294 Multi-omic Screening of Invasive GBM Cells in Engineered Biomaterials and Patient Biopsies Reveals Targetable Transsulfuration Pathway Alterations

INTRODUCTION: The poor prognosis of glioblastoma (GBM) is driven by tumor invasion; however, little is known of the metabolic alterations that drive invasion in glioblastoma. METHODS: We integrated spatially addressable hydrogel biomaterial platforms, patient site-directed biopsies, and multi-omics analyses to define metabolic drivers of invasive glioblastoma cells. A CRISPR metabolic gene screen was utilized to identify metabolic genes crucial for GBM invasion. We further targeted an identified hit from our high-throughput screens by utilizing a sgRNA to generate a knockdown cell line and assessing invasion in vitro and in vivo in a patient-derived xenograft orthotopic mouse model. RESULTS: Metabolomics and lipidomics revealed elevations in the redox buffers cystathionine, hexosylceramides, and glucosyl ceramides in the invasive front of both hydrogel-cultured tumors and patient site-directed biopsies, with immunofluorescence indicating elevated reactive oxygen species (ROS) markers in invasive cells. Transcriptomics confirmed upregulation of ROS-producing and response genes at the invasive front in both hydrogel models and patient tumors. Amongst oncologic ROS, hydrogen peroxide specifically promoted glioblastoma invasion in 3D hydrogel spheroid cultures. A CRISPR metabolic gene screen revealed cystathionine gamma lyase (CTH), an enzyme which converts cystathionine to the non-essential amino acid cysteine in the transsulfuration pathway, to be essential for glioblastoma invasion. Correspondingly, supplementing CTH knockdown cells with exogenous cysteine rescued invasion. Pharmacologic CTH inhibition suppressed glioblastoma invasion, while CTH knockdown slowed glioblastoma invasion in vivo. CONCLUSIONS: Our studies highlight the importance of ROS metabolism in invasive glioblastoma cells and support further exploration of the transsulfuration pathway as a mechanistic and therapeutic target.