SPHEROMAP CYTOMETRY: MAPPING INFILTRATION AND HYPOXIA-DRIVEN FUNCTIONAL MODULATION OF CAR-T CELLS IN THE TUMOR MICROENVIRONMENT
- 1Ribeirão Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo (FMRP-USP), Ribeirão Preto, São Paulo, Brazil
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).
Related
- Tissue Factor Expression Is Associated with Distinct Pattern of Immune-Cell Infiltration in the Ovarian Tumor Microenvironment
- Therapeutic potential of natural product polysaccharides in hepatocellular carcinoma: from microenvironment modulation to combination strategies
- p38α orchestrates BRAFV600E-melanoma progression and immunotherapy response via tumor fitness and immune microenvironment modulation.
- Mechanistic insights on mogrosides as bifunctional regulators of metabolic reprogramming and immune modulation in tumor microenvironment
- Immunoregenerative Microenvironment Modulation in Tendon-Ligament Regeneration: Evolution of Chitosan-Based Composite Biomaterials.
BibTeX
@article{moreto2026spheromap,
title = {{SPHEROMAP CYTOMETRY: MAPPING INFILTRATION AND HYPOXIA-DRIVEN FUNCTIONAL MODULATION OF CAR-T CELLS IN THE TUMOR MICROENVIRONMENT}},
author = {Giovana Beatriz Capuz MORETO and João Pedro Zanon MURAROLLI and Sara Evelyn de Castro SILVA and Larissa Ignácio da SILVA and Camila Cristina de Oliveira Menezes BONALDO and Amanda Mizukami MARTINS and Maristela Delgado ORELLANA and Patrícia Vianna Bonini PALMA and Sâmia Rigotto CARUSO and Rodrigo Alexandre PANEPUCCI},
journal = {Hematology, Transfusion and Cell Therapy},
year = {2026},
month = oct,
doi = {10.1016/j.htct.2026.107435},
url = {https://doi.org/10.1016/j.htct.2026.107435},
note = {CC BY},
}
RIS
TY - JOUR
TI - SPHEROMAP CYTOMETRY: MAPPING INFILTRATION AND HYPOXIA-DRIVEN FUNCTIONAL MODULATION OF CAR-T CELLS IN THE TUMOR MICROENVIRONMENT
AU - Giovana Beatriz Capuz MORETO
AU - João Pedro Zanon MURAROLLI
AU - Sara Evelyn de Castro SILVA
AU - Larissa Ignácio da SILVA
AU - Camila Cristina de Oliveira Menezes BONALDO
AU - Amanda Mizukami MARTINS
AU - Maristela Delgado ORELLANA
AU - Patrícia Vianna Bonini PALMA
AU - Sâmia Rigotto CARUSO
AU - Rodrigo Alexandre PANEPUCCI
PY - 2026
DA - 2026/10/23
JO - Hematology, Transfusion and Cell Therapy
DO - 10.1016/j.htct.2026.107435
UR - https://doi.org/10.1016/j.htct.2026.107435
AB - 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).
SN - 2531-1379
SN - 2531-1387
ER -