Background: Breast cancer is the most common malignant tumor among women. Recent studies have found that gut probiotics and their metabolic products play a significant role in activating the immune system, reshaping the tumor microenvironment, and inhibiting cancer progression.

Methods: We established a 4T1 tumor-bearing mice model and analyzed the proportions of CD4⁺ T and CD8⁺ T cells in the spleen using flow cytometry and immunohistochemistry. The expression levels of TNF-α, IL-6, and IL-10 were measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin staining was used to observe the tumor morphology. Selective protein blotting and quantitative real-time PCR were used to analyze the expression of Bax, Bcl-2, and Caspase-3. Cell proliferation was evaluated using the MTT assay, and apoptosis was detected by flow cytometry.

Results: The results indicated that oral administration of CB and AKK possesses the capability to inhibit the progression of 4T1 breast cancer; however, the combined treatment with both strains (CB-AKK) exhibited significantly superior effects compared to each individual strain. Further mechanistic analysis revealed that the CB-AKK combination could activate the antitumor immunity in mice and reshape the tumor microenvironment. Additionally, it was found that the live bacteria and their metabolites derived from CB-AKK could inhibit cell proliferation and promote tumor apoptosis by activating the Bcl-2/Bax signaling pathway.

Conclusion: This study is the first to demonstrate that orally administered live bacteria CB-AKK can inhibit the progression of 4T1 breast cancer, providing a promising new strategy for the development of innovative biotherapies for breast cancer.

Keywords: Akkermansia muciniphila; Clostridium butyricum; Bcl‐2/Bax signaling pathway; apoptosis; breast cancer; probiotics.

© 2025 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.

背景： 乳腺癌是女性中最常见的恶性肿瘤。最近的研究发现，肠道益生菌及其代谢产物在激活免疫系统、重塑肿瘤微环境和抑制癌症进展方面发挥着重要作用。

方法： 我们建立了一个4T1荷瘤小鼠模型，并使用流式细胞术和免疫组织化学分析脾脏中CD4⁺T细胞和CD8⁺T细胞的比例。通过酶联免疫吸附法测定TNF-α、IL-6和IL-10的表达水平。使用苏木精-伊红染色观察肿瘤形态。采用选择性蛋白印迹及实时定量PCR分析Bax、Bcl-2和Caspase-3的表达情况。通过MTT实验评估细胞增殖，并利用流式细胞术检测凋亡。

结果： 结果显示，口服给予CB和AKK能够抑制4T1乳腺癌的发展；然而，两种菌株联合使用（CB-AKK）的治疗效果显著优于单独使用任一菌株。进一步的机制分析表明，CB-AKK组合能够激活小鼠的抗肿瘤免疫并重塑肿瘤微环境。此外还发现，来自CB-AKK的活细菌及其代谢物可以通过激活Bcl-2/Bax信号通路抑制细胞增殖并促进肿瘤凋亡。

结论： 本研究首次证明口服活菌CB-AKK能够抑制4T1乳腺癌的发展，为开发新的生物疗法治疗乳腺癌提供了有前景的新策略。

关键词： Akkermansia muciniphila；Clostridium butyricum；Bcl‐2/Bax信号通路；凋亡；乳腺癌；益生菌。