Fig. 2

EV-dead promotes the metastasis and chemoresistance of TNBC cells by inducing macrophage M2 polarization. A EV-dead uptake by Raw264.7 macrophages was visualized using immunofluorescence labeling and quantified by flow cytometry. EV-dead was labeled with PKH67 (green). Raw264.7 macrophages were labeled with ActinRed (red) and DAPI (blue); Yellow arrows indicate EVs phagocytosed by Raw264.7 macrophages. n = 3. Scale bar: 5 μm. B The uptake of red fluorescent polystyrene beads (negative control of EV-dead) by Raw264.7 macrophages was quantified by flow cytometry. n = 3. C, D The polarization changes of Raw264.7 macrophages after EV-dead treatment (25–100 μg/ml) for 48 h; n = 3. E Diagram of 4 T1 and Raw264.7 cell co-culture using the Transwell system. Proliferation activity changes of the co-cultured 4 T1 cells after EV-dead treatment as indicated; n = 3. F Migration and invasion efficacy changes of the co-cultured 4 T1 cells after EV-dead treatment. Scale bars: 100 μm; n = 3. G Changes in apoptosis resistance of the co-cultured 4 T1 cells to paclitaxel after EV-dead treatment for 48 h, as determined by annexin V-FITC/PI staining and flow cytometry; n = 3. H The ALDH⁺ BCSC subpopulation and CD133⁺ BCSC subpopulation in 4 T1 cells after EV-dead treatment for 48 h were detected by flow cytometry. Diethylaminobenzaldehyde (DEAB) is a specific inhibitor of ALDH activity. n = 3. I The CD133⁺ BCSCs were sorted from 4 T1 cells, and the in vitro limiting dilution assay was conducted to further investigate the self-renewal activities of BCSCs after treatment with the CM of EV-dead-treated Raw264.7 cells; n = 8. Data are presented as mean ± SD. ^*p < 0.05, ^**p < 0.01