The combination gene therapy of HSV-TK/IL-12 inhibits non-small cell lung cancer through the bystander effect associated with Cx43 up-regulation and immune activation

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Background Lung cancer remains the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) representing a major subtype characterized by high incidence and mortality. Despite advances in targeted therapy and immunotherapy, treatment resistance and the immunosuppressive tumor microenvironment remain major clinical challenges. The combination of herpes simplex virus thymidine kinase and interleukin-12 (HSV-TK/IL-12) has demonstrated anti-tumor potential, but its role and mechanism of action in NSCLC remain poorly defined. This study aims to systematically evaluate the anti-tumor efficacy of HSV-TK/IL-12 combination gene therapy in NSCLC and to investigate the potential involvement of Connexin 43 (Cx43) in the bystander effect (BSE) alongside immune activation. Methods Overexpression vectors for HSV-TK and IL-12 were constructed to study their functions in NSCLC. Protein expression was measured by western blot. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry were employed to assess cell survival and apoptosis, respectively. In vitro , human peripheral blood lymphocytes were used to evaluate immune responses relevant to clinical immunotherapy. In vivo , xenograft mouse models were used to evaluate effects on tumor growth and experimental lung colonization. Results Overexpression of HSV-TK or IL-12 significantly increased their respective protein levels in A549-TK and H1299-TK cells ( P < 0.01). Ganciclovir (GCV) treatment reduced the viability of A549 and H1299 cells in a dose-dependent manner ( P < 0.05). Importantly, the combination therapy of HSV-TK and IL-12 induced apoptosis in A549 and H1299 cells ( P < 0.05). In vitro , the combination also enhanced the proliferation and cytotoxic activity of co-cultured human peripheral blood lymphocytes ( P < 0.05). A pronounced BSE was observed in the presence of HSV-TK/GCV, and its magnitude was positively correlated with the proportion of TK + cells and with Cx43 expression levels ( P < 0.05). In vivo , the combination of HSV-TK and IL-12 inhibited tumor growth and reduced lung nodule formation in a tail-vein colonization model ( P < 0.05). Conclusion The combination of suicide gene HSV-TK and immunomodulatory gene IL-12 exerts a stronger anti-tumor effect than either monotherapy in NSCLC, demonstrating significant potential for clinical translation as a novel gene therapy strategy for NSCLC patients, particularly those with treatment-resistant disease.

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