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bioRxiv · 10.64898/2026.01.30.702833

Coverslip Hypoxia High-Content Screening (CH-HCS): A 2D Imaging Platform for Spatially Resolved Analysis of CAR-T Function Under Oxygen Gradients

Murarolli, J. P. Z.·Beatriz Capuz, G. B. C.·Silva, S. E. d. C.·Arrojo, M. L.·Martins, A. M.·Caruso, S. R.·Orellana, M. D.·Panepucci, R. A.
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Abstract

Hypoxia within the tumor microenvironment profoundly limits the efficacy of immune and cellular therapies, yet most in vitro cytotoxicity assays neglect spatial oxygen heterogeneity. We developed Coverslip Hypoxia High-Content Screening (CH-HCS), a simple, scalable 2D co-culture platform that enables quantitative, region-resolved evaluation of CAR-T cell activity across controlled oxygen gradients within a single well. In CH-HCS, a 5 mm glass coverslip placed over tumor-stroma co-cultures restricts oxygen diffusion, generating concentric hypoxia-normoxia zones in standard 96-well plates. Fluorescently labeled CD19+ Raji or CD19- K562 tumor cells, stromal cells, and anti-CD19 CAR-T cells were analyzed using multiparametric fluorescence imaging with an ImageXpress Micro XLS system coupled to custom CellProfiler-KNIME pipelines, enabling segmentation of spatial Regions of Interest--InnerCore, OuterCore, Periphery, and Outside--and single-cell quantification of tumor death (SYTOX Green) and T-cell morphodynamics. The platform reproducibly established oxygen gradients that strongly shaped cellular behavior: CAR-T cytotoxicity and motility were maximal in normoxic regions but markedly suppressed within hypoxic cores, whereas effector cell survival increased under low oxygen. Unlike bulk cytotoxicity assays, CH-HCS directly visualizes spatial functional heterogeneity within the same well, allowing simultaneous comparison of matched hypoxic and normoxic compartments. Together, CH-HCS provides a cost-effective, high-throughput, and physiologically relevant tool for preclinical screening of CAR-T products and therapeutic strategies aimed at overcoming hypoxia-driven immune resistance at the tumor-stroma interface. Key PointO_LISpatially resolved quantification: Simultaneous measurement of cellular behavior in matched hypoxic and normoxic compartments within the same well. C_LIO_LIPhysiologically relevant gradients: Coverslip geometry generates reproducible oxygen diffusion profiles that emulate the in vivo tumor-stroma interface. C_LIO_LIMultiparametric single-cell readouts: High-content imaging coupled with automated segmentation provides data on viability, cytotoxicity, and effector morphology. C_LIO_LIHigh-throughput and low-cost: Fully compatible with 96-well formats, enabling parallel pharmacological or genetic screening without specialized hypoxia chambers. C_LIO_LIGeneralizable design: Applicable beyond CAR-T assays to study stromal adaptation, drug resistance, or immune suppression across controlled oxygen gradients. C_LI

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Murarolli, J. P. Z., Beatriz Capuz, G. B. C., Silva, S. E. d. C., Arrojo, M. L., Martins, A. M., Caruso, S. R., Orellana, M. D., Panepucci, R. A.. 2026-02-02. Coverslip Hypoxia High-Content Screening (CH-HCS): A 2D Imaging Platform for Spatially Resolved Analysis of CAR-T Function Under Oxygen Gradients. https://doi.org/10.64898/2026.01.30.702833

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