TMOD-27. Patient-derived glioblastoma organoid cultures for CAR T cell evaluation

Abstract We aim to develop clinically relevant human tumor organoid models of glioblastoma for optimizing chimeric antigen receptor (CAR) T cell therapy. We therefore have developed three-dimensional (3D) organoid cultures derived from patient tumors to preserve spatial cell-cell and cell-matrix interactions, offering advantages over traditional two-dimensional (2D) culture models. Although Matrigel is commonly used in 3D cultures, its batch variation, xenogeneic origin and non-brain relevant components raise concerns for modeling brain tumors and evaluating CAR T therapies. To address this, we evaluated brain-relevant hydrogels that contain hyaluronic acid, a major component of brain tumor extracellular matrix. We tested a panel of conditions and found one promising candidate, a mixture of PhoGel and PhotoHA (termed PGPH). PGPH allowed reliable tumor sphere migration, stable droplet formation, efficient sample recovery, and cost-effectiveness. Comparable to Matrigel, PGPH supports migration and growth of several patient-derived brain tumor spheroid cell lines. Transcriptomic analyses revealed similarities and differences between Matrigel and PGPH when culturing tumor cells or T cells. Notably, we demonstrated that T cells could migrate and kill organoid tumors in PGPH, and by incorporating soluble immune regulators, we were able to assess how these reconstructed tumor microenvironment (TME) compartments interact with CAR Ts and influence their killing potency. Moreover, PGPH enabled efficient and rapid tumor-derived explant culture (PDE) that closely resembled the native tumor ecosystem by preserving parental tumor heterogeneity and TME features, allowing for drug testing only 7 days after surgery. Lastly, we were able to evaluate CAR T cytotoxicity in PDE and are investigating the correlation of ex vivo CAR T responses with clinical responses in ongoing trials. In summary, this chemically defined, brain-relevant hydrogel offers opportunities to preserve the native or reconstructed TME, which could facilitate the translational success of CAR T treatments for brain tumors from bench to bedside.