Intratumoral heterogeneity (ITH) has increasingly being described for multiple cancers as the root cause of therapy resistance. Recent studies have started to explore the scope of ITH in glioblastoma (GBM), a highly aggressive and fatal form of brain tumor, to explain its inevitable therapy resistance and disease relapse. In this review, we detail the emerging data that explores the extensive genetic, cellular and functional ITH present in GBM. We discuss current experimental models of human GBM recurrence and suggest harnessing new technologies (CRISPR-Cas9 screening, CyTOF, cellular barcoding, single cell analysis) to delineate GBM ITH and identify treatment-refractory cell populations, thus opening new therapeutic windows. We will also explore why current therapeutics have failed in clinical trials and how ITH can inform us on developing empiric therapies for the treatment of recurrent GBM.

Purpose: Glioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of cancer stem cells (CSCs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here, we identify CD70 as a potential therapeutic target for recurrent GBM CSCs.Experimental design: In the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We use CD70 knockdown studies, subsequent RNAseq pathway analysis, andin vivoxenotransplantation to validate CD70’s role in GBM. Next, we developed and tested an anti-CD70 chimeric antigen receptor (CAR)-T therapy, which we validatedin vitroandin vivousing our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples.Results: CD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicityin vitroandin vivo. CD70 CAR-T therapy significantly improves prognosisin vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations, notably putative M1 macrophages and CD4 T cells.Conclusion: CD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable polytherapeutic avenue to co-target both GBM and its TIME.