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
- 1Faculdade de Medicina de Ribeirao Preto (FMRP), Universidade de São Paulo (USP), São Paulo, SP, Brazil
- 2Hemocentro de Ribeirão Preto, Ribeirão Preto, SP, Brazil
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.
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BibTeX
@article{murarolli2026highcontent,
title = {{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}},
author = {João Pedro Zanon MURAROLLI and Giovana Beatriz Capuz MORETO and Larissa Ignácio SILVA and Sara Evelyn Castro SILVA and Sâmia Rigotto CARUSO and Amanda Mizukami MARTINS and Maristela Delgado ORELLANA and Rodrigo Alexandre PANEPUCCI},
journal = {Hematology, Transfusion and Cell Therapy},
year = {2026},
month = oct,
doi = {10.1016/j.htct.2026.106823},
url = {https://doi.org/10.1016/j.htct.2026.106823},
note = {CC BY},
}
RIS
TY - JOUR
TI - 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
AU - João Pedro Zanon MURAROLLI
AU - Giovana Beatriz Capuz MORETO
AU - Larissa Ignácio SILVA
AU - Sara Evelyn Castro SILVA
AU - Sâmia Rigotto CARUSO
AU - Amanda Mizukami MARTINS
AU - Maristela Delgado ORELLANA
AU - Rodrigo Alexandre PANEPUCCI
PY - 2026
DA - 2026/10/23
JO - Hematology, Transfusion and Cell Therapy
DO - 10.1016/j.htct.2026.106823
UR - https://doi.org/10.1016/j.htct.2026.106823
AB - 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.
SN - 2531-1379
SN - 2531-1387
ER -