Fig. 6

CXCL1 knockdown in EV-dead or macrophage depletion inhibits EV-dead-induced TNBC growth and lung metastasis in vivo. A The successful generation of 4 T1/shCXCL1 cells was verified by western blotting assay. The difference in CXCL1 content between EV-dead and EV-dead^shCXCL1 was compared by ELISA. EV-dead^shCXCL1 was isolated from the supernatants of apoptotic 4 T1/shCXCL1 cells induced by paclitaxel treatment; n = 3. B Representative images of the tumors (n = 7) and the in vivo imaging assay (n = 3), and mouse weight and tumor volume curves (n = 7). Clodronate liposomes (CL) were used to deplete macrophages in the TME of the 4 T1-Luc xenograft model. Scale bar: 2 cm. C Representative images of the lungs (n = 3) and lung HE assay (n = 3) as well as the K-M curves of lung metastasis time (n = 10). Scale bar: 100 μm. D, E The infiltration levels of CD45⁺/F4/80⁺/CD206⁺ TAMs (D) and CD45⁺/F4/80⁺/PD-L1⁺ TAMs (E) in the TME of mice following treatment with EV-dead, EV-dead^shCXCL1, or the combination of EV-dead and CL; n = 3. F CD206 (green) and PD-L1 (red) expression levels in the TME. Arrows indicate PD-L1 expression in TAMs. Scale bar: 10 μm. G The quantity of CTCs in the peripheral blood of mice treated as indicated; n = 3. Data are presented as mean ± SD. ^*p < 0.05, ^**p < 0.01