Figure 2. Dysbiosis enhances mammary gland inflammation and myeloid cell infiltration.

A. Experimental design for B-E. C57BL/6 mice were orally gavaged for 14 days with a broad-spectrum cocktail of antibiotics or an equal volume of water as a vehicle control. Gavage was ceased four days prior to BRPKp110 tumor initiation into the abdominal mammary fat pad. Mice were euthanized at early (day 12) and advanced (day 27) tumor timepoints. Four days prior to each timepoint, a modified Whitten effect was used to synchronize estrus in these animals. Normal tumor-adjacent mammary glands were harvested, and infiltrating myeloid cell populations were quantitated by flow cytometry at early (B) and advanced (C) timepoints after tumor initiation. All populations were gated on live, singlet, CD45⁺CD11b⁺ cells. Numbers represent absolute numbers of cells quantitated using counting beads. M0 macrophages = F4/80⁺CD86⁻CD206⁻. M1 macrophages = F4/80⁺CD86⁺CD206⁻. M2 macrophages = F4/80⁺CD86⁻CD206⁺. Monocytic MDSC = Ly6C^hiLy6G⁻. Polymorphonuclear MDSC = Ly6C^midLy6G⁺. Arginase-1 and IL-6 expression were quantitated by intracellular staining in bulk CD45⁺CD11b⁺ cells from mammary glands at early (D) and advanced (E) timepoints after tumor initiation. Numbers represent absolute numbers of cells quantitated using counting beads. F. Experimental design for G. Similar to A, C57BL/6 mice were orally gavaged for 14 days with a broad-spectrum cocktail of antibiotics or an equal volume of water as a vehicle control. Gavage was ceased four days prior to BRPKp110 tumor initiation into the abdominal mammary fat pad. Mice were euthanized prior to tumor initiation (pre-tumor; day 0) and at an early point (early tumor; day 12) during tumor progression. Four days prior to each timepoint, a modified Whitten effect was used to synchronize estrus in these animals. Normal tumor-adjacent mammary glands were harvested, and protein levels of CXCL10, CCL2, and CXCL2 were quantitated by multiplex cytokine analysis (G).