{
  "abstract": "Although CAR-T cell therapy has transformed the treatment of hematological malignancies, overcoming the tumor microenvironment (TME) remains a major challenge. Hypoxia and other immunosuppressive features of the TME shape CAR-T cell infiltration, activation, persistence, and therapeutic efficacy. However, conventional in vitro models lack the spatial resolution required to investigate these processes. To recapitulate key features of the TME, we used SpheroMap Cytometry, a three-dimensional (3D) spatial flow cytometry platform that enables compartment-specific immunophenotyping of tumor, stromal, and immune cells in hypoxic (core) and normoxic (periphery). Spheroids were generated by co-culturing CD19+ Raji lymphoma cells and immunosuppressive HS-5 mesenchymal stromal cells (1:4; 4 × 10⁴ cells/spheroid) in ultra-low attachment U-bottom 96-well plates. After 24 h, CAR-T cells (∼4 8% transduction) or untransduced T cells (UTD) were added (4 × 10⁴ cells/spheroid). Conditions included untreated, Dasatinib (Src/Lck inhibitor), and vehicle control. After 12 h, hypoxic regions were labeled with Image-iT Green Hypoxia. After 48 h, spheroids were enzymatically dissociated, stained with FVS780 viability dye and antibodies against CD3, CAR, CD25 and CD19, and analyzed by flow cytometry. Microscopy confirmed compact spheroids at 24 h and a hypoxic core at 48 h. In untreated spheroids, CAR-T infiltration was approximately twofold higher than UTD (40% vs 23%), leading to near-complete tumor clearance (1% vs 11% CD19+ remaining cells), consistent with CAR-mediated tumor-specific cytotoxicity. Spatial analysis revealed that 76% of infiltrating T cells localized within the hypoxic core, where CAR⁺ cells represented 48.9% of the CD3⁺ population, compared with 33.4% in the normoxic periphery. Consistent with its established role as a pharmacological off-switch for CAR signaling, Dasatinib reduced global T-cell infiltration in CAR-T spheroids (40% to 20% CD3+ cells), accompanied by increased residual tumor burden (1.16% to 18% CD19+ cells). Moreover, Dasatinib reduced CAR+ cell accumulation within the hypoxic core from 48.9% to 30.9%, suggesting preferential inhibition of antigen-engaged CAR-T cells. CD25 expression was also markedly decreased following Dasatinib treatment, dropping from 68.9% to 26.5% in the hypoxic core and from 44% to 14% in the normoxic periphery, confirming effective suppression of CAR-mediated activation. Notably, CAR-T cells displayed higher CD25 expression in hypoxia than in normoxia. These findings reflect antigen-driven activation, as tumor cells accumulate within the spheroid core. However, they may also indicate a hypoxia-driven hyperactivated state associated with metabolic stress, which can compromise long-term effector function. Together, these findings establish SpheroMap Cytometry as a scalable spatial flow cytometry platform for investigating CAR-T cell behavior and evaluating next-generation immunotherapies in hypoxic and spatially organized tumor microenvironments. 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).",
  "abstract_shown": true,
  "affiliations": [
    "Ribeirão Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo (FMRP-USP), Ribeirão Preto, São Paulo, Brazil"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 10,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Giovana Beatriz Capuz MORETO"
    },
    {
      "affs": [
        0
      ],
      "name": "João Pedro Zanon MURAROLLI"
    },
    {
      "affs": [
        0
      ],
      "name": "Sara Evelyn de Castro SILVA"
    },
    {
      "affs": [
        0
      ],
      "name": "Larissa Ignácio da SILVA"
    },
    {
      "affs": [
        0
      ],
      "name": "Camila Cristina de Oliveira Menezes BONALDO"
    },
    {
      "affs": [
        0
      ],
      "name": "Amanda Mizukami MARTINS"
    },
    {
      "affs": [
        0
      ],
      "name": "Maristela Delgado ORELLANA"
    },
    {
      "affs": [
        0
      ],
      "name": "Patrícia Vianna Bonini PALMA"
    },
    {
      "affs": [
        0
      ],
      "name": "Sâmia Rigotto CARUSO"
    },
    {
      "affs": [
        0
      ],
      "name": "Rodrigo Alexandre PANEPUCCI"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/dcspseq9ae5cz.bib",
  "cited_by": 0,
  "doi": "10.1016/j.htct.2026.107435",
  "fulltext_url": "http://www.sciencedirect.com/science/article/pii/S253113792601182X",
  "id": "dcspseq9ae5cz",
  "issns": [
    "2531-1379",
    "2531-1387"
  ],
  "landing_url": "https://doi.org/10.1016/j.htct.2026.107435",
  "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": "W7223142999",
  "pdf_url": "https://www.sciencedirect.com/science/article/pii/S253113792601182X/pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-23",
  "publisher": "Elsevier",
  "ris": "https://research.draconitic.com/paper/dcspseq9ae5cz.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T13:43:21Z",
      "native_id": "b39d122e77114dbc8fe898a50932a581",
      "source": "doaj",
      "url": "https://doi.org/10.1016/j.htct.2026.107435"
    }
  ],
  "subjects": [
    {
      "field": "medicine",
      "origin": "openalex:2730|CAR-T cell therapy research",
      "subfield": "oncology"
    }
  ],
  "title": "SPHEROMAP CYTOMETRY: MAPPING INFILTRATION AND HYPOXIA-DRIVEN FUNCTIONAL MODULATION OF CAR-T CELLS IN THE TUMOR MICROENVIRONMENT",
  "title_text": "SPHEROMAP CYTOMETRY: MAPPING INFILTRATION AND HYPOXIA-DRIVEN FUNCTIONAL MODULATION OF CAR-T CELLS IN THE TUMOR MICROENVIRONMENT",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T13:43:21Z",
  "url": "https://research.draconitic.com/paper/dcspseq9ae5cz",
  "venue": "Hematology, Transfusion and Cell Therapy",
  "versions": []
}