Completed clinical trials of CAR-T cells in glioblastoma (GBM) have revealed key challenges that limit their efficacy. These include incomplete antigen coverage, downregulation of target antigen in response to therapy, exposure to immunosuppressive cells and cytokines in the tumor microenvironment and exhaustion of CAR-T cells. To overcome these challenges, CAR-T cells have been modified to maximize effector function and resist immunosuppression in the tumor while limiting toxicities to the host. Adoption of these novel CAR-T strategies in GBM can overcome the “cold tumor” phenotype of GBM and trigger an inflammatory cascade that maximizes tumor clearance and minimizes CAR-T dysfunction. To achieve this, understanding and harnessing the antigenic, metabolic and immunological composition of GBM is crucial. Here we review the findings from completed clinical trials of CAR-T cells in GBM as well as novel strategies that could improve CAR-T survival and function in the tumor.

Glioblastoma (GBM) is a highly aggressive brain tumor with poor prognosis and high recurrence rates. The complex immune microenvironment of GBM is highly infiltrated by tumor-associated microglia and macrophages (TAM). TAMs are known to be heterogeneous in their functional and metabolic states and can transmit either protumoral or antitumoral signals to glioma cells. Here, we performed bulk RNA sequencing and single-cell RNA sequencing on samples from patients with GBM, which revealed increased ATP synthase expression and oxidative phosphorylation activity in TAMs located in the tumor core relative to the tumor periphery. Both in vitro and in vivo models displayed similar trends of augmented TAM mitochondrial activity, along with elevated mitochondrial fission, glucose uptake, mitochondrial membrane potential, and extracellular ATP (eATP) production by TAMs in the presence of GBM cells. Tumor-secreted factors, including GM-CSF, induced the increase in TAM eATP production. Elevated eATP in the GBM microenvironment promoted glioma growth and invasion by activating the P2X purinoceptor 7 (P2X7R) on glioma cells. Inhibition of the eATP–P2X7R axis attenuated tumor cell viability in vitro and reduced tumor size and prolonged survival in glioma-bearing mouse models. Overall, this study revealed elevated TAM-derived eATP in GBM and provided the basis for targeting the eATP–P2X7R signaling axis as a therapeutic strategy in GBM. Significance: Glioblastoma-mediated metabolic reprogramming in tumor-associated microglia increases ATP secretion that supports cancer cell proliferation and invasion by activating P2X7R, which can be inhibited to attenuate tumor growth.

Regulatory T cells (Treg) are important players in the tumor microenvironment. However, the mechanisms behind their immunosuppressive effects are poorly understood. We found that CCR6–CCL20 activity in tumor-infiltrating Tregs is associated with greater glycolytic activity and ablation of Ccr6 reduced glycolysis and lactic acid production while increasing compensatory glutamine metabolism. Immunosuppressive activity toward CD8+ T cells was abrogated in Ccr6−/− Tregs due to reduction in activation-induced glycolysis. Furthermore, Ccr6−/− mice exhibited improved survival across multiple tumor models compared to wild-type mice and Treg and CD8+ T-cell depletion abrogated the improvement. In addition, Ccr6 ablation further promoted the efficacy of anti-PD-1 therapy in a preclinical glioma model. Follow-up knockdown of Ccl20 with siRNA also demonstrated improvement in antitumor efficacy. Our results unveil CCR6 as a marker and regulator of Treg-induced immunosuppression and identify approaches to target the metabolic determinants of Treg immunosuppressive activity.