大剂量维生素C与溶瘤腺病毒协同治疗乳腺癌的免疫学机制

苏旭; 邹洋; 蒋双鞠; 刘宁亿; 魏青青; 刘金星; 罗俊; 黄安亮; 杨帆

doi:10.12182/20260960203

大剂量维生素C与溶瘤腺病毒协同治疗乳腺癌的免疫学机制

Immunologic Mechanism of the Synergistic Antitumor Effects of High-Dose Vitamin C and Oncolytic Adenovirus in Breast Cancer

摘要

摘要:
目的探究大剂量维生素C（VitC）联合溶瘤腺病毒（oncolytic viruses, oAds）对乳腺癌的治疗效果，并阐明其是否通过诱导Gasdermin E（GSDME）依赖性细胞焦亡激活抗肿瘤免疫应答。
方法采用4T1乳腺癌细胞及BALB/c小鼠荷瘤模型，通过CRISPR-Cas9技术构建GSDME基因敲除（GSDME^－/－）细胞。设置对照组、VitC单药（2 mmol/L）组、oAds单药（感染复数（MOI）=10）组及联合治疗组。Western blot检测GSDME蛋白切割及caspase-3活化；流式细胞术区分焦亡与凋亡；免疫荧光检测肿瘤内CD8⁺ T细胞浸润；ELISpot及流式细胞术检测脾脏干扰素（IFN）-γ⁺ CD8⁺ T细胞比例。
结果联合治疗诱导78.3%的4T1细胞发生膜破裂，激活caspase-3并切割GSDME。GSDME敲除后，焦亡率由65.4%降至6.2%（P<0.001），转为凋亡为主。体内联合治疗组肿瘤体积（454.3 mm³）显著小于对照组（1339.4 mm³，P<0.0001）及单药组（约910 mm³，P<0.01），中位生存期延长至52 d。GSDME敲除部分逆转疗效（肿瘤体积增至773.9 mm³，P<0.01；生存期缩至43 d，P<0.05）。联合治疗显著增加瘤内CD8⁺ T细胞浸润（212 个/视野 vs. 58个/视野，P<0.0001）及脾脏IFN-γ⁺ CD8⁺ T细胞比例（15.8% vs. 7.3%，P<0.0001），该效应在GSDME敲除后消失。
结论大剂量VitC联合oAds通过诱导GSDME依赖性细胞焦亡，重塑肿瘤免疫微环境，激活系统性抗肿瘤免疫。GSDME是联合治疗的关键效应分子，本研究为三阴性乳腺癌的“病毒-免疫”联合治疗提供了新策略。

Abstract:
Objective To investigate the therapeutic effect of high-dose vitamin C (VitC) combined with oncolytic adenovirus (oAds) against breast cancer and to determine whether the combination therapy activates antitumor immune responses by inducing gasdermin E (GSDME)-dependent pyroptosis.
Methods The 4T1 murine mammary cancer cell line and a BALB/c mouse tumor-bearing model were used. GSDME-knockout (GSDME^－/－) cells were generated using CRISPR-Cas9. The 4T1 cancer cells and tumor-bearing mice were assigned to a control group, a VitC group receiving only VitC treatment (2 mmol/L), an oAds group receiving only oAds treatment (multiplicity of infection MOI = 10), and a combination treatment group. Western blotting was performed to assess GSDME cleavage and caspase-3 activation. Flow cytometry (annexin Ⅴ/propidium iodide PI staining) was used to differentiate pyroptosis and apoptosis. Immunofluorescence was performed to assess intratumoral CD8⁺ T cell infiltration. ELISpot and flow cytometry were performed to determine the proportions of interferon (IFN)-γ⁺ CD8⁺ T cells in the spleen.
Results Combination treatment induced membrane rupture in 78.3% of 4T1 cells, activated caspase-3, and cleaved GSDME. After GSDME knockout, pyroptosis rate decreased from 65.4% to 6.2% (P < 0.001), shifting to apoptosis. In vivo, combination group tumor volume (454.3 mm³) was significantly smaller than control (1339.4 mm³, P < 0.0001) and monotherapy groups (approximately 910 mm³, P < 0.01), with median survival extended to 52 d. GSDME knockout partially reversed efficacy (tumor volume increased to 773.9 mm³, P < 0.01; survival shortened to 43 d, P < 0.05). Combination significantly increased intratumoral CD8⁺ T cell infiltration (212 cells/field vs. 58 cells/field, P < 0.0001) and splenic IFN-γ⁺ CD8⁺ T cell proportion (15.8% vs. 7.3%, P < 0.0001); these effects were abolished by GSDME knockout.
Conclusion High-dose VitC combined with oAds induces GSDME-dependent pyroptosis, remodels the tumor immune microenvironment, and activates systemic antitumor immunity. GSDME is a key effector molecule in this combination treatment. These findings provide a new strategy for virus-immunotherapy combination treatment for triple-negative breast cancer.

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