Immune-driven stromal inflammation in pancreatic cancer within a microfluidic platform

Abstract Pancreatic ductal adenocarcinoma (PDAC) is defined by a highly fibrotic and immunosuppressive tumor microenvironment (TME). The inflammatory polarization of cancer-associated fibroblasts (CAFs), as well as the dense and early trafficking of myeloid-derived suppressor cells (MDSCs) contribute to tumor progression, immune exclusion, and therapy resistance. Yet, key mechanisms governing their interplay remain undefined. We developed a novel microfluidic platform that recapitulates the immuno–stromal PDAC microenvironment and enables controlled co-culture, high-resolution live imaging, and downstream molecular analyses. Spheroids composed of tumor cells, and dual reporter CAFs, were embedded in our engineered system and allowed to dynamically interact with MDSCs for 48 h. Dynamic interactions with MDSCs induce a significant increase in inflammatory CAF-specific reporter fluorescence compared to media controls. Gene set enrichment analysis demonstrated enrichment of inflammatory response pathways, and increased expression of iCAF related genes. RNA velocity, CellRank, and PAGA trajectory analyses collectively identify a constitutively fate-committed CAF progenitor population that is selectively expanded by MDSC exposure and preferentially transitions into an inflammatory iCAF state. Overall, our novel microfluidic platform recapitulated targeted MDSC-driven inflammatory CAF polarization and revealed a progenitor CAFs population as a potential precursor for iCAF differentiation, establishing its utility in mechanistic studies of stromal–immune interactions of the TME.