Spatial Biology and Organoid Technologies Reveal a Potential Therapy-Resistant Cancer Stem Cell Population in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies with a 5-year survival rate of less than 10%. Chemotherapy is the current standard-of-care (SOC) for advanced PDAC; however, treatment resistance driven by a complex and immunosuppressive tumor microenvironment (TME) limits its effectiveness. Cabozantinib (cabo), a multi-kinase inhibitor has shown promise in preclinical PDAC models and clinical trials, particularly when combined with immunotherapy, but its mechanisms of action within the PDAC TME remain unclear. Using CosMx Spatial Molecular Imaging and Orion Multiplex Immunofluorescence, we analyzed PDAC tissues collected from patients after first line systemic chemotherapy and surgery, or liver metastatic sites. Matched patient-derived organoid (PDO) co-cultures harboring cancer associated fibroblasts (CAFs), and autologous immune cells (IMM) (PDO/CAF/IMM) were generated and used to evaluate the effects of cabo and anti-PD1 immune checkpoint inhibitor pembrolizumab. Spatial analyses of post-chemotherapy and metastatic PDAC tissues revealed heterogeneous cell neighbor-hoods within the TME, including enrichment of Schwann cells, CAFs, T regulatory cells, and cancer stem cells (CSCs). Distinct niches were observed in metastatic liver tissues, characterized by CSCs expressing CD44 and TROP2. In PDO/CAF/IMM co-cultures, treatment with cabo in combination with pembrolizumab enhanced cancer cell death by depleting myeloid-derived suppressor cells and promoting cytotoxic T lymphocyte proliferation. Both patient tissues and treated co-cultures showed a persis-tent SOC-resistant CSC population that expressed CD44 variant 9 (CD44v9). This in-tegrative spatial and organoid-based approach demonstrates that cabo potentially re-models the PDAC TME to potentiate PD-1 immunotherapy, while revealing therapy-resistant CD44v9+ CSCs as a potential therapeutic target.