Fig. 6.

Etoposide inhibits the EMT/β-catenin/STT3/PD-L1 axis and downregulates PD-L1 of CSC and non-CSC populations. a Effect of etoposide (ETO) on the protein and mRNA expression of EMT markers, STT3, and PD-L1 in the general cell population of 4T1 cells. E-Cad E-cadherin, N-Cad N-cadherin, Vim vimentin. b Effect of etoposide and exogenous STT3A/B on PD-L1 expression in the general cell population of 4T1 cells. c Flow cytometric analysis of the influence of etoposide and exogenous STT3A/B on the frequency (left) and PD-L1 expression (right) of 4T1 CSC (CD44⁺CD24⁺ALDH1⁺) populations. Open histograms represent isotype IgG negative control. d Experimental workflow of preparing spheres derived from etoposide-treated 4T1 cells. e Western blot analysis (left), PD-1 binding assay (upper right), and in vitro PBMC-mediated tumor cell killing assay (bottom right) of tumorspheres derived from 4T1 cells treated with etoposide and exogenous STT3A/B. f–h Tumor-seeding ability (f), tumor onset curve (g), and tumor growth curve (h) of tumorspheres cultured from etoposide-treated 4T1 cells. i Representative images of tumors staining with PD-L1, CD8, granzyme b (GZMB), and Hoechst. Scale bar, 200 μm. The intensity of immunofluorescence signal was quantified from nine core biopsy sections, normalized relative to the control group, and shown as means ± s.d. Other error bars represent s.d. (n = 3). *P < 0.05, Student’s t-test. See also Supplementary Fig. 6