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

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