Cancer-associated fibroblast-derived extracellular vesicles loaded with GLUT1 inhibitor synergize anti-PD-L1 to suppress tumor growth via degrading matrix stiffness and remodeling tumor microenvironment

Cancer immunotherapy has transformed cancer treatment, demonstrating the potential for lasting responses in multiple solid and hematologic malignancies and thus has revolutionized cancer treatment in clinic. However, the intricate tumor microenvironment (TME), characterized by a rigid extracellular matrix (ECM) and robust immunosuppressive environment, presents substantial hurdles to the effectiveness of cancer immunotherapy. Thus, cancer-associated fibroblasts (CAFs), the most abundant stromal cells that mediate ECM remodeling and participate in immune suppression, represent promising therapeutic targets for combination immunotherapy. In this study, by using and analyzing single-cell RNA-sequencing (scRNA-seq) in the public datasets, we have identified the elevated expression of glucose transporter 1 (GLUT1) in activated CAF subgroups within tumor sites compared to normal tissues. Moreover, the recent literature has also demonstrated that CAFs undergoing high metabolic levels have been identified to show a better response to immunotherapy. Furthermore, extracellular vesicles (EVs) secreted by CAFs remain unexplored, and their role in drug transport systems and targeting efficiency towards tumorous cells remains uninvestigated. Herein, we identified the elevated expression of glucose transporter 1 (GLUT1) as a prognostic indicator for cancer associated with poor prognosis and investigated the vulnerability of lung tumor cell lines and CAFs to pharmacological GLUT1 inhibition with BAY-876. Based on the possibility of targeting the intrinsic TME-associated metabolism by GLUT1 inhibition, we have firstly employed CAFs-derived extracellular vesicles (cEVs) as a carrier for targeted delivery of BAY-876 into GLUT1-high CAFs and tumor cells. The cEV-BAY-876 (cEVB6) treatment significantly resulted in glucose-rich, low-lactate TME, reversed the activated CAFs phenotype, enabled stromal reprogramming, decreased ECM stiffness and enhanced the infiltration of CD3 + CD8+ T cells in tumor core, thereby achieving an excellent anti-tumor efficiency. Moreover, cEV-B6 treatment synergized anti-programmed death ligand 1 (antiPD-L1) to reinvigorate the exhausted lymphocytes and exerted strong anticancer effects against mice lung tumors. Our study provides the first evidence that tumor stroma-specific therapies by targeting glucose metabolism present a promising strategy of remodeling the extracellular matrix to reverse CAFs into normal type and potentiate cytotoxic T lymphocytes (CTLs) infiltration thereby improving anticancer immunotherapy.