Patient-derived 3D organoid platform for functional assessment of GPC3-targeted CAR T cell cytotoxic activity in head and neck squamous cell carcinoma

Background: Head and neck squamous cell carcinoma (HNSCC) remains a therapeutic challenge owing to its marked heterogeneity and limited immunotherapy efficacy, underscoring the need for improved therapeutic strategies and preclinical systems supporting clinical translation. Method: We established a simplified and optimized 3D head and neck cancer organoid (HNCO)–chimeric antigen receptor (CAR) T cell co-culture platform that maintains uniform spheroid architecture (>500 μm) to recapitulate physiological hypoxia while preserving the tumor secretome. CAR T cell cytotoxic activity was assessed through multimodal readouts—structural disruption, ATP-based viability, and granzyme B secretion. Following initial organoid seeding, the workflow proceeded without physical manipulation, enabling concurrent multimodal assessment of CAR T cell activity from a single co-culture. Results: Analysis of The Cancer Genome Atlas (TCGA) data revealed that high glypican-3 (GPC3) expression was associated with poor survival in patients with HNSCC. We generated GPC3-targeted CAR T (GPC3-CAR T) cells. Using our co-culture platform, we evaluated the cytotoxic activity of GPC3-CAR T cells against five HNCOs harboring diverse genetic alterations and variable GPC3 expression. Organoids with high or moderate GPC3 expression consistently exhibited structural disintegration, reduced viability, and increased granzyme B secretion, whereas GPC3-low organoids showed heterogeneous responses. Conclusions: This proof-of-concept study introduces a patient-derived 3D organoid platform for functional assessment of CAR T cell cytotoxic activity in HNSCC. Our findings suggest that GPC3-CAR T therapy may be clinically applicable to subsets of patients with HNSCC, while emphasizing the need for functional validation to account for interpatient heterogeneity in clinical translation.