Circulating tumor cells (CTCs) represent a systemic phase of the localised cancer disease. They can be distinguished and enriched from the peripheral blood and so from the surrounding leukocytes by either physical properties (e.g., density and size) or biological properties (e.g., expression of epithelial proteins such as EpCAM or cytokeratins) and are usually further characterized by immunostaining or RT-PCR assays. Selecting patients with the risk of disease relaps at the time of diagnosis is crucial for clinicians in deciding who should, and who should not, receive adjuvant chemotherapy. We know that CTCs are strong prognostic factor in patients with metastatic as well as localized breast cancer (BC). It is also known that the prognostic power of circulating tumor cells in women with BC is independent from the standard prognostic indicators. Testing of CTCs known recently as "liquid biopsy" could be informative not only as predictor of the disease relapse, but also as the predictor of therapy effectiveness. The clinical use of CTCs must be strictly encouraged by clinical trials results. Monitoring of CTCs in time could zoom in the mechanism of therapy resistance and/or may provide the identification of new druggable targets. The purpose of my work was therefore to assess the CTCs positivity rate...

No description available.

Epigenetic alterations are observed in virtually all cancer types, yet there is limited understanding of their role in tumorigenesis and evolution. The role of DNA methylation has been particularly elusive in this context. While this epigenetic mark has been extensively profiled in healthy and cancerous samples, our ability to understand its relationship with underlying biological processes is still limited. Moreover, recent advancements in the profiling of cell-free DNA in circulation have sparked renowned attention toward tissue-specific and cancer-specific DNA methylation patterns. In this thesis, I present results to improve and refine the computational characterization of DNA methylation in cancer, focusing on metastatic castration-resistant prostate cancer. The first contribution is the development and performance assessment of Rockermeth, a computational methodology to leverage large-scale DNA methylation profiling data to nominate robust differentially methylated regions (DMRs). Rocker-meth can retrieve biologically relevant DNA methylation changes, as demonstrated by extensive integrative analyses with gene expression, chromatin states, and genomic annotations. The second contribution is the generation of a map of DNA methylation changes across prostate cancer progression. The application of Rockermeth and other tailored methodologies can be used to trace the critical evolutionary steps of this disease, from the healthy tissue to the most lethal metastatic AR-independent counterpart. The main result is the evidence of the ability of DNA methylation to capture a snapshot of the active transcription factors in each state of the disease, offering orthogonal information compared to standard genomic sequencing. The third contribution is the design and development of NEMO, a tailored liquid biopsy sequencing panel approach to allow non-invasive neuroendocrine castration-resistant prostate cancer detection in patients with metastatic disease. Based on previous results and the comprehensive analysis of multiple datasets, I designed a set of informative genomic regions to estimate disease burden and evidence of neuroendocrine transdifferentiation. The actual implementation of the NEMO panel produced a scalable and cost-effective strategy, which has been extensively benchmarked using both in silico and in vitro approaches. The application of NEMO to patient-derived cfDNA samples demonstrated accurate tumor content estimation and robust detection of neuroendocrine disease, making it a promising instance of liquid biopsy for CRPC.

Liquid biopsy and next-generation sequencing of cell-free DNA (cfDNA) in cancer patients’ plasma offer a minimally-invasive solution to detect tumor cell genomic information to aid real-time clinical decision-making. Reliability and sensitivity in the detection of genomic alterations is crucial for patient management and it is particularly relevant in the context of targeted therapies. However, biological and technical factors, such as low cfDNA tumor fraction and sequencing errors, affect the correct interpretation of genomic data limiting the utility of non-invasive cfDNA-based tumor profiling. To address these issues, we designed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation of patients’ tumor. The framework also implements ABEMUS, an ad-hoc computational procedure we specifically designed for cfDNA samples to accurately detect somatic point mutations that could emerge under treatment pressure and as drug resistance mechanism. When applied on serial plasma samples from three patients receiving PARP inhibition harboring DNA repair gene aberrations, PCF_SELECT demonstrated high sensitivity in detecting BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. As a step towards a more sensitive detection of tumor features in circulation of cancer patients, we next hypothesized that recent WGS-based approaches exploiting cfDNA fragments characteristics could be extrapolated for targeted sequencing data and that gene-region specific measures could improve detection metrics. Preliminary results suggest an increased sensitivity compared to copy-number-based methods supporting the integration at no extra cost in our targeted assay. Overall, this work is relevant to the context of precision oncology. It provides an innovative platform for the management of cancer patients, delivering detailed patient-specific molecular information which could be applied to guide treatment and improve clinical outcomes.

Early detection of breast cancer is critical for increasing survival rates. However, currently available screening strategies provide ambiguous results, leaving invasive tissue biopsy procedures necessary for definitive diagnosis. Considerable efforts have investigated using nipple aspirate fluid (NAF), a liquid biopsy rich in proteins representative of the breast microenvironment, as a non-invasive source of early detection biomarkers. However, by using traditional two-dimensional discovery proteomic approaches, many technical challenges of using NAF have limited analysis of large sample sizing: such as low expressed volume (<10µL) or insufficient analytical material (<200µg protein). Following non-invasive collection by manual massage, we developed a one-dimensional sample preparation workflow that reduced sample handling steps, minimised sample losses and increased sample throughput to 96-samples by using a PVDF-membrane based system, which was ideally suited to the NAF proteome. Samples were prepared within a single working day, and results correlated significantly with conventional in-solution protocols. ​Proteins typically associated with the dysregulation of innate immune response and haemostatic pathways had a significantly altered proteome profile in response to breast cancer. Overall, our new workflow will allow future studies to take a more high-throughput approach, revealing biomarkers for breast cancer early detection, and providing a real impact.

Extracellular vesicles (EVs) are small membrane-surrounded structures containing transmembrane proteins and enclosing cytosolic proteins and nucleic acids. They are released in the extracellular space by both normal and neoplastic cells and play an important role in cell-cell communication in numerous physiological processes and pathological conditions, through the transfer of their functional cargo to recipient cells. EVs are highly abundant in biological fluids, and even more represented in cancer patients’ biofluids, therefore many studies suggested that they can be instrumental in liquid biopsies as prognostic markers or for early detection of tumors. Moreover, being secreted by potentially all the cells, they can serve in oncology to represent the tumor heterogeneity, which is underestimated by the current diagnostic tools. Given their small size, EVs are difficult to isolate in a high-throughput way and, therefore, one of the main obstacles to their clinical application, is that the existing isolation methods are impractical. During these years, I worked at the development and optimization of a novel technique that allows purification of heterogeneous EVs from biological fluids in an efficient, fast and reproducible way. This technique, named Nickel-Based Isolation (NBI), is a biochemical assay that allows obtaining polydisperse EVs in a physiological pH solution, therefore, preserving their morphology, heterogeneity, and stability. We tested and optimized this assay in protein-enriched systems and comparing it to the techniques currently used to characterize and measure EVs, such as flow cytometry and Tunable Resistive Pulse Sensing. We challenged the reproducibility of this method by isolating EVs from different biological fluids. Interestingly, the EVs purified with NBI result more intact and stable compared to the ones obtained with other methods, and can be studied in a clinical setting and used as an innovative tool for detection of molecules associated with diseases. We demonstrated the specificity of the procedure by using individual isolated vesicles in biochemical and molecular assay, optimized to characterize the biological content of EVs. We were able to detect picomolar concentration of PSMA on 105 EVs isolated from plasma of prostate cancer patients and BRAF-V600E transcript in just 103 EVs from the plasma of colon cancer patients, reaching unprecedented matching with tissue biopsy results. We also investigated the transcriptome of EVs isolated from glioblastoma cancer stem cells, in order to exploit the potential of EVs as diagnostic markers.

Ce travail de thèse s'est axé autour de deux approches technologiques issues du domaine de la microfabrication pour la sélection et la capture de biomarqueurs circulants dans le sang. A l'échelle moléculaire, un module d'assemblage capillaire dirigé a été implémenté dans un automate de tamponnage moléculaire puis validé en utilisant un modèle simple, permettant l'isolement et l'étirement de biomolécules individuelles de manière entièrement contrôlée et automatisée à large échelle. Nous avons ensuite appliqué cette technologie à des biomarqueurs moléculaires d'intérêt tels que les ADN libres contenus dans du sang complet, démontrant la capacité de la technique à isoler des acides nucléiques dans un fluide complexe contenant de nombreux éléments cellulaires . A l'échelle cellulaire, une approche innovante pour la sélection et la capture de Cellules Tumorales Circulantes a été développée. Le microdispositif mis au point est fabriqué par écriture laser à 3 dimensions et permet le piégeage physique de ces cellules dans du sang complet non traité tout en les préservant pour une récupération et analyse ultérieure. Après adaptation du microdispositif pour maximiser son efficacité de capture in vitro, une première preuve de concept de capture sélective de cellules cancéreuses dans du sang complet non traité a été réalisée. U n premier prototype pour une utilisation in vivo a été mi s au point et validé in vitro sur la capture de cellules cancéreuses dans du milieu de culture. / This research project focused on two technological approaches emerging from microfabrication for the selection and capture of circulating biomarkers from blood. At the molecular scale, this work was based on the automation of a directed capillary assembly protocol. A dedicated module was implemented into an automate for molecular stampin g and validated using a simple molecular model, allowing the elongation and large-scale assembly of single biomolecules in a controlled and automatized manner. The developed technology was then used for the assembly of relevant molecular biomarkers such as cell -free DNA (cf DNA) from untreated whole blood , evidencing the capabilities of this technology to single out nucleic acids from complex fluids composed of other cellular elements. At the cellular scale, an innovative concept for Circulating Tumor Cell s (CTCs) selection and capture was developed . The developed microdevice is fabricated using 30 direct laser writing and allows for a physical capture of cell s from untreated whole blood while preserving them for further recovery and analysis. After having optimized the design in vitro to maximize the capture efficiency of the system, a selective capture of cancer cell s from untreated whole blood was achieved . A first prototype for the in vivo use of this system was also developed and validated in vitro with cancer cells spiked into culture medium.

No description available.

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. [...]

<div>Rare circulating cells are becoming a subject of interest due to their potential clinical applications to replace invasive procedures. Due their low presence in blood (as low as 1 in 1 ml of blood) various platforms are developed to capture and isolate them. Common limitations of current platforms include the inability to process large volumes of blood without an initial volume reduction step such as centrifugation, reliance on a single antibody for the capture, and the difficulty of releasing and retrieving the captured cells with high purity. A rare cell retrieval platform with high throughput operation and high purity retrieval is needed to capture these rare cells by processing large volumes of blood.</div><div><br></div><div>In this thesis study, we have developed a two-tiered microchip system to capture and retrieve rare cells from blood samples with high purity. The first module of the system is a high throughput microfluidic interface that is used to immunomagnetically isolate targeted rare cells from whole blood, and discard > 99.999% of the unwanted leukocytes. The second module is a microwell array that furthers the purification by magnetically guiding each cell into a separate well concurrently, and allows individual retrieval of each cell. Even though the system we have developed is applicable to many fields pertaining to rare cell capture, here we demonstrate the proof-of-concept using model cell lines that represent circulating fetal trophoblasts. We describe the design, operation as well as the experimental characterization of the system. Our characterization results show that the process can be completed within 145 minutes from the very beginning till the retrieval of a target cell, and can provide efficiencies and purities that are as high as 100%. </div><div><br></div><div>In order to demonstrate a real-world use case for our device, we present preliminary experiments done with blood samples from pregnant women. We show that we are able to retrieve candidate fetal cells under 167 minutes. Future work will be focused on sequencing the candidate fetal cells retrieved from maternal samples to confirm their fetal origin as well as enhancing system performance in maximizing the number of cells captured.</div><div><br></div>