Engineered CAR-T extracellular vesicles loaded with miR-17-5p inhibitor suppress hepatocellular carcinoma progression
TL;DR
It is demonstrated that NKG2D-targeted third-generation CAR-T EVs exert combined anti-HCC activities through direct tumor cytotoxicity and restoration of NK cell function, providing a novel immunotherapeutic strategy for HCC.
Abstract
Hepatocellular carcinoma (HCC) immune escape is co-driven by tumor malignant proliferation and an immunosuppressive tumor microenvironment, characterized by frequently impaired natural killer (NK) cell function. Previous research indicated that HCC cell-secreted extracellular vesicles (EVs) deliver miR-17-5p, inhibiting the RUNX1 pathway and downregulating the activating receptor NKG2D in NK cells, which ultimately results in NK cell dysfunction. Although chimeric antigen receptor (CAR)-T therapy has achieved remarkable efficacy in hematological malignancies, it encounters significant challenges in treating solid tumors, including HCC. Recent studies have confirmed that CAR-T cell–derived EVs (CAR-T EVs) retain CAR targeting specificity and carry cytotoxic effectors such as granzyme B and perforin, demonstrating independent antitumor potential. This study aimed to develop engineered CAR-T EVs loaded with a miR-17-5p inhibitor (EV-miR inhibitor) and evaluate their combined therapeutic efficacy against HCC through direct tumor cytotoxicity and restoration of NK cell function. This study constructed NKG2D-targeted third-generation CAR-T cells, isolated CAR-T EVs via ultracentrifugation, characterized EV properties using multiple assays, and generated engineered CAR-T EVs loaded with a miR-17-5p inhibitor (EV-miR inhibitor). The therapeutic effects of this system against HCC were verified both in vitro and in a zebrafish HCC xenograft model. CAR-T EVs exhibited characteristic cup-shaped morphology and robust expression of canonical EV markers together with the engineered NKG2D extracellular domain. Results indicated that CAR-T EVs entered HCC cells and exhibited concentration-dependent cytotoxicity against HCC cells without affecting the viability of normal hepatocytes. The loading of the miR-17-5p inhibitor was efficient, and the procedure did not alter the morphology, particle size, or marker expression of CAR-T EVs. EV-miR inhibitor effectively restored NKG2D and RUNX1 expression in miR-17-5p-overexpressing NK cells, significantly enhancing NK cell cytotoxicity against HCC and promoting IFN-γ secretion in vitro . In vivo experiments further confirmed that EV-miR inhibitor treatment markedly enhanced the antitumor activity of NK cells, leading to significant tumor growth inhibition in zebrafish xenografts. In conclusion, this study demonstrates that NKG2D-targeted CAR-T EVs exert combined anti-HCC activities through direct tumor cytotoxicity and restoration of NK cell function, providing a novel immunotherapeutic strategy for HCC.