A quantitative MDSC infiltration assay in a tumor organoid-stromal microenvironment for immuno-oncology candidate evaluation

Background: Myeloid-derived suppressor cells (MDSCs) are key drivers of immunosuppression and therapy resistance in cancer, but practical, human-relevant assays to quantify MDSC function and trafficking remain limited. Methods: We established a THP-1-derived MDSC-like cell platform using a cytokine-driven differentiation condition and validated phenotype and immune-related gene expression by flow cytometry and qRT-PCR. Immunosuppressive function was tested using PBMC/T-cell clustering assays, including direct co-culture and conditioned-medium exposure. To model migration, we developed a Matrigel-coated transwell infiltration assay (5-µm pore) in which MDSC-like cells were seeded in the upper chamber and colorectal cancer (CRC) organoids and/or cancer-associated fibroblasts (CAFs) were placed in the lower chamber. Infiltrated cells were quantified by imaging-based counting and flow cytometry. Finally, candidate MDSC-targeting agents were profiled for selective effects using CellTiter-Glo 3D and for inhibition of MDSC infiltration in the transwell system. Results: THP-1-derived MDSC-like cells showed an MDSC-consistent phenotype and immune-regulatory gene signatures, and were distinct from conventional THP-1 macrophage differentiation conditions. Functionally, these cells suppressed T-cell clustering through both contact-dependent and soluble-factor-mediated mechanisms. The transwell assay enabled quantitative measurement of MDSC infiltration toward organoids, CAFs, or their combination, and provided orthogonal readouts by imaging and FACS. Drug profiling revealed differential activity across organoids, MDSC-like cells, and CAFs, and several candidates modulated the extent of MDSC infiltration in a concentration-dependent manner. Conclusions: We present an integrated organoid-based workflow that combines MDSC-like cell generation, immunosuppressive function assays, and quantitative infiltration readouts in a CRC organoid-CAF microenvironment. This platform can support mechanism-informed screening and preclinical evaluation of MDSC-modulating strategies in a human-relevant setting.