Fig 2: DNA topoisomerase II (TOP2) poisons induce mesenchymal–epithelial transition (MET). a Western blot analysis of epithelial marker E-cadherin (E-Cad) induction in mesenchymal-like 4T1 cells treated with indicated agents for 24 h (please check Supplementary Table 1 for compound concentrations). Compounds that significantly induce E-cadherin are labeled in red. b Relative cell number after treatment of indicated agents at a high-dose, short-term schedule. CTRL control, PTX paclitaxel (1 µM, 24 h), DOXO doxorubicin (2 µM, 24 h), ETO etoposide (10 µM, 24 h), MX mitoxantrone (2 µM, 24 h), DNR daunorubicin (1 µM, 24 h), EP epirubicin (1 µM, 24 h). c Cell viability of resting cells after treatment of indicated agents at a high-dose, short-term schedule. Cell viability was analyzed by trypan blue exclusion. d Western blot analysis of EMT markers (E-cadherin, N-cadherin, Vimentin) in resting cells after treatment of indicated agents at a high-dose, short-term schedule. e Relative cell number after treatment of indicated agents at a metronomic (low-dose, long-term) schedule. CTRL control, PTX paclitaxel (0.02 µM, 96 h), DOXO doxorubicin (0.05 µM, 96 h), ETO etoposide (0.2 µM, 96 h), MX mitoxantrone (0.05 µM, 96 h), DNR daunorubicin (0.02 µM, 96 h), EP epirubicin (0.02 µM, 96 h). f Cell viability of resting cells after treatment of indicated agents at a metronomic (low-dose, long-term) schedule. g Western blot analysis of EMT markers (E-cadherin, N-cadherin, Vimentin) in resting cells after treatment of indicated agents at a metronomic (low-dose, long-term) schedule. h Representative phase-contrast microscopy images of 4T1 cells treated with indicated agents. Scale bar, 50 µm. i Representative trajectories and quantification of motility speed of 4T1 cells treated with indicated agents. The box plots represent sample maximum (upper end of whisker), upper quartile (top of box), median (band in the box), lower quartile (bottom of box), and sample minimum (lower end of whisker). j Migration and invasion assays of 4T1 cells treated with indicated agents. All error bars represent s.d. (n = 3)

Fig 3: Etoposide inhibits the EMT/ß-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

Fig 4: Etoposide inhibits the EMT/ß-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear ß-catenin downregulation. a Effect of TOP2 isoforms knockdown on the expression of EMT markers, STT3 and PD-L1 in 4T1 cell. b Representative phase-contrast microscopy images of 4T1 cell treated with indicated sg-RNAs. Scale bar, 50 µm. c Influence of TOP2 isoforms knockdown on etoposide-induced MET and downregulation of STT3 and PD-L1. d Western blot analysis (left), PD-1 binding assay (upper right), and in vitro PBMC-mediated cancer cell killing assay (bottom right) of tumorspheres cultured from various sgRNA-treated 4T1 cells. e Effect of proteasome inhibitor (MG132) and caspase inhibitor (Z-VAD-FMK) on etoposide-induced TOP2B degradation, MET, and downregulation of STT3 and PD-L1. f Western blotting of whole-cell extracts analyzing the effect of etoposide and TOP2B knockdown on total and active (non-phospho) ß-catenin (ß-Cat). g Western blotting of whole-cell extracts (WCE), cytosolic (Cyto), membrane (Mem), or nuclear (Nuc) fractions from sgRNA-treated 4T1 cells analyzing the effect of TOP2B knockdown on ß-catenin subcellular localizations. Histone H3, E-cadherin (E-Cad), and tubulin were used as makers of nuclear, membrane, and cytosolic fractions, respectively. h Efficacy of ß-catenin knockdown (si-ß-Cat) in desensitizing cells to etoposide- and sg-TOP2B- induced MET and downregulation of STT3 and PD-L1. Nuc nuclear fraction, WCE whole-cell extracts. i Co-immunoprecipitation assay showing interaction between TOP2B and ß-catenin. j Duolink assay analyzing the interaction of TOP2B and active (non-phospho) ß-catenin in the nucleus. Red dots (indicated by arrows) represented TOP2B-ß-catenin interaction signals and nuclei were counterstained with DAPI (blue). The right column shows higher-magnification image of the area outlined in the left column. Scale bar, 5 µm. Error bars represent s.d. (n = 3). *P < 0.05, Student’s t-test. See also Supplementary Fig. 8

Fig 5: EMT induces a more robust upregulation of PD-L1 protein in CSCs than in non-CSCs without significant induction of PD-L1 mRNA levels. a, c Western blot analysis of PD-L1 and EMT markers in the general cell population of MCF-10A cells undergoing EMT driven by TGF-ß (a) or RasV12 (c). E-Cad E-cadherin, N-Cad N-cadherin, Vim vimentin. b, d Flow cytometric analysis of PD-L1 protein expression in breast stem-like cell (SC) (CD44+CD24-/low, red histograms) and non-SC (non-CD44+CD24-/low, blue histograms) populations from control cells and EMT cells driven by TGF-ß (b) or RasV12 (d). Open histograms represent isotype IgG negative control. The mean fluorescence intensity (MFI) of each cell population was quantified by FlowJo and compared. e, f Representative phase-contrast microscopy images and PD-L1 expression of spheres growing from control or EMT cells driven by TGF-ß (e) or RasV12 (f). Scale bar, 200 µm. g, h qRT-PCR analysis comparing PD-L1 (CD274) mRNA induction levels between SC (CD44+CD24-/low) and non-SC (non-CD44+CD24-/low) populations upon TGF-ß (g) or RasV12 (h) treatment. Error bars represent s.d. (n = 3). *P < 0.05; ns: non-significant, Student’s t-test. See also Supplementary Fig. 1