Etude de l'impact d'adenomatous polyposis coli sur la cytotoxicité des cellules T : modèles d'immuno-oncologie murin et humain dans des gouttes microfluidiques

Cytotoxic T lymphocytes (CTLs) are key actors of the anti-tumor immune response that can directly kill tumor cells and are often targeted by immunotherapies. Therefore, it is crucial to develop tools to evaluate their efficiency. Most of the existing immuno-oncology models either lack the 3D aspect of the tumor or are not suited for understanding individual cell interactions. During my PhD, I used a microfluidic system to coculture 3D tumor models with CTLs encapsulated in droplets. A multi-anchor microfluidic chip is used to add CTLs at a specific moment and to follow the droplet content in time. Coupled with live microscopy and live/dead assay, this platform allows to correlate CTL dynamics with tumor cell death in tens of identical replicates in parallel. This system is applied to evaluate the impact of adenomatous polyposis coli (APC) mutation on CTL anti-tumor function.APC is a large multi-domain protein involved in multiple cellular functions such as adhesion, proliferation, polarization, and differentiation. It is one of the key components of the Wnt/β-catenin pathway and is mutated in >80% of colorectal cancers (CRC). Recent evidence suggests that the germline mutation of APC, leading to the familial adenomatous polyposis syndrome, can cause defects in the T lymphocytes functions by decreasing the stability of the immunological synapse and affecting their migration. However, how these effects combine to affect CTL anti-tumor function remains unclear.I used the microfluidic droplet system to coculture primary OT-I CTLs from APC(Min/+) mice with 3D spheroids of B16-OVA murine melanoma cells. The results revealed decreased cytotoxicity of APC mutated T cells compared to WT T cells. Tracking of single-cell trajectories revealed no impact of APC on CTL migration in the extracellular matrix and accumulation on target tumors. However, once in contact with the tumor cells, mutated CTLs displayed a reduced engagement, measured from their trajectories. Realignment of CTL trajectories with apoptotic regions of the spheroids revealed a transition to high engagement in the 2 hours preceding the apoptosis. This result suggests that the low engagement of APC mutated cells is the cause of their reduced cytotoxicity, in line with the reported low stability of the immunological synapse.Then, I translated this system to the coculture of human CRC patient-derived organoids (PDO) with CAR-T cells. After validation of the protocol, I modified CRC PDOs using Crispr-Cas9 to create APC knock-out PDO lines and used this system to investigate the impact of APC mutation in CRC on CAR-T cell-mediated cytotoxicity. The impact of APC appears to be limited in this system, potentially due to an already overactivation of the Wnt/β-catenin pathway in CRC. Therefore, targeting downstream effector proteins is proposed as a potential promising way to increase CAR-T cell efficiency.Altogether, this PhD work shows how the use of microfluidic droplets, live imaging, cell tracking, and cell engineering can be coupled to understand the influence of a genetic modification on CTL anti-tumor function. The data analysis allows to disentangle complex interactions and to identify the steps that limit tumor destruction.Finally, in a separate project, an image analysis toolbox is proposed to segment and classify single cells in fetal liver tissues composed of tens of thousands of cells. These tools are used to describe the spatiotemporal organization of the organ as well as the interplay between the hematopoietic and the stromal compartment during liver development. The results suggest that the periphery of the tissue might be an important region supporting hematopoiesis during mouse development. This work offers relevant tools that can be used to understand spatial organization and cell-cell interactions in complex tissues.