Fig. 1

Gut microbiota is required for psychological stress-induced breast cancer progression and stemness. a Schematic of specific pathogen-free (SPF) and germ-free (GF) mice that were stressed (Stress) or served as control (Ctrl) (n = 10 mice per group). The stress group was exposed to chronic restraint stress for 7 days before inoculation with Py8119-pLVX-MCS-Luc2 or PY8119 cells, and Ctrl group was inoculated with Py8119-pLVX-MCS-Luc2 or PY8119 cells without stress exposure. b Representative bioluminescence images of Py8119-pLVX-MCS-Luc2 subcutaneous tumors in SPF and GF mice that were stressed or served as Ctrl. c Tumor volume curves in SPF/GF C57BL/6 J Ctrl or Stress mice (n = 10 mice per group). d Representative Hematoxylin-eosin (HE) photomicrographs, NANOG and Ki67 immunohistochemical (IHC) staining in Py8119-pLVX-MCS-Luc2 subcutaneous tumors (Scale bar, 50 μm). e Quantitative results of HE and IHC staining from indicated markers in indicated groups (n = 6 representative images). f Schematic of MMTV-PyMT mice in Ctrl and Stress groups treated with an antibiotic cocktail (Abx) or H₂O (n = 6 mice per group). The Abx and Abx+Stress groups received Abx treatment throughout the experiment for 32 days. g Representative images of the MMTV-PyMT mice treated with Abx or H₂O in Ctrl and Stress groups. h Tumor volumes curves in the Ctrl and Stress groups treated with Abx or H₂O (n = 6 mice per group). i Relative protein levels of NANOG and SOX2 in MMTV-PyMT tumors. j Schematic of the NOD/SCID mice under Ctrl or Stress conditions. Mice were orthotopically injected with 1×10⁶ MDA-MB-231 cells and treated with Abx or H₂O (n = 8 mice per group). The Abx and Abx+Stress groups received Abx treatment throughout the experiment for 32 days. NOD/SCID mice were orthotopically inoculated with MDA-MB-231 cells and treated with Abx or H₂O in the Ctrl and Stress groups. Representative images of MDA-MB-231 xenograft tumors (k) and tumor volumes curves (l) are shown (n = 8 mice per group). m ALDH⁺ cells in MDA-MB-231 primary tumors (n = 3 biological replicates). n Representative images of spheroid formed by a single MDA-MB-231 tumor primary cell (left). Data represent the number of spheres per 400 cells (d > 50 μm) (middle) and the diameter of spheres (right) (Scale bars, 100 μm). o Extreme limiting dilution assays (ELDA) were performed in MDA-MB-231 tumor primary cells. Representative sphere images are shown (Scale bars, 50 μm). Stemness frequency of cells with the upper and lower 95% confidence intervals that shows the frequency of one stem cell in the tumors. Spheres were counted from 24 replicate wells. All data represent the mean ± s.d. (c, h, l) by two-tailed, unpaired Student’s t-test at the ethical endpoint, e, m, n by one-way analysis of variance (ANOVA), o by the likelihood ratio test. P values are as indicated