Par définition, une métastase est la formation de foyers tumoraux secondaires liée à la capacité des cellules tumorales à se détacher de la tumeur primitive, de s’implanter dans un autre organe et de proliférer. A partir du foyer primitif, il peut y avoir une dissémination micrométastatique c’est-à-dire la libération dans le sang ou le réseau lymphatique de cellules tumorales isolées ou de petits amas cellulaires. Ces micrométastases peuvent proliférer et donner naissance à des métastases. La difficulté est de déceler ces cellules tumorales isolées ou en micro-amas, et également d’évaluer leur valeur pronostique, ainsi que leur potentiel métastatique. Dans cette étude, nous allons nous intéresser plus particulièrement au processus métastatique lié à l’acte chirurgical dans les cancers epdermoïde des voies aérodigestive supérieures (CEVADS). En effet, dans ce type de cancers, la meilleure méthode pour établir un diagnostic est l’analyse anatomo-pathologique du prélèvement réalisé par biopsie. La principale barrière physique empêchant les cellules tumorales de migrer est la membrane du tissu atteint. Lors de la chirurgie, ces barrières sont détruites, ce qui facilite l’invasion du système vasculaire. Les cellules tumorales peuvent ainsi se loger dans les vaisseaux et proliférer à distance du site primitif pour former des tumeurs secondaires. Généralement, les métastases sont détectées par imagerie ou sérologie à un stade très avancé du cancer. L’objectif de cette étude est de détecter la présence de cellules isolées ou disséminées (CTCs) de CEDVADS dans la circulation sanguine a travers trois axes de recherche des métastases : 1) Étude des marqueurs moléculaires pour le diagnostic d’envahissement ganglionnaire des carcinomes épidermoïdes des voies aérodigestives supérieures réalisée par PCR quantitative en temps réel et en OSNA. 2) Recherche des cellules tumorales disséminées dans les drains de Redon après curage cervical 3)Recherche des cellules tumorales circulantes après acte chirurgical pour carcinomes épidermoïdes des VADS stade III et IV. Ce protocole nous permettra de valider la recherche des CTCs en situation clinique et par la suite de développer des études prospectives diagnostiques et pronostiques des CTCs des CEVADS / Metastasis is defined as the development of secondary tumor sites related to the ability of tumor cells to detach from primary tumor, to implant in another organ and to proliferate. From the primary site, a micrometastatic dissemination can occur through the release in blood stream or lymph system of isolated tumor cells or of small cell clusters. These micrometastases can proliferate and grow into metastases. The detection of isolated or microclustered tumor cells, the evaluation of the prognosis value, and their metastatic potential encounter difficulties. In this study, we focused mainly on the metastatic process related to surgery in epidermoid cancers of the upper aerodigestive tract (…). Indeed, in this type of cancers, the best method to establish diagnosis is the biopsy assessed by the anatomo-pathological analysis of a sample. The main physical barrier preventing cells from migrating is the membrane of the malignant tissue. During surgery, these barriers are destroyed, facilitating the invasion of the vascular system. Therefore tumor cells can locate in vessels and proliferate at distance from the primitive site, thus forming secondary tumors. Generally, metastases are detected by imaging or serology at a very advanced stage of cancer disease. The aim of this study was to detect isolated or disseminated cells (CTCs) of CEDVADS in blood stream by three different approaches : 1) the study of molecular markers for the diagnosis of node involvement of epidermoid carcinomas of the upper aerodigestive tract using quantitative PCR in real time and OSNA. 2) Screening of tumoral cells disseminated in Redon drains after cervical curettage 3) Detection of circulating tumor cells after surgery for epidermoid carcinomas of stage III and IV VADS. This protocol will allow to validate the detection of CTCs in clinic setting and to develop prospective studies for the diagnosis and prognosis of CTCs of CEVADS

The improvement of blood cell sorting techniques in recent years have attracted the attention of many researchers due to the possible benefits that these methods can lead in biology, regenerative medicine, materials science and therapeutic area. In this work a cell sorting technique based on filtration is described. The separation occurs by means of a microfluidic device, suitably designed, manufactured and tested, that is connected to an external experimental set-up. The fabrication process can be divided in two parts: at first it is described the manufacturing process of a filtering membrane, with holes of specific size that allow the passage of only certain cell types. Following the microfluidic device is fabricated through the mechanical micromilling. The membrane and the microdevice are suitably bonded and tested by means of an external connection with syringe pumps that inject blood samples at specific flow rates. The device is designed to separate blood cells and tumor cells only by using differences in size and shape. In particular during the first experiments red blood cells and platelets are sorted from white blood cells; in the other experiments red blood cells and platelets are separated from white blood cells and tumor cells. The microdevice has proven to be very efficient, in fact a capture efficiency of 99% is achieved. For this reason it could be used in identification and isolation of circulating tumor cells, a very rare cancer cell type whose presence in the bloodstream could be symptom of future solid tumor formation. The various experiments have also demonstrated that tumor cells survive even after the separation treatment, and then the suffered stress during the sorting process does not harm the biological sample.

Les cellules tumorales circulantes (CTCs) sont la principale voie de dissémination du cancer dans le corps humain au travers de la circulation sanguine. Ces cellules ont la capacité de se détacher de la tumeur primaire, de rejoindre la circulation sanguine et de survivre dans cet environnement. Une sous-population spécifique de ces cellules a la capacité de coloniser de nouveaux tissus et de former des métastases. L'importance de ces cellules rares dans la circulation sanguine a été intensément étudiée au cours des dernières décennies, et il a été constaté que les informations phénotypiques et génomiques qu'elles contiennent pourraient être corrélées avec celles obtenues à partir d'une biopsie tissulaire. De plus, le nombre et l'incidence des CTC chez les patients métastatiques pourraient être utilisés comme indicateurs pronostics. Ainsi, leur isolement à partir d'échantillons sanguins et leur analyse a été proposé en remplacement des biopsies conventionnelles, comme une alternative moins invasive et permettant un échantillonnage plus répété. In fine, la détection et l'analyse des CTC en routine clinique pourraient être utilisées pour le suivi en temps réel des thérapies et de leur efficacité pour améliorer la prise en charge des patients, un pas de plus vers une médecine de précision. Dans ce projet de thèse, nous avons développé de nouveaux micro-dispositifs pour la capture, sous flux, de cellules cancéreuses à partir de sang complet humain. Nous avons exploité les propriétés physiques des CTC, plus grandes et moins déformables que les cellules sanguines normales, pour discriminer ces cellules rares (<1 cellule par mL aux premiers stades de la maladie). Des micro-dispositifs ont été conçus tels des tamis à trois dimensions pour filtrer sélectivement les cellules cancéreuses tout en préservant l'intégrité et la viabilité des cellules. De plus, les dispositifs ont été conçus pour permettre l'accès au matériel biologique isolé et effectuer ainsi une identification des cellules in situ, e.g. par immunocytochimie, mais aussi potentiellement pour servir de plateforme pour une analyse fonctionnelle de ces cellules. Nous avons proposé deux approches totalement compatibles avec la routine clinique. La première consiste en un guide équipé de microdispositifs, conçu pour être introduit directement dans la circulation sanguine au travers d'un cathéter médical et effectuer la capture des cellules cancéreuses in vivo. La deuxième approche vise à réaliser l'isolement des CTCs en utilisant des microdispositifs intégrés à des plateformes ex vivo compatibles avec les consommables médicaux de prélèvement sanguin.[...] / Circulating tumor cells (CTCs) are believed to represent the main pathway of cancer dissemination in the human body through the circulatory system. These cells have the ability to detach from the primary tumor, enter into the bloodstream, and survive in this environment. A specific subpopulation of these cells possesses the capacity of colonizing new tissues and forming metastases. The relevance of these rare cells in the bloodstream has been intensively investigated during the last decades, finding that phenotypic and genomic information they carry could be correlated with that of solid biopsies. Moreover, the number and incidence of CTCs in metastatic patients could be used as an indicator for prognosis. Thus, their isolation from blood samples and analysis has been proposed as a surrogate to solid biopsies, having the added value of being a less invasive procedure and allow a more repeated measure. In fine, the routine analysis of CTCs in clinical practice could be used for the real-time monitoring of therapies and the adaptation of treatment in order to improve the outcome of patients, a step forward towards so-called precision medicine. In this PhD project, we have developed novel micro- devices for the capture, in flow conditions, of tumor-derived cells from human whole blood. CTCs being larger and less deformable than normal blood cells, we exploited theses physical traits to discriminate them. Sieve-like micro-devices were engineered to selectively sort out tumor-derived cells having as a priority the preservation of cell integrity and viability. In addition, devices were designed to allow direct access to the isolated biological material and thus perform in situ cell identification, such as immunocytochemistry, but also to potentially serve as a platform for functional analysis. We proposed two approaches compatible with clinical routine. The first approach consists in a customized guiding-strip equipped with integrated microfilters, designed to be introduced directly within the bloodstream through a conventional medical catheter to perform the capture of tumor-derived cells in vivo. The second approach aims to perform CTC isolation ex vivo through the integration of microfilters into a platform compatible with blood collection medical sets. [...]

Attachment of circulating tumor cells in microfluidic devices functionalized with proper antibodies was studied. Under static experimental conditions, microchambers were utilized to study the parameters such as cell suspension concentration, incubation time or ambient temperature that may affect the binding of cell to the functionalized surfaces. Specific capture of cells from suspensions increases exponentially with incubation time and linearly with concentration within the tested range. Functionalizing a surface with counter-receptors enables capture of almost 100% of cells within 15 minutes incubation time at ambient temperature higher than 25°C. Suspending cells with different receptors, changing the counter receptors immobilized on the surface, or incubation the cell suspension at low ambient temperature result in a poor capture ratio. To illustrate the specific binding of target cells, various binary mixtures of target cancer and blood cells were incubated in the microchambers. The microsystem sensitivity, specificity and accuracy were determined as a function of the incubated cell concentrations. In general, the system specificity increases while the sensitivity decreases with increasing cell concentration; the accuracy of the system depends weakly on cell concentration within the tested range. The cell attachment dynamics in shear flow was studied by driving the MDA-MB- 231 or BT-20 cells through microchannels functionalized with EpCAM antibodies. The cell attachment ratio was experimentally determined at different flow rates. A modeling system based on Stokesian as well as cell-adhesive dynamics is adopted to analyze the cell motion. The cell motion is modeled as a rigid sphere, with receptors on its surface, moving under shear flow above a surface immobilized with ligands. The system is described mathematically by the Langevin equation, in which the receptor-ligand bonds are modeled as linear springs. Primarily depending on the applied flow rate, three distinct dynamic states of cell motion have been observed: free motion, rolling adhesion, and firm adhesion. The fraction of cells captured due to firm adhesion, defined as attachment ratio, depends on the applied flow rate with a characteristic value that increases with either cellreceptor or surface-ligand density. Utilizing this characteristic flow rate as a scaling parameter, all measured and calculated attachment ratios for different receptor and ligand densities collapse onto a single exponential curve. Binary mixtures of target MDA-MB-231 cells and non-target BT-20 cells were driven through anti-cadherin-11 functionalized microchannels to study the selective isoaltion of target cells from binary mixtures. The system sensitivity is very high, above 0.95, while the specificity is only moderately high, about 0.85, essentially independent of the relative concentration of the target and non-target cells in the binary mixture. An attachment/detachment flow field pattern is proposed to enhance the system specificity. Utilizing this flow pattern with a 1:1,000 MDA-MB-231:BT-20 binary cell mixture, the microfluidic system specificity increased to about 0.95 while the sensitivity remained above 0.95. In order to obtain high experimental throughput allowing lower relative concentration of target cells, a microchannel array which enables processing samples containing about 510⁵ cells with a minimum target cell concentration ratio of 1/100,000 was designed and fabricated. To demonstrate selective isolation of target cells, binary mixtures of BT-20 cells and MIA PaCa-2 cells were driven through microchannel arrays functionalized with EpCAM antibodies; the EpCAM positive BT-20 cells served as target cells and the EpCAM negative MIA PaCa-2 cells as non-target cells. The relative concentration ratio of target/non-target cells varied from 1:1 to 1:100,000. The sensitivity was close to 1.0 while the specificity was also high, about 0.95. The additional detachment step, with a faster flow rate, enhanced the specificity to about 0.985. Initial results of two sets of experiments are reported as preliminary studies for future work. In the first set of experiments, whole blood samples from healthy donors were spiked with a known number of BT-20 cells at a concentration of 500 CTCs per milliliter blood or 50 CTCs per milliliter blood. After a pretreatment to enrich the CTCs, the samples were driven through microchannel arrays functionalized with anti-EpCAM. For both samples, around 55% of the target CTCs were captured in the microchannel arrays. The second set of experiments was dedicated to characterization of target cells exposed to applied shear stress. BT-20 or MDA-MB-231 cells were driven through microchannels functionalized with EpCAM antibodies to allow target cell attachment; then, a high flow rate was applied to detach the captured cells. The detached cells were collected and cultured in an incubator to test their viability. For both cell lines, the majority of the captured CTCs collected from the microchannels were viable. The images taken after three and seven days of culture demonstrate continuous cell growth and division.

Circulating tumor cells (CTCs) are a potential predictive surrogate marker for disease monitoring. Due to the sparse knowledge about their phenotype and its changes during cancer progression and treatment response, CTC isolation remains challenging. Here we focused on the mechanical characterization of circulating non-hematopoietic cells from breast cancer patients to evaluate its utility for CTC detection. For proof of premise, we used healthy peripheral blood mononuclear cells (PBMCs), human MDA-MB 231 breast cancer cells and human HL-60 leukemia cells to create a CTC model system. For translational experiments CD45 negative cells—possible CTCs—were isolated from blood samples of patients with mamma carcinoma. Cells were mechanically characterized in the optical stretcher (OS). Active and passive cell mechanical data were related with physiological descriptors by a random forest (RF) classifier to identify cell type specific properties. Cancer cells were well distinguishable from PBMC in cell line tests. Analysis of clinical samples revealed that in PBMC the elliptic deformation was significantly increased compared to non-hematopoietic cells. Interestingly, non-hematopoietic cells showed significantly higher shape restoration. Based on Kelvin–Voigt modeling, the RF algorithm revealed that elliptic deformation and shape restoration were crucial parameters and that the OS discriminated non-hematopoietic cells from PBMC with an accuracy of 0.69, a sensitivity of 0.74, and specificity of 0.63. The CD45 negative cell population in the blood of breast cancer patients is mechanically distinguishable from healthy PBMC. Together with cell morphology, the mechanical fingerprint might be an appropriate tool for marker-free CTC detection.

Nous proposons, dans cette thèse, d'utiliser les techniques de modélisation en pharmacométrie selon l'approche de population afin de décrire les cinétiques de plusieurs marqueurs tumoraux sériques, et d'analyser leurs potentielles applications. Dans un premier temps, nous avons construit un modèle reliant les cinétiques de taille tumorale et de CA-125 dans le cancer de l'ovaire. Nous avons ensuite évalué son application pour : i) la prévision de la réponse tumorale au niveau individuel ; ii) la prédiction précoce de la survie au niveau d'une population dans le développement du médicament. Dans un second temps, nous avons réalisé un travail plus méthodologique sur la modélisation des cinétiques conjointes de PSA et d'un nouveau marqueur, le nombre de cellules tumorales circulantes (CTCs), dans le cancer de la prostate. Un modèle atypique combinant plusieurs innovations en pharmacométrie a été développé. En perspective, un lien va être établi avec la survie. En conclusion, la modélisation mathématique est un outil efficace pour l'évaluation précoce de l'efficacité des traitements / Our thesis project aimed at building mathematical models, using population approach, for different serum tumor markers, in order to describe their kinetics and to assess their potential applications. In a first intent, we built a semi-mechanistic model linking tumor size changes and CA-125 kinetics induced by chemotherapy in ovarian cancer patients. This model allowed assessment of CA-125 as: i) a biomarker for tumor size dynamics and treatment efficacy for clinical purposes; ii) an early predictor of clinical benefit during drug development. Then, we realized a more fundamental work by developing a semi-mechanistic model for characterizing the relationships between PSA kinetics and circulating tumor cell count dynamics during treatment in metastatic prostate cancer patients. This is an atypical model combining several advanced features in pharmacometrics. We have planned to assess a link with survival. In conclusion, mathematical modeling could be an efficient tool for the early prediction of treatment efficacy