{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T06:04:49Z","timestamp":1789538689020,"version":"build-2803163510"},"reference-count":40,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T00:00:00Z","timestamp":1789516800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100020783","name":"Fujian Provincial Federation of Social Sciences","doi-asserted-by":"publisher","award":["2025J011170, 2025J01206"],"award-info":[{"award-number":["2025J011170, 2025J01206"]}],"id":[{"id":"10.13039\/501100020783","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>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\u2013derived EVs (CAR-T EVs) retain CAR targeting specificity and carry cytotoxic effectors such as granzyme B and perforin, demonstrating independent antitumor potential.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Objective<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:italic>in vitro<\/jats:italic>\n                      and in a zebrafish HCC xenograft model.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      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-\u03b3 secretion\n                      <jats:italic>in vitro<\/jats:italic>\n                      .\n                      <jats:italic>In vivo<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  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