StacheStrong Award - Sampling, Molecular Characterization and Modeling of Infiltrating Glioblastoma Cells

Introduction: Gross total resection of the contrast-enhancing portion of glioblastoma remains the standard of care, but infiltrating cells persist along the resection cavity in non-enhancing regions. Adjuvant therapy for glioblastoma theoretically should be developed to target these infiltrating cells, but most laboratory studies on glioblastoma use samples from the contrast-enhancing bulk of tumor, which given intratumoral heterogeneity may not best represent the targets for potential therapy.

Methods: In patients undergoing surgery for newly diagnosed IDH wild-type glioblastoma (n=37), after complete resection of contrast-enhancing tumor, tissue along the resection cavity in non-eloquent areas of potential T2 hyperintense non-enhancing tumor were assessed. Samples underwent immunohistochemical staining with neuropathological review (n=37), single cell sequencing (n=17), and transplantation into a cortical organoid model (n=10).

Results: Unresected non-enhancing infiltrating tumor was confirmed on H&E in 37/37 (100%). There was a negative correlation between cell density of these resection cavity biopsies and progression free (p=0.022) and overall survival (p=0.039). Spatial tagging showed areas of clear infiltrative tumor were most likely regions of recurrence at the resection cavity (p=0.001). Single-cell sequencing showed presence of infiltrative tumor in 100% of patients. Differential gene expression analysis showed significant differences from contrast-enhancing tumor. Specifically, there was upregulation of invasion genes (GPM6A, LHFPL3, DCLK2), ciliary proteins, unique growth factors (PTPRZ1) (FDR p< 0.0001 each). Implantation into an infiltrating tumor cortical organoid model showed initial growth slower than that from contrast-enhancing regions but eventual increase in growth rate similar to that of contrast-enhancing regions (p < 0.05 each).

Conclusion : Infiltrating glioblastoma cells exist in the resection cavity of all patients with “gross total resection” of tumor. These cells have unique properties highlighted by invasive characteristics that persist in in vivo modeling. We describe a pipeline for sampling, molecular studies, in vitro modeling, and in vivo modeling of infiltrative glioblastoma cells.