Fig. 3

TIM knockdown inhibits breast cancer oncogenic behavior through enhancing CD8⁺ cytotoxic T lymphocytes (CTLs) infiltration in vivo and in vitro. a The Western Blot analyses showed the stable interference efficiency of TIM via shRNA in mouse TNBC cell line 4T1. b Image of syngeneic tumors at the end of the experiment (4T1 shNC and shTIM groups, n = 4 in each group). The scale bar is 1 cm. c Tumor volume in each group was measured every five days and tumor weight at the end of the experiment was measured in each group. Values are means ± SD. d Representative Ki-67 IHC images of the syngeneic mice tumor tissue in 4T1 shNC mice and 4T1 shTIM mice. The scale bar is 50 μm. e The hematoxylin–eosin staining of lung metastasis in the 4T1 NC group and the 4T1 shTIM group of syngeneic mice. The scale bar is 100 and 500 μm. f Flow cytometry analysis was used to evaluate the percentage of CD8⁺ T cells in syngeneic mice tumor tissue from 4T1 shNC mice and 4T1 shTIM mice (n = 3). g Immunostaining of CD8 (CTL marker, pseudo-color: green) and granzyme B (activity of T cell, pseudo-color: red) in the 4T1 NC mice and 4T1 shTIM mice. Hoechst: nuclear counterstaining (pseudo-color: blue). The scale bar is 20 and 50 μm. h Immunostaining of cleaved caspase 3 (CCA3, pseudo-color: red) in the 4T1 NC mice and 4T1 shTIM mice. Hoechst: nuclear counterstaining (pseudo-color: blue). The scale bar is 20 and 50 μm. i Representative flow cytometric analysis figures of apoptosis levels on mouse breast cancer cell line 4T1 co-cultured with their specific CTLs or CD8⁺ T cells in the siNC group and siTIM group. Significant differences in the average apoptosis percentage were seen between siTIM + CTL group and siTIM + CD8 or siNC + CTL group. j Cell viability curve of 4T1 co-cultured with their specific CTLs or CD8⁺ T cells in the siNC group and siTIM group. Significant differences were seen between siTIM + CD8 group and siTIM + CTL or siNC + CTL group. k Enzyme-linked immunosorbent assay (ELISA) for IFN-γ secretion by CD8 + CTLs after co-culture of 4T1 with their specific CTLs or CD8⁺ T cells in the siNC group and siTIM group. Significance was tested using Mann–Whitney U-test: ns, p-value no significance; *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001. TNBC triple negative breast cancer, IHC Immunohistochemistry, CTL cytotoxic T lymphocytes, CCA3 cleaved caspase 3, ELISA enzyme-linked immunosorbent assay