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Background Ovarian cancer (OVCA) remains a therapeutic challenge. Folate receptor alpha encoded by the folate receptor 1 gene (FOLR1) provides a target for chimeric antigen receptor (CAR) T-cell therapy. To date the dynamicity and the spatiotemporal dynamics of CAR T-cell interactions with tumor cells are poorly resolved. Here, we employ high-resolution timelapse microscopy to accurately measure the cytotoxicity kinetics and behavioral patterns of CAR T cells against OVCAR4 cells, cell clusters, and patient-derived avatars based on 3D microtumor models.Methods CAR T cells stained for mitochondria and membrane were co-cultured with folate receptor alpha expressing OVCA cells stained for actin and mitochondria in culture media (RPMI + 10% FBS, 400 U/mL IL2). Serial images were collected over 3 hours at 1-minute intervals using a NIKON AXR confocal microscope with NSPARC super-resolution detector - the effective magnification was100X. All experiments were performed in a controlled incubator.Results Rapid cytotoxicity was repeatedly measured, with engagement triggering actin depolymerization in cancer cells within minutes. The mitochondrial networks collapsed monotonically over 60 minutes and culminated in complete apoptosis after 90 minutes. Single CAR T cells repeatedly demonstrated potent serial cytotoxicity moving rapidly between cancer cells and in some instances killing up to 3 OVCA cells within 3 hours. Approximately 30% of CAR T cells exhibited aggressive morphology and high motility (>15 µm/min) at any time, and clusters of up to 5 CAR T cells could be found engaging a single tumor cell and displayed cooperative behaviors. Bystander CAR T cells would occasionally become activated during attacks and thereby amplify cytotoxicity.Conclusions This study captured a wide array of cell dynamics, revealing rapid target elimination, cooperative swarm-like behaviors, and on-site proliferation. These findings underscore the potential of high-resolution imaging to optimize CAR T-cell design for functionality and performance.