EXTH-99. Tumor mechanics mediated suppression against CAR T cells in diffuse midline gliomas

Diffuse midline glioma (DMG) is a universally fatal pediatric brain tumor with severely limited therapeutic options. Recently, chimeric antigen receptor (CAR) T cell therapies have demonstrated promising clinical results in a phase 1 trial for patients with DMGs. These extremely encouraging results highlight that continuous efforts should be made to improve CAR T cell strategies. To that end, substantial efforts have been made to understand how cellular components and biochemical cues within the tumor microenvironment (TME) dysregulate CAR T cells. Within solid tumors like DMGs, CAR T cells also experience significant mechanical resistance. However, it remains unknown how DMG-specific mechanical cues regulate CAR T cell function. In this study, we first determined that DMG tumors exhibit a unique mechanical property known as viscoelasticity, which is a time-dependent response to deformation and is characterized by stress relaxation. We next established a biomaterials-based hydrogel system using hydrazone crosslinking chemistry that recapitulates DMG-specific viscoelasticity, specifically the DMG fast-relaxing stress relaxation properties. Moreover, this hydrogel design allows tuning stress relaxation while mimicking DMG tumor stiffness, thereby avoiding stiffness as a cofounding factor. Using this engineered model, we found that CAR T cells had impaired cytotoxicity and expansion against DMG cells on DIPG-mimicking, fast-relaxing viscoelastic substrates compared to both a control slow-relaxing viscoelastic substrates and 2D tissue culture plastic. Strikingly, when pre-exposed to DIPG-mimicking fast-relaxing viscoelastic hydrogels, CAR T cells exhibited significantly impaired in vivo tumor control, which was further corroborated within a Medulloblastoma in vivo model. Using single-cell RNA sequencing, we are currently working to identify negative molecular biophysical regulators driving CAR T cell dysfunction in response to DMG-specific tumor viscoelasticity. In summary, the present study is the first work identifying that DMG tumors are viscoelastic and uniquely demonstrates that DMG-specific tumor mechanics suppress CAR T cell in vitro and in vivo antitumor efficacy.