{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "Faculdade de Medicina de Ribeirao Preto (FMRP), Universidade de São Paulo (USP), São Paulo, SP, Brazil",
    "Hemocentro de Ribeirão Preto, Ribeirão Preto, SP, Brazil"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 8,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "João Pedro Zanon MURAROLLI"
    },
    {
      "affs": [
        0
      ],
      "name": "Giovana Beatriz Capuz MORETO"
    },
    {
      "affs": [
        0
      ],
      "name": "Larissa Ignácio SILVA"
    },
    {
      "affs": [
        0
      ],
      "name": "Sara Evelyn Castro SILVA"
    },
    {
      "affs": [
        1
      ],
      "name": "Sâmia Rigotto CARUSO"
    },
    {
      "affs": [
        1
      ],
      "name": "Amanda Mizukami MARTINS"
    },
    {
      "affs": [
        1
      ],
      "name": "Maristela Delgado ORELLANA"
    },
    {
      "affs": [
        1
      ],
      "name": "Rodrigo Alexandre PANEPUCCI"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/6mvwkxj3mqpf0.bib",
  "cited_by": 0,
  "doi": "10.1016/j.htct.2026.106823",
  "fulltext_url": "http://www.sciencedirect.com/science/article/pii/S2531137926005705",
  "id": "6mvwkxj3mqpf0",
  "issns": [
    "2531-1379",
    "2531-1387"
  ],
  "landing_url": "https://doi.org/10.1016/j.htct.2026.106823",
  "language": "en",
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "lcc",
      "RC633-647.5"
    ],
    [
      "openalex",
      "2730|CAR-T cell therapy research"
    ]
  ],
  "openalex": "W7223096219",
  "pdf_url": "https://www.sciencedirect.com/science/article/pii/S2531137926005705/pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-23",
  "publisher": "Elsevier",
  "ris": "https://research.draconitic.com/paper/6mvwkxj3mqpf0.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T13:43:21Z",
      "native_id": "b32e280d7c28412593c5339d3f6618b4",
      "source": "doaj",
      "url": "https://doi.org/10.1016/j.htct.2026.106823"
    }
  ],
  "subjects": [
    {
      "field": "medicine",
      "origin": "openalex:2730|CAR-T cell therapy research",
      "subfield": "oncology"
    }
  ],
  "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",
  "title_text": "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",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T13:43:21Z",
  "url": "https://research.draconitic.com/paper/6mvwkxj3mqpf0",
  "venue": "Hematology, Transfusion and Cell Therapy",
  "versions": []
}