Unraveling the spatial distribution of CAF subsets in PDAC spheroids through a novel spatial flow cytometry approach

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stromal compartment, predominantly composed of cancer-associated fibroblasts (CAFs), that contributes to immune exclusion and therapeutic resistance. Although the phenotypic and functional diversity of stromal cells within the TME is well characterized, their spatial distribution and the mechanisms driving this heterogeneity have not been thoroughly investigated. Here, we present SpheroMap Cytometry, an innovative spatial flow cytometry method that enables high-resolution analysis of spatially organized cellular phenotypes within spheroids. CAPAN-1 pancreatic tumor cells and either HS-5 bone marrow stromal or umbilical cord-derived mesenchymal stromal cells (UC-MSCs) were co-cultured in ultra-low-adhesion 96-well plates. After 48 hours aggregation, spheroids were incubated with Image-iT Green Hypoxia to mark hypoxic cells, and after 72 hours spheroids were dissociated and stained with antibodies against CD73 and CD140B. SpheroMap Cytometry revealed that the hypoxic core was significantly enriched for CD73 ⁺ and myCAF-like populations (CD140B ⁺ CD73 ^high ), indicating a functional link between hypoxia and this CAF subpopulation. Moreover, while hypoxia alone was sufficient to drive myCAF differentiation in heterotypic and monotypic spheroids, we found that normoxic induction of myCAF occurred only in the presence of tumor cells supporting the hypothesis that proximity to tumor cells synergizes with hypoxia to regulate CAF differentiation. Our findings demonstrate that hypoxia drives a distinct stromal architecture in PDAC. SpheroMap Cytometry provides a scalable, high-resolution method to dissect the spatial immunophenotype of 3D tumor models, overcoming limitations of static imaging and conventional flow cytometry, opening new avenues for preclinical assessment of stroma-targeting therapies and the development of immunotherapeutics that reprogram the TME.