Development of a patient-derived pancreatic cancer organoid-CAF coculture model to study tumor microenvironment influence on radiotherapy response

Introduction: Radiotherapy (RT) is a fundamental treatment modality for pancreatic ductal adenocarcinoma (PDAC) at all disease stages. However, local relapse, driven by treatment resistance, remains a critical issue for improving patient outcomes. While tumor cell-intrinsic factors contribute to resistance, evidence indicates that the tumor microenvironment (TME), particularly cancer-associated fibroblasts (CAFs), plays a crucial role through different mechanisms. Current patient-derived organoid (PDO) models fail to authentically recapitulate CAF-tumor interactions and their influence on RT response, limiting their translational relevance. We developed a coculture system to better mimic these interactions and enable physiologically relevant radiobiological studies. Methods: PDOs of PDAC and primary CAFs were cocultured in 3D suspension in ultra-low attachment plates with 300ug/mL of Matrigel. The structural integrity and composition of the resulting aggregates were characterized using H&E, Masson's trichrome, PAS-Alcian blue staining, and immunofluorescence (IF) for vimentin, pan-cytokeratin, phalloidin, and DAPI. To test the utility of this model for RT studies, cocultures were exposed to a single radiation dose of 8 Gy. Radiobiological effects were evaluated 12 days post-treatment using H&E, immunohistochemistry for Ki-67 and p16, IF (gamma-H2AX), and calcein-AM/propidium iodide viability assay. Results: The established protocol generated aggregates with organized epithelial-stromal compartmentalization, confirmed by IF and histological analysis. Following a single 8 Gy radiation dose, cocultures exhibited reduced aggregate size and decreased cellular density within the first 12 days post-treatment, with significantly impaired growth kinetics compared to untreated controls. Histological analysis of irradiated samples showed disrupted matrix integrity and increased cellular fragmentation. Elevated gamma-H2AX foci indicated DNA double-strand breaks, while reduced Ki-67 and increased p16 staining reflected decreased proliferation and increased senescence. Conclusion: This work establishes a direct coculture model of patient-derived PDAC organoids with CAFs, providing a platform to characterize RT response in a compartmentalized tumor-stroma context. Our findings reveal compartment-specific responses and differential growth dynamics, highlighting the utility of integrated tumor-stromal models for investigating the effects of RT and advancing toward more physiologically relevant preclinical approaches. Funding: ANID/FONDECYT Grants #1241269 and 1221253, ANID/FONDAP Grant #152220002.