Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: EV-dead induces TNBC lung metastasis and promotes TAM infiltration in vivo. A Diagram of the EV-dead and EV-alive separation procedures and their representative transmission electron microscopy (TEM) images. Scale bar: 100 nm. The sizes of the EVs were detected using a Flow NanoAnalyzer, and their protein markers were identified by Western blotting analysis. B NTA analysis was conducted to detect the relative secretion content of EV-dead and EV-alive; n = 3. C Representative images of zebrafish breast cancer xenotransplantation model assay. The effects of EV-alive (100 μg/ml) and EV-dead (50–100 μg/ml) on the metastasis of 4 T1 cells in the presence or absence of M0 co-injection were investigated; n = 6. D Schematic diagram of the animal assay and representative pictures of the in vivo imaging assay and tumors. Peritumoral injection with EV-dead (200 μg/20 g weight, q3d) promoted tumor growth of 4 T1-Luc xenografts, as evidenced by increases in both tumor volume and weight, but had no significant effect on the weight of the mice; n = 8. Scale bar: 1 cm. E Representative images of the lungs and the lung HE staining assay. Metastatic foci were identified by HE staining of the lung sections. Scale bar: 100 μm; n = 3. F Infiltration levels of CD45 + /F4/80 + /CD206 + TAMs in mammary tumors; n = 3. G Expression levels of ARG1 and iNOS in mammary tumor tissues; n = 3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C.

Techniques: In Vivo, Transmission Assay, Electron Microscopy, Western Blot, Injection, In Vivo Imaging, Staining, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: CXCL1 EV-dead promotes TNBC growth and lung metastasis in vivo by activating TAM/PD-L1 signaling. A The successful generation of 4 T1/rCXCL1 Flag cells was validated by immunoblotting and immunofluorescence assays. Scale bar: 5 μm. B The difference in CXCL1 content between EV-dead and EV-dead rCXCL1-Flag was compared by ELISA. EV-dead rCXCL1-Flag was isolated from the supernatants of apoptotic 4 T1/rCXCL1 Flag cells induced by paclitaxel treatment; n = 3. C , D Peritumoral injection with EV-dead rCXCL1 (200 μg/20 g weight, q3d) significantly accelerated TNBC growth (C) and lung metastasis (D) compared to that of the EV-dead group (200 μg/20 g weight, q3d); Tumor volume: n = 6; Number of metastatic lesions, n = 3; K-M curves of lung metastasis time, n = 10. Scale bar: 1 cm. E Tumor tissue immunofluorescence experiment showed that flag-tagged CXCL1 (red) from EV-dead rCXCL1-Flag was predominantly phagocytosed by CD206 + macrophages (green) in the TME. Scale bar: 5 μm. F The infiltration levels of CD45 + /F4/80 + /CD206 + TAMs (up) and CD45 + /F4/80 + /PD-L1 + TAMs (down) in the TME of mice following treatment with saline, EV-dead, or EV-dead rCXCL1-Flag ; n = 3. G QPCR assay was conducted to investigate the CTC quantity in the peripheral blood of mice following treatment with saline, EV-dead, or EV-dead rCXCL1-Flag ; n = 3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C.

Techniques: In Vivo, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Isolation, Injection, Saline

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: 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

Article Snippet: Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C.

Techniques: Knockdown, In Vivo, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, In Vivo Imaging, Liposomes, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: TPCA-1 chemosensitizes TNBC to paclitaxel and inhibits CXCL1 EV-dead -induced TNBC growth and lung metastasis in vivo . A Representative images of tumors and the tumor volume curves ( n = 6). TPCA-1 (10 mg/kg/d) and paclitaxel (10 mg/kg/3d) were administered. B Representative images of the in vivo imaging assay and lung HE staining assay; n = 3. Scale bar: 100 μm. C , D The infiltration levels of CD45 + /F4/80 + /CD206 + TAMs and CD45 + /F4/80 + /PD-L1 + TAMs in the TME of mice following treatment with TPCA-1, paclitaxel, or the combination of TPCA-1 and paclitaxel; n = 3. E The CTC quantity in the peripheral blood of mice when they were treated as indicated; n = 3. F Representative images of tumors and the tumor volume curves ( n = 6). EV-dead and EV-dead rCXCL1 (200 μg/20 g weight, q3d) were administered by peritumoral injection. G Representative images of the in vivo imaging assay and lung HE staining assay; n = 3. Scale bar: 100 μm. H , I The infiltration levels of CD45 + /F4/80 + /CD206 + TAMs ( n = 3) and CD45 + /F4/80 + /PD-L1 + TAMs ( n = 6) in the TME of mice when they were treated as indicated. J The CTC quantity in the peripheral blood of mice when they were treated as indicated; n = 3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C.

Techniques: In Vivo, In Vivo Imaging, Staining, Injection