Pancreatic ductal adenocarcinoma is characterized by a dense stroma predominantly formed by cancer-associated fibroblasts (CAFs). These cells are key drivers of tumor progression, immune exclusion and therapy resistance. While CAFs exist as two functionally distinct and reversible phenotypes—myofibroblastic (myCAFs) and inflammatory (iCAFs)—their spatial distribution and the mechanisms underpinning this heterogeneity remain poorly understood.

Here, we present SpheroMap Cytometry, an innovative spatial flow cytometry method that enables high-resolution analysis of spatially organized cellular phenotypes within spheroids. This approach leverages Image-iT Green Hypoxia, a fluorescent probe that accumulates in hypoxic cells, enabling spatial mapping of cells dissociated from spheroids.

Therefore, heterotypic spheroids composed of CAPAN-1 pancreatic tumor cells and HS-5 bone marrow stromal or umbilical-cord-derived mesenchymal stromal cells (UC-MSCs); or homotypic spheroids composed solely of HS-5 or UC-MSCs cells were cultured in ultra-low-adhesion 96-well plates. After 48h aggregation, spheroids were incubated with the hypoxia probe and after 72h they were dissociated and stained with CD73 and CD140B antibodies.

We identified a robust enrichment of CD73⁺/CD140B⁺ myCAF-like cells within the hypoxic core. In tumor-HS-5 spheroids, co-expression of CD73 and CD140B reached 59.27% in hypoxia vs 49.37% in normoxia (p < 0.01). Similarly, UC-MSC spheroids showed even stronger co-expression in hypoxia 80.57% vs 38.5% in normoxia (p < 0.0001). We observed the presence of a distinct subpopulation of small stromal cells (FSClow/SSClow) lacking expression of CD73 and CD140B. Upon exposure to hypoxia, these cells acquired CD73 and CD140B expression transitioning toward a myCAF phenotype as they also increased in size and complexity (FSChigh/SSChigh). Our findings point to the hypoxic tumor microenvironment (TME) not only as a trigger for phenotypic reprogramming, but also as a niche where immature or quiescent fibroblast cells are converted into immunosuppressive, tumor-supportive myCAFs. Moreover, while hypoxia alone was sufficient to drive myCAF differentiation in heterotypic (∼70%) and monotypic spheroids (∼40%), we found that normoxic induction of myCAF occurred only in tumor cells presence, reaching 47% (tumor–HS-5) and 30% (tumor–UC-MSCs), but dropping to ∼10% and ∼1% in monotypic spheroids.

Our results raise the possibility that the spatial proximity of myCAF cells to tumor cells may not result solely from paracrine signaling. Instead, it may reflect their shared occupancy of hypoxic tumor regions that actively promote a myCAF phenotype. This has profound implications for understanding how CAF heterogeneity is shaped and how spatial organization influences immunosuppressive dynamics within the TME. By preserving hypoxia-derived spatial information post-dissociation, SpheroMap Cytometry bridges the gap between functional imaging and high-throughput phenotyping. Overcoming limitations of both static microscopy and conventional flow cytometry, opening new avenues for preclinical assessment of stroma-targeting therapies and the development of immunotherapeutics that reprogram the TME. This study was funded by the São Paulo Research Foundation (FAPESP), Brasil, process #2022/12856-6; the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES); and the National Council for Scientific and Technological Development (CNPQ).