Andrés Alcover

Evaluating the ability of cytotoxic T lymphocytes (CTLs) to eliminate tumor cells is crucial, for instance, to predict the efficiency of cell therapy in personalized medicine. However, the destruction of a tumor by CTLs involves CTL migration in the extra-tumoral environment, accumulation on the tumor, antigen recognition, and cooperation in killing the cancer cells. Therefore, identifying the limiting steps in this complex process requires spatio-temporal measurements of different cellular events over long periods. Here, we use a cancer-on-a-chip platform to evaluate the impact of adenomatous polyposis coli (APC) mutation on CTL migration and cytotoxicity against 3D tumor spheroids. The APC mutated CTLs are found to have a reduced ability to destroy tumor spheroids compared with control cells, even though APC mutants migrate in the extra-tumoral space and accumulate on the spheroids as efficiently as control cells. Once in contact with the tumor however, mutated CTLs display reduced engagement with the cancer cells, as measured by a metric that distinguishes different modes of CTL migration. Realigning the CTL trajectories around localized killing cascades reveals that all CTLs transition to high engagement in the 2 h preceding the cascades, which confirms that the low engagement is the cause of reduced cytotoxicity. Beyond the study of APC mutations, this platform offers a robust way to compare cytotoxic cell efficiency of even closely related cell types, by relying on a multiscale cytometry approach to disentangle complex interactions and to identify the steps that limit the tumor destruction.

Evaluating the ability of cytotoxic T lymphocytes (CTLs) to eliminate tumor cells is crucial, for instance to predict the efficiency of cell therapy in personalized medicine. However, the destruction of a tumor by CTLs involves CTL migration in the extra-tumoral environment, accumulation on the tumor, antigen recognition, and cooperation in killing the cancer cells. Therefore, identifying the limiting steps in this complex process requires spatio-temporal measurements of different cellular events over long periods. Here, we use a cancer-on-a-chip platform to evaluate the impact of adenomatous polyposis coli (APC) mutation on CTL migration and cytotoxicity against 3D tumor spheroids. The APC mutated CTLs are found to have a reduced ability to destroy tumor spheroids compared with control cells, even though APC mutants migrate in the extra-tumoral space and accumulate on the spheroids as efficiently as control cells. Once in contact with the tumor however, mutated CTLs display reduced engagement with the cancer cells, as measured by a new metric that distinguishes different modes of CTL migration. Realigning the CTL trajectories around localized killing cascades reveals that all CTLs transition to high engagement in the two hours preceding the cascades, which confirms that the low engagement is the cause of reduced cytotoxicity. Beyond the study of APC mutations, this platform offers a robust way to compare cytotoxic cell efficiency of even closely related cell types, by relying on a multiscale cytometry approach to disentangle complex interactions and to identify the steps that limit the tumor destruction.

Familial adenomatous polyposis (FAP) is an inherited disease characterized by the development of large number of colorectal adenomas with high risk of evolving into colorectal tumors. Mutations of the Adenomatous polyposis coli (APC) gene is often at the origin of this disease, as well as of a high percentage of spontaneous colorectal tumors. APC is therefore considered a tumor suppressor gene. While the role of APC in intestinal epithelium homeostasis is well characterized, its importance in immune responses remains ill defined. Our recent work indicates that the APC protein is involved in various phases of both CD4 and CD8 T cells responses. This prompted us to investigate an array of immune cell features in FAP subjects carrying APC mutations. A group of 12 FAP subjects and age and sex-matched healthy controls were studied. We characterized the immune cell repertoire in peripheral blood and the capacity of immune cells to respond ex vivo to different stimuli either in whole blood or in purified T cells. A variety of experimental approaches were used, including, pultiparamater flow cytometry, NanosString gene expression profiling, Multiplex and regular ELISA, confocal microscopy and computer-based image analyis methods. We found that the percentage of several T and natural killer (NK) cell populations, the expression of several genes induced upon innate or adaptive immune stimulation and the production of several cytokines and chemokines was different. Moreover, the capacity of T cells to migrate in response to chemokine was consistently altered. Finally, immunological synapses between FAP cytotoxic T cells and tumor target cells were more poorly structured. Our findings of this pilot study suggest that mild but multiple immune cell dysfunctions, together with intestinal epithelial dysplasia in FAP subjects, may facilitate the long-term polyposis and colorectal tumor development. Although at an initial discovery phase due to the limited sample size of this rare disease cohort, our findings open new perspectives to consider immune cell abnormalities into polyposis pathology.

Adenomatous polyposis coli (APC) is a tumor suppressor whose mutations underlie familial adenomatous polyposis (FAP) and colorectal cancer. Although its role in intestinal epithelial cells is well characterized, APC importance in T cell biology is ill defined. APC regulates cytoskeleton organization, cell polarity, and migration in various cell types. Here, we address whether APC plays a role in T lymphocyte migration. Using a series of cell biology tools, we unveiled that T cells from FAP patients carrying APC mutations display impaired adhesion and motility in constrained environments. We further dissected the cellular mechanisms underpinning these defects in APC-depleted CEM T cell line that recapitulate the phenotype observed in FAP T cells. We found that APC affects T cell motility by modulating integrin-dependent adhesion and cytoskeleton reorganization. Hence, APC mutations in FAP patients not only drive intestinal neoplasms but also impair T cell migration, potentially contributing to inefficient antitumor immunity.

Adenomatous polyposis coli (APC) is a tumor suppressor whose mutations underlie familial adenomatous polyposis (FAP) and colorectal cancer. Although its role in intestinal epithelial cells is well characterized, APC importance for anti-tumor immunity is ill defined. APC regulates cytoskeleton organization, cell polarity and migration in various cells types. Here we address whether APC plays a role in T lymphocyte migration, a key step of anti-tumor immune responses. Using a series of cell biology tools, we demonstrated that T cells from FAP patients carrying APC mutations display adhesion and migration defects. Concomitantly, they presented lower expression of the integrin VLA-4. To further dissect the cellular mechanisms underpinning these defects, we depleted APC in the CEM T cell line. We found that APC is critical not only for VLA-4-dependent adhesion but also for actomyosin and microtubule organization in migrating T cells. Finally, APC-silenced CEM cells preferentially adopt an ameboid-like migration featuring unstructured pseudopodia and blebbing. These findings underscore a role of APC in T cell migration via modulation of integrin-dependent adhesion and cytoskeleton reorganization. Hence, APC mutations in FAP patients not only drive intestinal neoplasms, but also impair T cell migration, potentially leading to inefficient T cell-mediated anti-tumor immunity.

Adenomatous polyposis coli (Apc) is a cell polarity regulator and a tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Apc involvement in T lymphocyte functions and antitumor immunity remains poorly understood. Investigating Apc-depleted human CD8 T cells and CD8 T cells from Apc^Min/+ mutant mice, we found that Apc regulates actin and microtubule cytoskeleton remodeling at the immunological synapse, controlling synapse morphology and stability and lytic granule dynamics, including targeting and fusion at the synapse. Ultimately, Apc tunes cytotoxic T cell activity, leading to tumor cell killing. Furthermore, Apc modulates early TCR signaling and nuclear translocation of the NFAT transcription factor with mild consequences on the expression of some differentiation markers. In contrast, no differences in the production of effector cytokines were observed. These results, together with our previous findings on Apc function in regulatory T cells, indicate that Apc mutations may cause a dual damage, first unbalancing epithelial cell differentiation and growth driving epithelial neoplasms and, second, impairing T cell–mediated antitumor immunity at several levels.