Fig. 1
FAM114A1 promotes TNBC immune evasion. a Schematic diagram illustrating tumor/immune coculture-based CRISPRa screening used to identify genes that promote immune evasion. Py8119-OVA-dCas9-P65/HSF1 cells were used for screening. b The enrichment status of genes was plotted in comparison with that of the non-coculture group. c Py8119 cells labeled with ovalbumin and luciferase (Py8119-OVA-Luc) were used to generate stable endogenous FAM114A1-knockdown cell lines. Cells with stable FAM114A1-knockdown (KD#1 and KD#2) and the corresponding control (shCoo2) were collected for western blotting to test the efficacy of FAM114A1-knockdown. d, e Py8119-OVA-Luc cells with and without FAM114A1-knockdown were injected into the mammary fat pads of female OT-I mice. Tumor growth was monitored and measured weekly (d). The tumors were dissected, and tumor weight was measured at the endpoint (e). Representative tumors are shown (e, left panel). n = 12 mice per group. Bar, 1 cm. f Tumors from the above groups were collected, formalin fixed, and paraffin embedded (FFPE). FFPE tumors were subjected to single-cell RNA sequencing. UMAP plots of the cell populations from the indicated groups. The T cells in each group are highlighted with red circles. g Average cell type proportions in the control (shCoo2) and FAM114A1-knockdown groups (FAM114A1-KD). The red start indicates the T-cell population. h Heatmap showing activated and nonactivated marker genes expressed in CD8+ T cells from the shCoo2 and FAM114A1-KD groups. i Fresh tumors from d were collected for flow cytometry analysis. The percentages of the indicated populations are shown. shCoo2, control group; KD#1 and KD#2, tumors with endogenous FAM114A1-knockdown. n = 6 tumors per group. j FFPE samples from TNBC patients were subjected to immunohistochemistry (IHC) staining with anti-FAM114A1 or anti-CD8 antibodies. Representative images are shown. The expression levels of FAM114A1 were quantified with ImageJ, and positive ratios of CD8+ T cells were determined. High- and low-FAM114A1 expression was determined by the median expression. Samples with ≥5% CD8+ T-cell infiltration were defined as CD8+ T-cell high, and those with <5% infiltration were defined as CD8+ T-cell low. n = 109 TNBC patients. Bar, 500 µm. The data represent the means ± SEMs. P-values were determined by one-way ANOVA (d, e, i) or the χ2 test (j)
