[摘 要] 目的：探讨麻疹-流行性腮腺炎-风疹联合减毒活疫苗（MMR）通过诱导免疫原性细胞死亡（ICD）对三阴性乳腺癌 （TNBC）的免疫调节机制。方法：体外实验采用CCK-8法检测MMR对TNBC细胞4T1和MDA-MB-231存活率的影响，免疫荧光法检测MDA-MB-231细胞中钙网蛋白（CALR）表达，磷钼酸比色法检测MDA-MB-231细胞内ATP的含量，ELISA检测细胞上清液中高迁移率族蛋白B1（HMGB1）的表达水平，通过GEPIA网站分析CALR和HMGB1对乳腺癌患者生存期的影响。体内实验中经皮下注射4T1-Luc细胞于雌性BALB/c小鼠乳腺脂肪垫构建TNBC小鼠模型。设Control组、盐酸阿霉素（Dox）组、MMR组，按照分组每两天静脉给药1次，连续给药3次。动物活体成像系统观察肿瘤体积变化；记录小鼠体质量及各器官指数初步评估疫苗安全性；H-E染色观察肿瘤病理变化；免疫组化和Western blotting检测肿瘤中CALR和HMGB1蛋白的表达。流式细胞术分析小鼠脾T淋巴细胞亚群及肿瘤树突状细胞和巨噬细胞比例；免疫组化检测肿瘤CD8 + T淋巴细胞浸润；ELISA检测血清中促炎细胞因子IL-6和TNF-α 含量。结果：MMR对4T1细胞的活性抑制作用较弱；MMR对MDA-MB-231细胞抑制率达90.39% （P < 0.001）。与Control组相比，MMR组MDA-MB-231细胞中ICD相关蛋白（CALR、ATP、HMGB1）表达上调（P < 0.001）。 GEPIA网站分析表明，CALR和HMGB1高表达的患者总生存期有所延长。MMR延缓了荷瘤小鼠肿瘤体积增长（P < 0.001），延长小鼠的生存期，且28 d内MMR组荷瘤小鼠体重与Control组无显著性差异（P > 0.05）；H-E染色显示，与Control组相比，MMR组小鼠肿瘤组织坏死区域扩大且有免疫细胞浸润；MMR上调肿瘤ICD相关蛋白CALR、HMGB1的表达水平；MMR组脾CD4 +、 CD8 + T淋巴细胞及肿瘤树突状细胞和M1型巨噬细胞的比例增加（P < 0.05），CD8 + T淋巴细胞在肿瘤组织中的浸润增加（P < 0.001），且促炎细胞因子IL-6和TNF-α水平升高（P < 0.01）。结论：MMR减毒活疫苗对TNBC的抗肿瘤作用可能与诱导肿瘤发生ICD进而激活抗肿瘤免疫相关。

[Abstract] Objective: To investigate the immunomodulatory mechanisms of the live attenuated measles, mumps and rubella vaccine (MMR) against triple-negative breast cancer (TNBC) via induction of immunogenic cell death (ICD). Methods: In vitro experiments: The CCK-8 assay was used to evaluate the effect of MMR on the viability of TNBC cell lines 4T1 and MDA-MB-231. Immunofluorescence was performed to detect the expression of calreticulin (CALR) in MDA-MB-231 cells. ATP was measured using a phosphomolybdic acid colorimetric assay, and high mobility group box 1 (HMGB1) levels in the cell supernatant were determined by ELISA. The GEPIA database was used to analyze the correlation between CALR/HMGB1 expression and the overall survival of breast · · 641 [[PAGE_INDEX=24 FILE=08b612ef-59ff-47e1-960e-f599d97bfff7.pdf]] 中国肿瘤生物治疗杂志, 2026, 33(6) cancer patients. In vivo experiments: A TNBC mouse model was established by subcutaneous injection of 4T1-Luc cells into the mammary fat pad of female BALB/c mice. The mice were divided into three groups: Control group, doxorubicin hydrochloride (Dox) group, and MMR group. According to the grouping, intravenous administration was performed once every two days for a total of three consecutive doses. Tumor volume changes were monitored using an in vivo imaging system. Body weight and organ indices were recorded for preliminary safety assessment of vaccine. Histopathological changes in tumors were observed by hematoxylin-eosin (H-E) staining. The expression of CALR and HMGB1 in tumor tissues was detected by immunohistochemistry and Western blotting. Flow cytometry was used to analyze T lymphocyte subsets in the spleen, as well as the proportions of dendritic cells and macrophages in tumor. Immunohistochemistry was performed to detect CD8 + T lymphocyte infiltration in tumor tissues. The levels of pro-inflammatory cytokines IL-6 and TNF-α in serum were measured by ELISA. Results: MMR showed weak inhibitory effect on the viability of 4T1 cells but inhibited MDA-MB-231 cells by 90.39% (P < 0.001). Compared with the Control group, the MMR group exhibited upregulated expression of ICD-related proteins CALR, ATP and HMGB1 in MDA-MB-231 cells (P < 0.001). GEPIA database analysis indicated that patients with high expression of CALR and HMGB1 had prolonged survival. In vivo experiments, MMR delayed tumor volume growth (P < 0.001) and extended mouse survival. Within 28 days, there was no significant difference in body weight between the MMR group and the Control group (P > 0.05). H-E staining showed enlarged necrotic areas and immune cell infiltration in tumor tissues from the MMR group compared with the Control group. MMR upregulated the expression levels of the ICD-related proteins CALR and HMGB1. In the MMR group, the proportions of splenic CD4 + T and CD8 + T lymphocytes, as well as tumor-infiltrating dendritic cells and M1-type macrophages, were increased (P < 0.05). CD8 + T lymphocyte infiltration into tumors was also increased (P < 0.001), and the level of the pro-inflammatory cytokines IL-6 and TNF-α was upregulated (P < 0.01). Conclusion: The tumor- suppressive effect of the MMR vaccine on TNBC may be associated with the induction of ICD in tumors, thereby activating anti-tumor immunity.

[基金项目] 贵州省科技厅基础研究项目（黔科合作基础-ZK[2024]125）；贵州省卫生健康委-科学技术基金（gzwkj2023-280）；贵州省重点实验室项目（ZDSYS[2023]004），贵州省中央引导地方科技发展资金项目（黔科合[2025]024），国家科技部及教育部高等学校学科创新引智计划（D20009）；2024年国家级、省级大学生创新训练项目（2024106600555，S2024106601437X，S2024106601441X）