CellCage™ Technology Enables High-throughput Isolation of Defined Cell Combinations to Resolve the Determinants of CAR-T mediated Cytotoxicity

Yau, R. G.; Deshmukh, S.; Sacca, M.; Gupta, R.; Yang, Y.; Mohseni, M.; Wu, L. Y.; Yasar, F. G.; Sabri, S.; Ai, T.; Khurana, T. K.; Levy, S.; Gherardini, P. F.; Mason, C.; Jin, M. M.; Schroth, G. P.

doi:10.64898/2026.08.21.746043

bioRxiv · 10.64898/2026.08.21.746043

CellCage™ Technology Enables High-throughput Isolation of Defined Cell Combinations to Resolve the Determinants of CAR-T mediated Cytotoxicity

Yau, R. G.·Deshmukh, S.·Sacca, M.·Gupta, R.·Yang, Y.·Mohseni, M.·Wu, L. Y.·Yasar, F. G.·Sabri, S.·Ai, T.·Khurana, T. K.·Levy, S.·Gherardini, P. F.·Mason, C.·Jin, M. M.·Schroth, G. P.

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Abstract

Understanding the cellular determinants of CAR-T cell-mediated cytotoxicity at the single-cell level remains a critical challenge in developing effective cellular immunotherapies. Conventional bulk co-culture assays provide only population-level measurements of cytotoxic activity and cannot resolve the functional heterogeneity of individual effector-target interactions. Here, we demonstrate the Cellanome platform, which enables the formation of CellCage Enclosures (CCEs) through spatially controlled photopolymerization, as a tool for high-resolution, high-throughput characterization of CAR-T cell-mediated cytotoxicity. By isolating defined effector-target combinations within individual CCEs and coupling encapsulation with longitudinal time-lapse imaging and automated image analysis, we resolve cytotoxic activity across thousands of individually tracked CCEs. Using anti-CD19 CAR-T effectors and NALM6-GFP targets, we show that individual CD8+ CAR-T effectors exhibit substantial functional heterogeneity, with serial killing capacity, effector-target contact dynamics, and intrinsic motility each independently correlating with cytotoxic potency. Cytotoxic efficacy scaled positively with effector number, and CD4+ T helper cells augmented CD8+-mediated killing in a dose-dependent manner, providing direct evidence for cooperative T cell behavior during tumor cell clearance. Extending the platform to a solid tumor model using AIC100 anti-ICAM-1 CAR-T cells and HeLa-GFP targets, we show that individual AIC100 effectors are insufficient to mediate effective killing, while multi-effector CCEs exhibit robust cytotoxicity scaling with effector abundance and CD4+:CD8+ composition. These data establish the Cellanome platform as a broadly applicable system for dissecting the determinants of CAR-T cell cytotoxicity at single-cell resolution, with direct translational relevance to the optimization of cellular immunotherapy.

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Yau, R. G., Deshmukh, S., Sacca, M., Gupta, R., Yang, Y., Mohseni, M., Wu, L. Y., Yasar, F. G., Sabri, S., Ai, T., Khurana, T. K., Levy, S., Gherardini, P. F., Mason, C., Jin, M. M., Schroth, G. P.. 2026-08-25. CellCage™ Technology Enables High-throughput Isolation of Defined Cell Combinations to Resolve the Determinants of CAR-T mediated Cytotoxicity. https://doi.org/10.64898/2026.08.21.746043

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