Overcoming the immunosuppressive tumor microenvironment (TME) remains a critical bottleneck for the success of cellular immunotherapies, including chimeric antigen receptor (CAR) cells. Hypoxia-driven adenosinergic signaling is a major immune evasion mechanism, restricting immune cell infiltration, activation, and persistence. Within the TME, stromal cells expressing CD39 and CD73 convert extracellular ATP into immunosuppressive adenosine, while the CD26/ADA complex counteracts this by converting adenosine into inosine. Hypoxia further upregulates CD39/CD73, increasing adenosine accumulation and reinforcing immune suppression. Targeting this pathway, either pharmacologically or through CAR engineering to bypass or modulate these mechanisms, is an emerging frontier for next-generation cellular therapies.

Conventional in vitro assays lack the spatial resolution needed to capture hypoxia-driven phenotypic changes within the TME. To overcome these limitations, we developed SpheroMap Cytometry, a spatially resolved 3D flow cytometry platform that reproduces the structural, metabolic, and stromal complexity of the TME. The system enables compartment-specific immunophenotyping of tumor, stromal, and immune cells within the hypoxic (core) and normoxic (peripheral) regions of heterotypic tumor–stroma spheroids.

Spheroids were generated by co-culturing CellTrace Violet (CTV)-labeled Raji lymphoma cells with human umbilical cord-derived mesenchymal stromal cells at a 1:4 ratio in ultra-low-attachment 96-well plates, and aggregating them by centrifugation. At 20 h, hypoxic regions were labeled with Image-iT Green Hypoxia reagent (HypoxG). At 36 h, spheroids were enzymatically and mechanically dissociated into single-cell suspensions, stained with viability dye and antibodies against CD39, CD73, and CD26, acquired on a BD FACSymphony A1, and analyzed using FlowJo. After doublet exclusion (FSC-A vs FSC-H), dead cell removal and separation of Raji (CTV⁺) from MSC (CTV⁻) populations, each population was subdivided into HypoxG^High and HypoxG^Low fractions to assess compartment-specific expression profiles.

Fluorescence microscopy confirmed the formation of compact spheroids by 12 hours and the presence of a hypoxic core by 24 hours. Flow cytometry showed that in the hypoxic compartment, 90% of MSCs and 11% of Raji cells displayed a CD73highCD26high phenotype, versus 6% and 0% in normoxia. CD39 mean fluorescence intensity in hypoxic Raji cells was 25% higher than in normoxic counterparts. These results highlight the platform’s ability to resolve microenvironment-driven phenotypic heterogeneity that bulk assays cannot detect.

SpheroMap Cytometry recapitulates key immunosuppressive mechanisms of the lymphoma TME, offering a robust, scalable, and quantitative platform for preclinical functional screening of CAR designs. The platform can be readily adapted to evaluate diverse engineering strategies, with applicability across hematologic and solid tumor models. By combining spatial resolution with multiparametric flow cytometry, SpheroMap provides a powerful tool to accelerate the rational design and optimization of CAR-based therapies capable of maintaining activity and infiltration under hypoxic stress.

This study was financed by the São Paulo Research Foundation (FAPESP), Brazil. Process Number #2022/12856-6 and the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES).