Fig. 3: Landscape of the Hepa1-6 tumour immune microenvironment after the consumption of different diets and supplements.

Tumour-infiltrating immune cells isolated from the CD-fed, HFD-fed, or HFD + B. breve-treated mice were assessed by multicolour flow cytometry on Day 14 after tumour cell inoculation (n = 8 mice/group). A, F NK cells, NKT cells, T cells, Tc cells, and Th cells among total single live cells in Hepa1-6 tumours. B, G Proportions of Tc cells and Th cells among the total population of T cells. C, H DCs, TAMs and their subsets, and MDSCs of total single live cells in Hepa1-6 tumours. D, I Proportions of proinflammatory (M1) TAMs and alternatively activated (M2) TAMs among the total population of TAMs. E, J Representative images of H&E-stained tumour tissues are shown in the left panel. Immunohistochemical staining of the tumour sections revealed that the main tumour-infiltrating immune cells were recognized by detecting the expression of corresponding surface markers, such as CD3, CD4, CD8, F4/80, CD86 and CD206 (left panel; scale bar = 50 μm). The quantitative analysis was performed with ImageJ, and the number of positive cells is presented in column plots (right panel). The data are presented as the means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns: not significant. CD control diet, HFD high-fat diet, B. breve Bifidobacterium breve, NK natural killer, Tc cytotoxic CD8⁺ T cell, Th helper CD4⁺ T cell, DCs dendritic cells, TAMs tumour-associated macrophages, MDSCs myeloid-derived suppressor cells.