A High-content Screening Approach to Assess the Impact of Hypoxia-driven Adenosinergic Signaling on Car-t Cell Function Using a 2d Tumor–stroma Model
Year: 2026
Type: article
Abstract: The tumor microenvironment (TME), particularly hypoxia and associated adenosinergic signaling, limits CAR-T efficacy in solid tumors and hypoxic niches of hematological malignancies. However, conventional in vitro assays fail to capture spatial oxygen gradients required to evaluate these mechanisms. Here, we employed a heterotypic 2D tumor–stroma co-culture coupled to High-Content Screening (CH-HCS) to spatially quantify the impact of hypoxia on CAR-T cell function and assess whether A₂A receptor modulation could restore antitumor activity within hypoxic niches. HS-5 stromal cells (10⁴/well) were co-cultured with CD19⁺ Raji or CD19⁻ K562 tumor cells (10⁴/well, eFluor 670-labeled) and either anti-CD19 CAR-T or non-transduced T cells (2 × 10⁴/well, CellTrace Violet-labeled), in triplicate. Radial hypoxia gradients were generated using a 5 mm glass coverslip (CS). In parallel, co-cultures were treated with the A₂A receptor antagonist Ciforadenant to evaluate the contribution of A₂A receptor signaling to CAR-T function. Whole-well images (25-field montage) were acquired using an ImageXpress MICRO XLS HCS system at 0 and 24 h and analyzed in CellProfiler. Hypoxia Green fluorescence intensity was used to define four spatially distinct oxygen-dependent regions of interest (ROIs): CS_InnerCore (severe hypoxia at CS center), CS_OuterCore (moderate hypoxia), CS_Periphery (transition at CS edge), Outside (normoxia beyond CS), enabling spatially resolved quantification of tumor and effector cell death (SYTOX™ Green). Data were analyzed by two-way ANOVA with multiple comparisons. In Raji co-cultures, CAR-T cells induced marked tumor death after 24h, highest in Outside (∼40%; p < 0.01) and No-CS (∼60%; p < 0.0001) regions, but minimal in hypoxic regions (< 10%). Non-transduced T cells showed negligible cytotoxicity, with only modest increases (∼20%) in Outside and No-CS regions. By contrast, CD19− K562 co-cultures showed negligible tumor death across all regions (< 5–10% in normoxia), supporting antigen specificity. Effector cell death increased markedly after 24 h in normoxic Outside regions (∼35%; p < 0.01) versus hypoxic regions (∼5–10%), suggesting reduced activation-associated cell death under hypoxia. Pharmacological inhibition of A₂A signaling with Ciforadenant consistently enhanced CAR-T-mediated tumor killing, with the strongest effect in hypoxic InnerCore and OuterCore regions, where tumor death reached ∼60–70%, compared with ∼35–45% in vehicle-treated controls; two-way ANOVA revealed a significant interaction between time and oxygen availability (p < 0.01). These findings show that hypoxia gradients selectively impair CAR-T cytotoxicity while preserving antigen specificity. Furthermore, A₂A receptor blockade partially restored CAR-T-mediated tumor killing within hypoxic regions, supporting a contribution of hypoxia-associated adenosinergic signaling to CAR-T dysfunction. This approach provides a robust preclinical platform for investigating mechanisms of CAR-T resistance and evaluating CAR engineering strategies and combination therapies aimed at overcoming hypoxia-driven immunosuppression. This study was financed, in part, by the São Paulo Research Foundation (FAPESP), Brazil. Process Number #2022/12856-6.
Institution: Universidade de São Paulo
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: diamond
OpenAlex ID: W7223096219