Glioblastoma (GBM) is an extremely aggressive and incurable primary brain tumor with a 10-year survival of just 0.71%. Cancer stem cells (CSCs) are thought to seed GBM’s inevitable recurrence by evading standard of care treatment, which combines surgical resection, radiotherapy, and chemotherapy, contributing to this grim prognosis. Effective targeting of CSCs could result in insights into GBM treatment resistance and development of novel treatment paradigms. There is a major ongoing effort to characterize CSCs, understand their interactions with the tumor microenvironment, and identify ways to eliminate them. This review discusses the diversity of CSC lineages present in GBM and how this glioma stem cell (GSC) mosaicism drives global intratumoral heterogeneity constituted by complex and spatially distinct local microenvironments. We review how a tumor’s diverse CSC populations orchestrate and interact with the environment, especially the immune landscape. We also discuss how to map this intricate GBM ecosystem through the lens of metabolism and immunology to find vulnerabilities and new ways to disrupt the equilibrium of the system to achieve improved disease outcome.

INTRODUCTION Glioblastoma is a challenge for neuro-oncologists and current therapies are minimally effective. Standard-of- care treatment is almost inevitably followed by disease recurrence. Adoptive T cell transfer has emerged as a viable therapeutic for brain malignancies. While promising, the efficacy of this approach is often limited by a complex immunosuppressive tumor microenvironment. These complexities mean that more sophisticated T cell products are required. Objectives: The brain tumor microenvironment provides local restraints via metabolic competition suppressing antitumor immunity, specifically inhibiting infiltration and tumoricidal functions of host and adoptively transferred tumor-reactive T cells. The overall goal of this project is to test new treatments to reverse immune dysfunction in cancer through the regulation of T cell metabolic signaling. We propose that modulating the glucose pathway in T cells can potentiate their anti-tumor activity once adoptively transferred. METHODS The glucose metabolic pathway of T cells was modulated via overexpression of glucose transporters. The functionality of metabolically modified T cells was investigated in murine and human models. RESULTS We demonstrated the existence of a competition for glucose between T cells and tumor cells, with tumor cells imposing glucose restriction mediating T cell hyporesponsiveness. Overexpression of glucose transporters such as Glut1 and Glut3 increased T cell glucose utilization and provided a survival/growth advantage and enhanced T cell activation in glucose-restricted conditions. We also established that glucose transporter overexpression improves intratumoral infiltration and expansion of adoptively transferred T cells, resulting in improved survival. CONCLUSION This project integrates fundamental concepts of tumor and immune metabolism in the design of immunotherapy and confirms that immunometabolism represents a viable target for new cancer therapy to treat brain tumors.

INTRODUCTION Glioblastoma (GBM) contains cell populations with distinct metabolic requirements, with fast-cycling cells harnessing aerobic glycolysis, and treatment-resistant slow-cycling cells (SCCs) preferentially engaging lipid metabolism. The interaction between immune and tumor cells, and how their metabolic heterogeneity shapes the immune landscape in GBM has yet to be understood. Objectives: The primary objective of this project is to spatially and molecularly decode the GBM microenvironment heterogeneity with a specific focus on unraveling the metabolic links that underlie the interaction of SCCs with the immune compartment. METHODS Multiple murine glioma cell lines coupled with geospatial profiling were used to establish metabolic heterogeneity and communications, while various genetic and pharmacological approaches were applied to assess the effect of disrupting the metabolic interplay between SCCs and the immune system. RESULTS We determined that SCCs exhibit distinct metabolic dependencies, involving preferential lipid metabolism supported by enhanced fatty acid uptake. We also found that SCCs exhibit specific geospatial distribution and that tumor progression is regulated by their interactions with immune suppressive cells, which in turn work against tumor immune rejection by inhibiting T cell anti-tumor activity. The immune microenvironment shaped by SCCs is marked by specific metabolic features enhancing lipid exchange capacities that are exploited by SCCs to support their survival and functions. Importantly, disrupting lipid metabolic exchange sensitized tumors to chemotherapy. CONCLUSION Our results reveal that metabolic interactions between SCCs and tumor-associated immune suppressive cells within the GBM microenvironment play a critical role in the development of drug and immune resistant tumors. This study delineates these metabolic communications and assesses the potential therapeutic effect of disrupting these interactions to treat GBM. The insights generated from this project uncover fundamental principles of the emerging connections between the tumor microenvironment, cell metabolism, anti-tumor immunity, and associated therapeutic vulnerabilities.

Glioblastoma represent a great challenge and current therapies are negligibly effective, with disease recurrence being inevitable. T cell therapy has emerged as a viable treatment for brain malignancies. While promising, the efficacy of this approach is often limited by a complex immunosuppressive tumor microenvironment. These complexities mean that more sophisticated T cell products are required. The brain tumor microenvironment provides local restraints via metabolic competition suppressing antitumor immunity, specifically inhibiting infiltration and effector functions of host and adoptively transferred tumor-reactive T cells. The objective of this project is to test new treatments to reverse immune dysfunction in brain cancer through the regulation of T cell metabolic signaling. We propose that modulating glucose signaling can potentiate T cell anti-tumor activity. The glucose signaling pathway of T cells was modulated through overexpression of glucose transporters and the function of metabolically modified T cells was investigated using murine and human models. We revealed a competition for glucose between T cells and tumor cells, with tumor cells imposing glucose restriction mediating T cell hyporesponsiveness. Overexpression of glucose transporters such as Glut1 and Glut3 enhanced T cell glucose utilization and provided a survival/growth advantage and greater activation, specifically in glucose-restricted conditions. We established that glucose transporter overexpression improves intratumoral infiltration and expansion of adoptively transferred CAR T cells, resulting in improved survival. Our study integrates fundamental concepts of tumor and immune metabolism in the design of immunotherapy and confirms that immunometabolism represents a viable target for new cancer therapy to treat brain tumors. Citation Format: Tanya Ghosh, Avirup Chakraborty, Linchun Jin, Diana Feier, Aryeh Silver, Maryam Rahman, Catherine Flores, Matthew Sarkisian, Jianping Huang, Duane A. Mitchell, Loic P. Deleyrolle. Optimizing CAR T therapy via metabolic engineering for the treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 904.