The failure of immunotherapies in solid tumors, including pancreatic ductal adenocarcinoma (PDAC), is often attributed to poor immune cell infiltration and a highly immunosuppressive tumor microenvironment (TME). PDAC, in particular, is characterized by a dense stromal matrix and hypoxic gradients that create physical and functional barriers to immune cell entry. Developing therapies that enhance T-cell infiltration into solid tumors requires preclinical platforms that recapitulate key features of the immunocompetent TME.

Here, we present SpheroMap Cytometry, an innovative spatial flow cytometry method designed to quantitatively assess immune cell localization within tumor spheroids under physiologically relevant conditions.

Heterotypic spheroids were generated by co-culturing CAPAN-1 pancreatic tumor cells with either HS-5 bone marrow stromal or umbilical cord-derived mesenchymal stromal cells (UC-MSCs). After 24 hours aggregation 100,000 PBMCs (pre-activated or not) were added to infiltrate spheroids. The Image-iT Green Hypoxia probe was applied to define hypoxic regions, and after 72 hours spheroids were enzymatically dissociated and analyzed for T-cell markers (CD3, CD4, CD8, CD25, CD127) using flow cytometry.

In tumor-HS-5 spheroids, pre-activation of PBMCs increased CD3⁺ infiltration into the hypoxic core (79.3% vs. 66.2% p < 0.01), doubled the proportion of CD8⁺ cells (46.1% vs. 24.8%), and significantly raised CD8⁺CD25high cells (39.8% vs. 9.7% p < 0.001). However, this was accompanied by a rise in CD4⁺CD25⁺CD127⁻ regulatory T cells (Tregs) from 18.3% to 48.9% (p < 0.001), indicating that T-cell activation enhances cytotoxic infiltration but also promotes suppressive phenotypes. In contrast, spheroids formed with UC-MSCs showed reduced CD8⁺ infiltration and enhanced Treg induction, consistent with the known immunomodulatory effects of mesenchymal cells. Notably, CD8⁺ infiltration and activation profiles differed significantly between normoxic and hypoxic compartments, demonstrating that oxygen gradients critically modulate immune cell function and localization. In another study (abstract is also being presented at this event) using SpheroMap Cytometry technique, we found that hypoxia also modulates the stromal component, favoring the emergence of cancer-associated fibroblasts with a myofibroblast profile, which contributes to desmoplasia through extracellular matrix deposition, leading to immunosuppression by excluding immune cells.

These findings highlight the complex interplay between hypoxia, stromal context and immune activation in determining the quality and distribution of infiltrating lymphocytes. SpheroMap Cytometry enables precise quantification of these dynamics, making it an ideal platform for testing therapies aimed at overcoming immune exclusion. The ability to track lymphocyte phenotypes within spatially defined tumor compartments makes this approach well-suited to evaluate the impact of checkpoint inhibitors, CD73/adenosinergic pathway blockers and next-generation CAR-T cell therapies in immunocompetent models. This study was funded by the São Paulo Research Foundation (FAPESP) Brazil, 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).