Repressed Blautia-acetate immunological axis underlies breast cancer progression promoted by chronic stress

Chronic stress is a known risk factor for breast cancer, yet the underlying mechanisms are unclear. This study explores the potential involvement of microbial and metabolic signals in chronic stress-promoted breast cancer progression, revealing that reduced abundances of Blautia and its metabolite acetate may contribute to this process. Treatment with Blautia and acetate increases antitumor responses of CD8+ T cells and reverses stress-promoted breast cancer progression in female mice. Patients with depression exhibit lower abundances of Blautia and acetate, and breast cancer female patients with depression display lower abundances of acetate, decreased numbers of tumor-infiltrating CD8+ T cells, and an increased risk of metastasis. These results suggest that Blautia-derived acetate plays a crucial role in modulating the immune response to breast cancer, and its reduction may contribute to chronic stress-promoted cancer progression. Our findings advance the understanding of microbial and metabolic signals implicated in cancer in patients with depression and may provide therapeutic options for female patients with breast cancer and depression.


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We enrolled 52 female patients with breast cancer, out of which 24 were also diagnosed with depression. Given that breast cancer primarily affects females, our study exclusively included female participants.
In addition, we recruited 40 patients with major depressive disorder, comprising 12 males and 28 females. We also had 30 healthy donors, consisting of 7 males and 23 females. We did not perform sex-and gender-based analyses. In this study, we excluded sex as a variable in both the study design and analysis, as our primary focus was to investigate whether reduced Blautia-acetate levels observed in depressed mice could be replicated in depressed patients. Since sex was not a targeted variable in our study, and data pertaining to sex-specific differences were neither collected nor analyzed separately.
No significant covariate factors, such as age, sex, past diagnoses, or treatments, were identified in this study.
Participants were recruited according to the study protocol for each project. Notably, there was no evident self-selection bias identified in this study.
Human stool samples were collected from both healthy controls and depression patients following ethical guidelines and with the approval of the Institutional Review Board of Southeast University Affiliated Zhongda Hospital (Reference 2018ZDSYLL119-P01). Human blood and breast tissue samples were collected with the approval of the Institutional Review Board of Nanfang Hospital (Reference NFEC-2018-038).
For our in vivo studies, we determined the sample size based on our extensive experience with animal models and endpoints. We selected appropriate sample sizes, as outlined in the manuscript, to ensure the generation of reproducible results with a significance level of less than 0.05 and a power of over 90%.
In the case of in vitro experiments, we chose sample sizes, with a minimum of three independent experimental replicates, in accordance with the standard practices of the research field (N=3). This selection was made to produce reproducible results with a significance level of less than 0.05 and a power exceeding 90%. Regarding human samples, the total sample size was contingent upon the availability of samples and the characteristics of the patients.
No data were excluded from the analyses.
We repeated experiments involving chronic stress and tumor induction in mice 2-3 times, consistently achieving satisfactory reproducibility. In vitro experiments conducted with 4T1 cells and/or CD8+ T cells were successfully replicated at least twice.For human research participants, we denoted the experimental points as 'n'-values, serving as biological replicates, in each figure legend. We utilized biological replicates for our statistical analyses, and we confirmed that all replication attempts in this study were successful.
Allocation was random. Detailed definitions and descriptions were provided in the manuscript.
Investigators were blinded to group allocation during data collection and analysis wherever possible. This was not possible during real-time treatment of live animals in mouse studies, as the treatment of each mouse would need to be known to the person handling the mice.

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Six-week-old female balb/c and C57 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). Mice (4-5 mice per cage) were housed under specific pathogen-free condition with controlled temperature at 22°C-26°C, humidity of 45 ± 5%, and 12 hour light/dark cycle.
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Female mice. We exclusively utilized female mice in our study, as breast cancer predominantly occurs in women.
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To analyze the immune cells infiltrating the tumor, the tumor tissue was minced on ice and digested in RPMI 1640 medium containing 3mg/ml collagenase II/IV and 10 $g/$l DNase I at 37°C℃ for 60 min. The digestion was stopped by adding RPMI 1640 supplemented with 1% penicillin-streptomycin, 0.5 mM EDTA and 10% FBS. The digested tumor tissue was passed through a 70 $m cell strainer (BD Biosciences) to abtain a single cell suspension. Similarly, the spleen tissue was homogenized by grinding with the plunger of a syringe (2.5 mL) and then filtered through a 70 $m cell strainer. To prevent non-specific binding, the cells were incubated with mouse CD16/CD32 monoclonal antibody (0.25 ug/100 ul) for 15 minutes at room temperature. Subsequently, the cells were stained with appropriate antibodies against s[ecific antigens in a blocking buffer on ice, following the manufacturer's instructions. A viability dye was used to exclude dead cells from the analysis.
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