Engineered CAR-T extracellular vesicles loaded with miR-17-5p inhibitor suppress hepatocellular carcinoma progression.

Authors

Zhifeng Zhou, Huiling Wu, Shen Zhao, Mingshui Chen, Wansong Lin, Jieyu Li, Qingfeng Zheng

Published in

Frontiers in immunology. Volume 17. Pages 1919449. Epub Sep 16, 2026.

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.

PMID:
42819479
Bibliographic data and abstract were imported from PubMed on 02 Oct 2026.

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