Abstract 4223: The dual immunestimulatory and immunesuppressive role of MRC5 fibroblast cells in 3D coculture with T cells and tumor cells

Abstract

Abstract Introduction: Cancer-associated fibroblasts (CAFs) alter anti-tumor immune responses and modulate resistance to immune checkpoint inhibitors. The mechanisms of crosstalk between tumor cells, CAFs, and T cells remain not fully understood, and studying these multicellular interactions requires adoption of novel tools in preclinical research. Here, we established a spheroid co-culture model comprising of tumor cell lines, a fibroblast cell line, and primary human T cells, for studying the immunomodulatory effects of fibroblasts on T cell responses in vitro, in the context of immunotherapies. Methods: For setting up the 3D co-culture, A375 or OV-90 tumor cells were seeded alone or with MRC-5 fibroblasts in 96-well ultra-low attachment round bottom plates in complete growth medium supplemented with 1% Geltrex matrix. The cells were centrifuged, and spheroids were allowed to form for 24 hours. After spheroid initiation, activated T cells and treatments with anti-PD1 antibody or an immune stimulatory drug were added. Spheroid co-cultures were live imaged by Incucyte SX5 for 72 hours taking images every 4 hours, after which treatment responses were evaluated by flow cytometry and measuring IFN-γ accumulation with ELISA. Results: To mimic fibroblast and T cell interactions in tumor microenvironment we built and optimized an in vitro 3D co-culture assay using various tumor cell lines. With this assay we found that depending on the tumor cell interaction, fibroblasts MRC-5 cell line have either immune-suppressive or immune-promoting properties. IFN-γ secretion of T cells in the assay indicated that MRC-5 cells suppressed T cells when co-cultured with A375 cells, while with OV-90 cells T cells activation was enhanced. In addition to the IFN- γ secretion, T cells in MRC-5 OV-90 interaction significantly upregulated 4-1BB expression on both CD4 & CD8 T cells. Interestingly we noticed that MRC-5 fibroblasts could modulate T cell responses to anti-PD-1 treatment in the co-culture depending on the tumor cell used. Conclusion: Here, we established a spheroid co-culture model, which will allow studies of the immunomodulatory crosstalk occurring in tumor microenvironment. Our model revealed a bifunctional role of MRC-5 fibroblasts in altering T cell phenotype. This finding is in line with literature highlighting the ability of fibroblasts to act as either tumor promoters or tumor suppressors depending on tumor microenvironment. We believe that this model, would aid in elucidating mechanisms of interaction between CAFs and T cells, potentially leading to discoveries of novel targets for immuno-oncology drug development. Additional studies, such as gene expression analysis using single cell RNA sequencing, are warranted to further validated the model and to better understand the kinetics of these interaction occurring in the co-culture with various tumor cell lines. Citation Format: Ilona Arnkil, Can Hekim, Elli Narvi, Emmi-Leena Ihantola, Endrit Elbasani, Anil Thotakura. The dual immunestimulatory and immunesuppressive role of MRC5 fibroblast cells in 3D coculture with T cells and tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4223.