seifrtova et al. (2012 -2013) ont également supposer que les DPSC peuvent être une source ultérieure pour l'approche thérapeutique (chapitre 5). prolifération des MCF7, comme vue précédemment, mais ici dans un modèle de co-culture directe, résultats avec la lignée cellulaire de MCF7.
	montré que tous les effets génotoxiques des médicaments sur les DPSC et blocage de la prolifération L'effet du traitement avec le CM (concentration 100 %) a été mesuré sur la base des analyses de la dès le ratio 1 DPSCsPTX pour 16 MCF7. Les DPSC non traitées ne sont inhibitrices de la prolifération
	étaient plus visibles après 72 heures et dépendaient de la dose du médicament. Le test de cytotoxicité viabilité et de la prolifération des cellules. Dans le chapitre 5, nous avons testé aussi peu que 1% en des MCF7 qu'à partir du ratio 2 DPSC pour 1 MCF7. L'observation en confocale des co-cultures
	in-vitro a été utilisé pour observer le rétablissement de la prolifération sur le long terme après le retrait concentration de CM des différentes DPSC amorcées par des médicaments (DPSCsPTX, DPSCs5FU, montre que les DPSC projettent des expansions cellulaires au contact des cellules cancéreuses et que
	du PTX. Les DPSC ayant reçu une forte dose de médicament ont été suivies pendant 21 jours. La DPSCsOXP). Les trois types de CM entraine une perte de viabilité très significative pour les cellules la morphologie de ces MCF7 est altérée en présence des DPSCsPTX au ratio de 2:1. Comme le confirment tous nos résultats, des ratios DPSCsPTX élevés ne sont pas nécessaires capacité de prolifération des DPSC a été partiellement, mais pas totalement, rétablie après trois du cancer du sein MCF7, et moins significative pour les cellules issues du cancer colorectal HCT116, Au moyen d'un microscope à fluorescence confocal, la co-culture de cellules DPSCsPTX (en pour expliciter une cytotoxicité énorme. Un effet synergique est supposé pour l'interaction de DPSC semaines. par rapport aux conditions contrôles. L'absorption des médicaments par les cellules cancéreuses a été rouge) et MCF7 (en vert) a révélé l'initiation de communications cellulaires directes. Une altération de et de paclitaxel libéré. Peu d'observations en tant qu'hypothèses sont mentionnées pour le puissant effet
	Notre étude est la première étude -d'après notre connaissance -jetant la lumière sur la migration confirmé par la méthode Raman, confirmant l'efficacité des DPSC comme véhicules et transporteurs la morphologie cellulaire et du noyau des cellules est montrée. De plus, seules les MCF7 résistantes anticancéreux de DPSCsPTX :
	des DPSC vers les cellules cancéreuses. La migration des DPSC exposées aux médicaments n'a jamais de différents types de médicaments anti-cancéreux. au PTX cultivées en sphéroïde pour mimer les conditions physiopathologiques, voient leur 1-Augmentation de la biodisponibilité et de l'effet du paclitaxel par l'administration du été abordée dans la littérature. Les MSC peuvent migrer vers une tumeur, en attirant plusieurs facteurs prolifération inhibée en présence de DPSC±PTX. Enfin, alors que le milieu de culture des DPSCsPTX médicament en vésicules extracellulaires DPSC-EVs (chapitre 3). de croissance, cytokines et protéases existant dans la tumeur, et en réagissant aux dommages tissulaires, à l'hypoxie, et à l'inflammation trouvés dans les microenvironnements des tumeurs (de la inhibe la dissémination des sphéroïdes (modèle mimant les possibilités métastasiques) et atténue * * * * * * * * * * totalement la dissémination des cellules sphéroïdes, le CM des DPSC non traitées semble favoriser la 2-Le phénotype modifié de la DPSCsPTX peut augmenter la capacité de destruction et l'effet pro-
	Torre et al., 2020). Nos résultats ont indiqué un tropisme tumoral inchangé des DPSC lorsqu'elles sont prolifération des cellules cancéreuses et leur dissémination à partir des sphéroïdes dès 48h de culture. apoptotique de la DPSC, comme le montre nos résultats de l'augmentation de MMP1 intracellulaire
	chargées de PTX et, par conséquent, confirme leur aptitude à être utilisées pour des applications de (le mécanisme n'est pas entièrement connu, chapitre 4).
	délivrance de médicaments. Avec les limitations de notre étude, le tropisme des cellules de DPSCsPTX

. Il n'est pas évident que cette augmentation de la MMP1 dans le DPSC soit suffisante pour induire une transformation tumorigène. Des tests supplémentaires devraient être effectués pour exclure ce risque. * * * * * * * * * * Le chapitre 5 démontre que les DPCS ne sont pas seulement résistantes à un traitement au paclitaxel, mais aussi aux 5-fluorouacyl et à l'oxaliplatine aux doses de 10μM pendant 24h. Il a été également prouvé que les DPSC sont extrêmement résistantes aux effets cytotoxiques du paclitaxel, mais il est à noter des réductions observées du nombre de cellules DPSC après 48h. Un modèle original « «

Résistance au traitement »

	en comparant avec les cellules sensibles (chapitre 6). Une réponse plus lente peut être justifiée par la CONCLUSIONS DPSCsPTX ont confirmé une efficacité migratoire comparable ou plus élevée par rapport aux cellules cellules souches de celles tumorales, et aussi pour détecter les mécanismes de réponse cellulaire aux
	plus lente pénétration de DPSC ±PTX aux sphéroïdes résistants (chapitre 5). Dans cette thèse, nous avons évalué la thérapie combinée en utilisant les cellules souches de la non chargées, vers les cellules cancéreuses. médicaments (ou à tout agent exogène) par incorporation avec d'autres tests biologiques.
	Enfin, une augmentation de la dose d'administration de PTX entraînera le développement de pulpe dentaire (DPSC) et des agents chimio-thérapeutiques pour traiter le cancer. L'approche de -Nos études ont montré une grande preuve que l'efficacité du médicament est améliorée par
	phénotypes de cancer plus résistants, ce qui provoque des échecs thérapeutiques importants. Notre l'amorçage des DPSC avec des agents anti-cancéreux est une nouvelle recherche prometteuse, utilisant l'incorporation de DPSC (très probablement due au sécrétome de DPSC) en contact direct avec le La conclusion finale de la thèse replace le travail dans le contexte des traitements des cancers et
	approche de traitement, donc, par les DPSCsPTX pourra viser à échapper à des doses très élevées de les propriétés uniques de ces cellules pour obtenir l'objectif thérapeutique ciblé pour les tissus du cancer. L'effet synergétique a révélé une efficacité à la fois supérieure et plus rapide que la de la régénération des tissus lésés. Elle rappelle l'intérêt de comprendre les mécanismes de migration
	la chimiothérapie et leurs effets secondaires, ce qui est très important dans le cadre clinique. cancer. monothérapie. des cellules souches vers les tumeurs afin de pouvoir répondre à terme au défi thérapeutique posé par
	La thèse a examiné le rôle des DPSC elles-mêmes dans l'application de l'administration et la -Les DPSC ont des effets modulateurs sur la prolifération cellulaire observés par l'interaction la chimiothérapie. Toutefois, avant d'envisager le transfert en clinique chez l'homme des données
	* * * * * * * * * * thérapie du cancer, a fourni une incorporation avec des méthodes d'aide physico-technique pour la directe DPSC-cancer. Une modulation directe du fonctionnement des cellules cancéreuses par obtenues, il sera nécessaire de déterminer précisément la chronologie et les relations entre d'une part
	résolution des nouvelles thérapies développées, a suggéré une compréhension de la chimiorésistance interaction avec les extra-vésicules (DPSC-EV) a également été signalée. l'environnement des cellules tumorales (rôles des cytokines due à l'inflammation chronique …etc) et
	Les interactions cellulaires dans les cultures en organisation 3D contribuent à une résistance à certaines classes de la chimiothérapie. Nos résultats indiquent que la résistance au PTX provenant de l'agrégation sphéroïdale des cellules de carcinome épithélial humain était surmontée par les facteurs sécrétés des DPSC. D'autre part, la supériorité de l'effet des DPSCsPTX était évidente à J7 par rapport Le chapitre 7 (article publié dans l'international journal of nanomedicine) porte sur les effets de basée sur les DPSC et l'interaction avec les agents cytotoxiques et aussi les cellules cancéreuses, et a d'autre part les cellules souches pulpaires. De même, la question des propriétés immunosuppressives -La thèse a mis en évidence le contexte des DPSC "naïves" et celles "engagées envers la nanoparticules au cobalt sur les MCF7, sur la lignée de cellules de cancer colorectal (HCT116), sur détaillé l'utilisation de la thérapie combinée par DPSCsPTX pour la thérapie du cancer du sein. des DPSC sur le développement des tumeurs doit être posée. Enfin, l'hétérogénéité des cellules thérapie", qui était clair tout au long de nos enquêtes. L'interaction directe avec les cellules cancéreuses une ligne d'adénocarcinome (PPC-1), sur des cellules humaines embryologiques de reins (HEK) et sur L'objectif de la thèse était en gros "d'évaluer les résultats en considérant les cellules, le sécrétome présentes dans les tumeurs et leur plasticité avec la transformation épithelio-mesenchymateuses (EMT) a permis de moduler les fonctions et activités thérapeutiques des cellules DPSC vers l'inhibition du les DPSC observées par microscopie Raman. L'incorporation rapide des nanoparticules par les cellules et les 3-dimensions comme un tout intégré". Cela a ajouté de la complexité à notre étude in-vitro, car des cellules cancéreuses nécessitent une grande prudence quant à l'interprétation des résultats de ce cancer. est décrite. Les tests MTT montrent une faible toxicité du cobalt sur les HEK et une forte toxicité sur une attention particulière doit être portée à la mise en place de l'expérience en fonction de l'objectif travail.
	à toutes les autres conditions en monothérapie (chapitre 6). les cellules PPC-1. La combustion du cobalt internalisé par les cellules peut être déclenchée par une -Les facteurs DPSC ont contribué à surmonter la résistance à la chimiothérapie, un obstacle
	spécifique, et à l'interprétation des données.
	irradiation laser. Dans cet article, la thérapie ciblée de cellules cancéreuses est envisagée par la énorme dans la voie du traitement du cancer. Avec des doses de chimiothérapie très faibles, un effet
	Les modèles de culture 3D de cellules MCF7 étaient évidemment résistants à une dose forte du
	photothérapie, thérapie thermale, ou délivrance de nanoparticules par les MSC. Les cellules DPSC synergétique important se reproduit qui aide à traiter les cellules résistantes.
	médicament anticancéreux PTX. Cependant, les cellules cultivées en 3D étaient moins résistantes
	(p<0.01) aux cellules DPSC activées par l'hypoxie (80% contre 88 % de viabilité après J5 pour le PTX servent à maximiser la charge de médicaments anticancéreux dans la nanoparticule et utiliser les En considérant toutes limitations des études établies, les principales conclusions suivantes ont pu être -La microscopie Raman confocale est une technique puissante pour la détection non-invasive et
	et DPSCsPTX, respectivement), où la consommation d'oxygène actif et la diffusion limitée de propriétés migratoires de DPSC par un système combiné de DPSC-médicament-nanoparticule. résumées : non-marquée de la diffusion de médicaments dans les cellules. En outre, la méthode multi variante
	l'oxygène ont créé un noyau hypoxique et nécrotique, ce qui contribue à l'augmentation de la migration -Plusieurs tests devront être fait pour comprendre les mécanismes de migration des cellules utilisée pour analyser les données ont permis d'identifier l'apoptose cellulaire induite par les
	et l'efficacité des DPSC -chargées au médicament-vers les sphéroïdes (chapitre 5). souches vers les tumeurs afin de pouvoir répondre à terme au défi thérapeutique posé par la médicaments, telle que celle qui se produit par une voie mitochondriale.
	Concernant les cellules cancéreuses qui ont acquis une résistance au PTX, les cellules MCF7 dites chimiothérapie. Cette propriété est particulièrement pertinente pour l'utilisation de DPSC amorcées -Les applications cliniques potentielles de la spectroscopie Raman comme un outil de diagnostic
	résistantes, des ratios plus élevés de DPSCsPTX sont nécessaires pour induire une réponse plus rapide pour cibler les tumeurs. Les DPSC ont montré un tropisme intrinsèque aux cellules cancéreuses, et les complémentaire (CD) pour détecter l'internalisation des médicaments et nanoparticules, distinguer les

  or treat the symptoms of the disease, and side effects caused by treatment of the disease ; II) Manage psychological, social, and spiritual problems related to the disease or its treatment ; III) Improve the patient's quality of life.

Several new strategies have been added in the last years, which can increase survival rate and reduces mortality rate; stem cell therapy is one of these strategies. Stem cells to be used can be from mostly autologous (same patient) or allogenic (matching family donor) sources. There are three MSC-based treatment approaches for cancer patients, which are MSC complementary therapy, MSC replacement or restoration therapy, and MSC reducing or inhibition therapy. The development of MSC cellular therapy for cancer can include three main elements (from oldest to newest concept):

MSC replacement or restoration therapy

  Stem cell tissue replacement or restoration therapy employs the reuse of stem cells to construct the tissue damage caused by cancers therapy, for instance after surgical tumor resection. Tissue reconstructions vary from the placement of a simple implant to elaborate reconstructions using natural tissue from the body. A reconstruction phase (so far as prosthetic surgery) is needed especially for breast, head and neck, and dental prosthesis. Careful attention is given to achieve the best possible cosmetic result and quality of life, by incorporating advanced prosthetic techniques without compromising the chance of cure. In each case, the approach is customized to best fit each patient's lifestyle and goals. Besides the MSC's tissue replacement application, MSC's regenerative properties can have potential utility after the cancer therapy: replacement and tissue regeneration therapy (ongoing studies).

	evidence of enhancement either. No increased relapse rate of the underlying malignancy was
	identified. MSC immunomodulatory functions in cancer patients have been studied over time as one
	gateway for MSC therapy: supporting (palliative) to other transplantation therapy.

Lazerus et al. represented the first application of ex-vivo expanded, related-donor MSCs in human allogeneic SCT. Safety was established in this study with no infusion toxicities or late MSC-associated adverse effects identified. There was no deleterious impact on engraftment but there was no clear (b)

Table 1 :Summary of main Raman system characteristics and the used data analysis. System CRM alpha 300R (WITec)

 1

	Spatial resolution	300 x 300 nm
	Depth resolution	1 µM
	Acquisition time	0.5 second (average)
	Laser used	532 nm, 50 mW (max)
	Points per image
	Average	150 x 150
	(minimum used)	75 x 75
	(maximum used)	200 x 200

Table 2 : Summary of our tests for cell cytotoxicity and apoptosis detection

 2

	Category	Apoptosis sign	Cell(s) tested Type of test
	Morphological	Signs of organelle destruction	MCF7	Raman subcellular clustering
	Landmarks	or membrane fragmentation		analysis
		Change in cell form	MCF7	Light scatter analysis (flow
				cytometry)
	Biochemical	Cytochrome-C translocation	MCF7	Raman data analysis, by
	Landmarks		DPSC	KMCA (mitochondria) and Pearson correlation (cyt-C
				map)
		Cleavage of caspases	MCF7	Caspase 3/7 test
		(Late) mitochondrial	MCF7	MTT reduction measured by
		dysfunction	DPSC	spectrophotometer

Table 3 : Summary table of all migration-related assays in the thesis.

 3

	Category of test	Cell tested Analysis method	Variable measured
	(model)
	Homing into	DPSC	Imaging RFP-DPSC in	Fluorescence intensity
	spheroids (3D)		one spheroid zone
	O-Ring migration	DPSC	Imaging RFP-DPSC in	Fluorescence count
	(horizontal 2D)		the gap +cancer cell zone	Percent gap healing
				(% cell area)
	Transwell migration	DPSC	Imaging RFP-DPSC	Fluorescent count
	(vertical 3D-like)		inside pores +bottom of
			insert
	Qualitative cell	DPSC	High magnification	Development of cell
	morphology (2D)		fluorescence confocal	protrusions, Cytoplasmic
			imaging	membrane extensions
	Spheroid cell	MCF7	Measuring whole	Dissemination area
	dissemination (3D-like)		spheroid cell diameter	Dissemination attenuation or growth

Table 1 : MMP1 ratio calculation in DPSC stem cells

 1

		DPSC-CTR
				MMP	MMP/cell	Max.	30% of	Max -
		Total area	Cell area DPSC-CTR	pixel	ratio	correlation	max	30%
	1	804.15	398.39	78.00	0.20	62	18.60	43.40
	2	544.70	158.34	1426.00	high	39	11.70	27.30
	3	3101.25	1195.07	65.00	low	53	15.90	37.10
	4	965.43	266.18	69.00	0.26	26	7.80	18.20
	5	621.87	263.56	254.00	0.96	48	14.40	33.60
		DPSC CTRL		0.47
		SEM			0.30
		DPSC 1µM 1h PTX
				MMP	MMP/cell	Max.	30% of	Max -
		Total area Cell area DPSC 1µM 1h PTX	pixel	ratio	correlation	max	30%
	1	752.24	339.41	337.00	0.99	31	9.30	21.70
	2	1775.03	774.10	1604.00	2.07	40	12.00	28.00
	3	1502.27	532.81	299.00	0.56	54	16.20	37.80
		DPSC 1µM		1.21
		SEM			0.58

DPSC 10µM 1h PTX Total area Cell area DPSC 10µM 1h PTX MMP pixel MMP/cell ratio Max. correlation 30% of max

		(B) Tables in Annex
							Max -
							30%
	1	1939.72	646.71	390.00	0.60	14.40	33.60
	2	1801.28	1004.46	146.00	0.15	15.60	36.40
	3	564.18	289.50	327.00	1.13	16.50	38.50
	4	2074.38	1498.39	96.00	low	17.70	41.30
	5	1667.41	581.19	30.00	low	17.70	41.30
	6	2806.76	1353.18357 no	or 130	1.18	9.60	22.40
	7	422.38	209.98	high5769	1.16	14.70	34.30
	8	1311.15	503.20	3490	6.94	16.50	38.50
	9	1632.81	849.22	415	0.49	18.30	42.70
	10	580.00	348.61	5731	0.56	17.70	41.30
	11	2346.63	1218.559	440.00	0.36	23.70	55.30
	12	2859.18	1122.91	127.00	0.11	18.30	42.70
		DPSC 10µM		1.27
		SEM			1.05

Table 3 : MMP1 ratio calculation in resistant cancer cells

 3 co-cultured with DPSCsPTX (11% of control co-culture). (b) The same number of DPSCs

	MCF7r-CTRL
	Total	Cell area MCF7r-CTRL MMP	MMP/cell	Max.	10% of	Max -
	area		pixel	ratio	correlation	max	10%
	1 1078.34	530.079	3505	6.61	63		56.70
	2 2088.98	1478.536	92	low	48		43.20
	3 2740.78	1154.751	162	0.14	57		51.30
	4 1102.42	899.887	134	0.15	35		31.50
	5 3394.42	2537.022	399	0.16	51		45.90
	6 650.95	330.961	964	2.91	63		56.70
	7 199.56	114.798	1126	high	50		45.00
	8 441.31	230.131	5020	high	64		57.60
	9 3501.38	2115.82	42	low	63		56.70
		MCF7r CTRL		1.99
		SEM		1.27
	MCF7r+PTX
	Total	Cell area MCF7r+PTX MMP	MMP/cell	Max.	10% of	Max -
	area		pixel	ratio	correlation	max	10%
	1 2433.68	1443.786	160	0.11	60		54.00
	2 2588.47	1338.864	1166	0.87	70		63.00
	3 1145.76	742.746	836	1.13	67		60.30
	4 2352.05	1454.478	66	low	64		57.60
	5 3281.52	1842.288	411	0.22	70		63.00
	6 3434.90	1680.306	4098	2.44	66		59.40
	7 2939.15	2212.753	566	0.26	66		59.40
	8 3192.50	1825.043	1419	0.78	64		57.60
	9 2241.73	750.30	1522.0	2.03	70		63.00
		MCF7r +PTX		0.98
		SEM		0.30

Acknowledgments

First and foremost, all praises and thanks to Allah, the Most Merciful, the Most Compassionate. I deeply thank Professor Frédéric CUISINIER for welcoming me since the beginning in this laboratory.

I thank you for giving me the opportunity to accomplish this work. I extend my thanks to Professor Csilla GERGELY for being president of the jury. I really appreciate your undenied support and kind heart all along my masters and Ph.D. I thank Professor Philippe GIBERT, Doctor Hamideh SALEHI and Professor Valérie ORTI for supervision and assistance throughout my thesis. Thank you Dr. Salehi for your kindness and encouragement during the five years. I give my thanks to Professor Brigitte ALLIOTT-LICHT for being the reviewer in the dentistry, stem cells speciality. I thank Doctor Youri ARNTZ for being the reviewer for the biomedical sciences' field. Thank you for accepting to evaluate this work, I am proud to have worked with you. Special thanks to Doctor Ahmad HAM DAN, my professor from the University of Jordan, for your permanent support and for accepting to be a member of the examination committee. My gratitude goes always to the UNIVERSITY OF JORDAN and CAMPUS FRANCE for giving my masters and Ph.D. scholarships during the five years. Enfin et surtout, merci pour La France.

CHAPTER 1: INTRODUCTION

The results are divided into six main chapters, in the form of six interdisciplinary articles representing the full work.

4. Methodology Introduction

Generally, certain challenges of cell therapy development for solid tumors necessities the use of multiple types of in-vitro assays and analytical techniques, beneath summarized in few points : I-the need to prolong well-being and persistence of the cell applied in the cell therapy (DPSCs), for example by optimizing DPSC isolation and expansion, and optimization of dosing/ timing/ delivery for the priming approach ; II-the need for high throughput phenotypic and functional assays to identify and understand existing mechanisms, for example: Raman spectroscopy for intracellular detection, in-vitro cytotoxicity and apoptosis assays ; III-the need for 3D assays for in-vivo like study of cell affinity and cancer resistance and metastasis, for example: cancer spheroid growth, cell penetration into spheroids, dissemination of spheroids ;

IV-the need for diverse co-culture assays and controlled experiments to improve predictability of cell therapy, for example: direct, indirect, and minimal interaction assays. The main methods are described beneath: (1) DPSC cell preparation (isolation and transfection), (2) Raman methods (acquisition and analysis), (3) cell apoptosis assays, and ( 4) cell migration assays used in the thesis.

4. DPSC cell preparation 4. DPSC isolation and expansion

Human wisdom teeth extracted for orthodontic reasons were recovered from healthy patients (15-18 years old). Written informed consent was obtained from the parents of the patients. This protocol was approved by the local ethical committee (Comité de Protection des Personnes, Montpellier hospital,

Data analysis CRR (cosmic ray removal) SS (spectral smoothening)

Sum-filter (C-H region 2800-3000 cm -1 ) K-Mean Clustering (WITec ImagePlus) Pearson Correlation (MatLab)

4. 2. 2. Raman data analysis

Examples of the processing analysis that we have used throughout the thesis:

1-Cosmic ray removal (CRR)

Cosmic rays are high-energy particles from outer space. They are mainly charged type of particles which reach the surface of the Earth and interact with molecules in the Earth's atmosphere. If a cosmic ray hits a CCD detector, it will create a false signal in a form of a very sharp peak. Two mathematical methods can be used to remove the cosmic rays' false signals: the spectral or the temporal cosmic ray removal. The principle of spectral cosmic ray removal method is that each pixel is compared to its adjacent pixels and if it passes a certain threshold, it will be identified as a cosmic ray. In temporal cosmic ray removal method, each spectrum is compared to the others recorded before and each pixel is compared to its variation in the time domain. This method is not valid in the cases where the sample changes its spectral signature rapidly.

2-Spectral smoothing (SS)

Smoothing is a common method to treat Raman spectra in order to reduce the noise associated with a recorded spectrum. The algorithms rely on gradually changing in spectral data from one point to another, whereas in noise typically a very quick change is observed. Smoothing can be applied by replacing each value with a value calculated from its surroundings. Median filter is the most famous filter used for this purpose.

3-Sum filter

The WITec Project software provides a Sum Filter. By choosing the specific spectra region in Sum to decipher the mechanisms underlying mesenchymal stem cells' intrinsic resistance, allowing their potential application for drug delivery. We hereby, using confocal Raman micro-spectroscopy coupled with Pearson correlation analysis, could quantify the intracellular "fibroblastic collagenase" Matrix MetalloProteinase (MMP-1) in cell types with three different pathologic traits. Differentially expressed MMP1 in cancer cells was observed compared to the dental pulp stem cells (DPSC). An increased MMP1 level in DPSCs when exposed to paclitaxel is associated with a transformed phenotype, and is suggested to enhance their resistance to PTX as well as improve these cells' inhibitive ability when co-injected with cancer cells, according to literature. Our study highlights the potential application of Raman spectroscopy as an analytical technique in preclinical development to predict drug's effect and/ or mechanism of action, when incorporated with other tests, in order to improve the understanding of molecular responses to anti-cancer drug and/or to predict cellular resistance pathway.

with stem cells however instead of originating cell types with the cancerous potential, stem cells can differentiate into different types of somatic cells that are present in several tissues. We have studied the MMP1 in DPSC cells, and compared it to the sensitive breast cancer cell line and its PTX-resistant subline (MCF7 and MCF7 TAX19 , respectively).

Matrix metalloproteinases are related to zinc-dependent endopeptidases family, having various types of proteins which play essential roles in physiological processes such as organogenesis, tissue wound healing, angiogenesis, apoptosis, cell proliferation, and motility [19]. In principle, MMPs are requisite in intracellular as well as extracellular matrix remodeling. Some types of MMPs degrade extracellular proteins and the extracellular matrix; for instance, MMP-1, MMP-8, and MMP-13 degrade collagen types I, II and III [20]. MMPs are also involved in some abnormal processes including childhood infections, cardiovascular, immune disorders, and tumor formation [21].

Materials and Methods

Cell culture

Human wisdom teeth extracted for orthodontic reasons were recovered from healthy patients (15-18 years old). Written informed consent was obtained from the parents of the patients. This protocol was approved by the local ethical committee (Comité de Protection des Personnes, Montpellier hospital, France). DPSCs were isolated and characterized as previously described [6,22]. DPSCs were cultured in αMEM (Modified Eagle's Medium, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 100 μg/mL penicillin and streptomycin (PS, ThermoFisher), with the addition of 1 ng/mL bFGF (basic fibroblast growth factor, R&D system) at 37°C and 5% CO2. DPSCs used for all experiments were between passages 2 and 8.

Two cancer cell lines are tested as well in correlation measurements, in addition to the DPSC cells.

The MCF7 (ATCC-HTB22), derived from a metastatic breast cancer patient, is a standard cell line model in cancer research [23]. The paclitaxel-resistant subline MCF7 TAX19 (offered from Dr. Peter Coopman, IRCM, Montpellier) was previously established [24] by culturing the parental MCF7 cells in increasing concentrations of paclitaxel, and then maintained in paclitaxel-free medium.

New multivariate methods have been used in a classification method in number of studies which include diagnostic or cellular studies [26]. Although commonly performed in an unsupervised manner, correlation measurement can also be performed in a supervised or semi-supervised manner.

Unsupervised correlation analysis is based on statistical similarity of the data but does not take any of the experimental variables such as treatment, phenotype, tissue, etc. into account while finding correlation [27]. In contrast, supervised or semi-supervised clustering utilizes such additional information to "guide" or "adjust" the clustering process [28].

Vibrational spectroscopic data of biological components within cells are highly correlated; therefore, 10% correlation thresholds (between 0.9 and 0.98) are typically used in order to produce clusters that adequately differentiate the spectra of the different biological components in the cell [25]. We have used this threshold for quantification in cancer cells (10% of maximum correlation obtained). In DPSC stem cells, the correlation was found considerably less. This may indicate that maybe the enzyme is in most of its form in latent (pro-MMP1) form, compared to the active form used as a spectrum reference.

A higher threshold, though, was tested as 30% correlation (of the maximum), to be able to compare with the cancer controls (Fig. 2). Maximum correlation value was always maintained, while the lowest was set to zero for all correlation maps. Exclusion criteria were applied for final ratios (pixel/ µm 2 )

where very low values, less than 0.1, or very high ones, higher than 10, were rejected. The minimum and maximum values accepted were around 0.11 and 6.9 pix/µm 2 . The methodology is well explained in detail in appendix (see Appendix; Fig. 7). All data are shown in supplementary tables (see Supplementary; Tables 23-4) for cancer and stem cell calculations.

Conclusion

Supported by their high adaptability and maintained migration/ penetration ability after drug priming, we conclude for the further testing of DPSCs application as targeted drug vector towards cancer cells.

Discussion

Being considered for different biotechnology applications and perspectives, it is essential to have information also on drug sensitivity of dental pulp MSCs against different anti-cancer drugs. In fact, in literature, exists heterogeneous data concerning mesenchymal stem cells sensitivity to cytotoxic agents, and the mechanisms of their relative resistance are largely unknown. Several studies have shown that dental pulp MSCs in patients under whole body irradiation and/or high-dose chemotherapy seem mainly to be affected [45][46][47][48].

We have examined three classical anticancer drugs belonging to different classes characterized by a unique cytotoxic activity. The first class, the anti-metabolites (5-fluorouracil), are structural analogs to precursors of nucleobases of DNA -the pyrimidine-and are responsible for the induction of apoptosis during the DNA synthesis (S-phase). 5-Fluorouracil is a nucleoside inhibitor associated with the silencing of transcription and translation in breast cancer [13]. The second class includes platinumbased agents; they are DNA alkylating agents (oxaliplatin) which interact with DNA and block its replication and therefore arrest cell division. The last class are the anti-microtubules or antimitotic (paclitaxel); they affect particularly the microtubules leads to cell cycle arrest during the mitosis (Mphase) and subsequently apoptosis. Our results related to the three drug classes influence on DPSC's proliferation and viability conform with that for different other MSCs reviewed by Rühle et al. [14].

On the other hand, it was noted earlier that the kinetics of incorporation and release of PTX by bone marrow MSCs has a higher trend of drug release during the first 24h of incubation [15], hence our choice for drug priming was between 12h (mainly for DPSCsPTX) or 24h (for other cells/ drugs comparison) in the different experiments.

Our results confirm other studies stating that MSCs are resistant to PTX direct cytotoxicity, but are greatly sensitive to its anti-proliferative effect [15][16][17][18]. DPSCs although resistant to all 3 classes, were influenced by their proliferation ability when exposed to drugs. For the priming of MSCs, a relatively high drug dose is used that intentionally block cell proliferation, while at the same time maintain high cell viability necessary to uptake/ release the drugs [15]. In a period of 21 days, DPSCs were recovering their proliferation, but did not attain full recovery until 3 weeks. Constant exposure or post-withdrawal impairment effect on DPSCs proliferation are comparable (for the same conditions, compared to drugfree cells). This indicates that paclitaxel does strongly impair proliferation, till 14 days, after which DPSC cells start to gradually recover.

We also determined whether the reduction in cell numbers by PTX was not the result of loss of viability Reproduced from (de Pizzol Júnior et al., 2018). Briefly, DPSCs or MCF7 were plated in 75 cm 2 culture flasks until 90% confluency in complete medium, as described above. Then, as transduction reagent, SureENTRY (Qiagen) was added at a concentration of 8 μg/mL together with the RFP/GFP-containing viral particles at a concentration of 1.4×10 7 transducing units (TU)/ mL (or using a multiplicity of infection MOI of 50) with an antibioticfree medium. Following overnight incubation, medium containing viral particles was removed and replaced with fresh medium containing 2 μg/mL (active concentration) of puromycin (Sigma-Aldrich) in order to isolate the puromycin-resistant colonies. The cancer cell line was infected with three spin infection cycles for 3-4 days, whereas primary stem cells were infected with one spin infection.

APPENDICES APPENDIX A : PROOF-OF-PRINCIPLE

Spectral Analysis by Raman Microscope

Transduced cells underwent fluorescence-activated cell sorting (FACS) analysis after the last infection cycle, for the presence of fluorescence proteins. Sorted and puromycin-selected polyclones were subsequently used for the assays. Green detection reagent and DAPI (blue) nuclear stain. Cells treated with both conditioned media show increase in signal from apoptotic cells (green), with no significant difference between both groups. Scale bar:

500µm.