Real-time monitoring of CAR T cell dynamics in tumor patient-derived organoids using the OrganoIDNet algorithm

Abstract Background Patient-derived organoids (PDOs) provide physiologically relevant 3D tumor models for preclinical drug testing, yet robust and automated methods to quantify dynamic responses to immunotherapies remain limited. OrganoIDNet is a deep learning-based image analysis framework that enables automated, label-free segmentation and longitudinal quantification of organoid morphology. Here, we extend the application of OrganoIDNet to evaluate chimeric antigen receptor (CAR) T cell activity against pancreatic ductal adenocarcinoma (PDAC) PDOs targeting the tumor-associated antigen CD318. Results CD318-directed CAR T cells were co-cultured with PDAC PDOs using a Matrigel-based sandwich system and monitored by time-lapse bright-field imaging. OrganoIDNet enabled accurate single-organoid segmentation and continuous quantification of organoid number and area across multiple effector-to-target ratios. CAR-318 T cells induced robust, antigen-dependent cytotoxicity, characterized by progressive reductions in organoid number and size and were accompanied by increased T cell activation marker expression and changes in TIM-3 expression at the endpoint. Dynamic imaging and T cell spatial analysis, further revealed close T cell-organoid interactions and early tumor cell elimination, providing time-resolved information on organoid responses and T cell proximity that complements conventional endpoint assays. Conclusions By integrating organoid-immune co-cultures with OrganoIDNet-driven live-cell imaging, we established an automated longitudinal imaging assay for assessing CAR T cell-mediated responses in PDAC PDOs. The assay enabled continuous quantification of organoid number and area, together with image-based assessment of T cell proximity, across multiple effector-to-target ratios. These measurements provide time-resolved information on antigen-dependent cytotoxicity and spatial association during the observation period, supporting its potential utility as a preclinical platform for CAR T cell development and future personalized immunotherapy studies.