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

Journal: Nature Communications

Article Title: Synthesis of bioactive (1→6)-β-glucose branched poly-amido-saccharides that stimulate and induce M1 polarization in macrophages

doi: 10.1038/s41467-022-32346-5

Figure Lengend Snippet: Fold change of median fluorescence intensity of a CD80, b CD206, and c CD163 compared to an unstained control. Two-tailed unpaired t test were performed for a LPS vs P5 ( p = 0.0002), LPS vs Lentinan ( p = 0.0002), LPS vs Laminarin ( p = 0.0132). Percentage of positive cells of d CD80, e CD206, and f CD163 compared to an unstained control. Error bars represent standard deviation. Two-tailed unpaired t test were performed for d LPS vs P5 ( p < 0.0001), LPS vs Lentinan ( p = 0.0003), LPS vs Laminarin ( p = 0.0296). Unless otherwise stated, data are means ± SD of three separate experiments ( N = 3). ns p > 0.05, * p < 0.05, **** p ≤ 0.0001 compared using two-tailed unpaired t test. Abbreviations used: Ctrl = Control, Dex = Dextran, Lent = Lentinan, Lam = Laminarin.

Article Snippet: After 24 h of incubation, cells were lifted from the plate with cold PBS and Accutase cell detachment solution and stained for CD68 (StemCell 60105FI) and either CD80 (Biolegend 333611), CD163 (Biolegend 305221), or CD206 (R&D Systems FAB25342P).

Techniques: Fluorescence, Control, Two Tailed Test, Standard Deviation

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Combinatorial macrophage induced innate immunotherapy against Ewing sarcoma: Turning "Two Keys" simultaneously.

doi: 10.1186/s13046-024-03093-w

Figure Lengend Snippet: Fig. 5 Characterization of markers for ES-phagocytic macrophages. A Gating strategy for evaluating cell surface protein expression on phagocytic versus non-phagocytic macrophages. In-vitro phagocytosis assay was carried out incubating A673 cells (Non- or DOX- or MAG- or D&M-treated) labeled with CMFDA (detected by FITC channel) and M-CSF Mφ. Cells were harvested and stained with APC-CD11b and PE-CD38 antibodies followed by flow cytometry analysis. The viable cells were plotted in FITC (CMFDA) x APC view to visualize cell populations in the in-vitro phagocytosis assay. A gate was drawn to include cells that were FITC positive and APC positive as the phagocytic macrophage population, and another gate was drawn to include cells that were FITC- APC + as the non-phagocytic macrophage population. The FITC + APC + and FITC- APC + cells were then plotted separately in a PE x APC view to visualize expression of CD38 in both cell populations. A quat gate was drawn to visualize APC + PE + as the CD38 + and APC + PE- as the CD38- macrophages. B Comparison of percent of CD14, CD38, CD163, CD206 or CD209 positive and negative cells in the phagocytic and non-phagocytic macrophages incubated with non- or DOX- or MAG- or D&M-treated A673 cells. Macrophages alone (no A673) served as a control. *p < 0.05, **p < 0.01, ***p < 0.001 (Fisher’s Exact test). Shown are results in a representative experiment. The same trend was seen in three independent biological repeats

Article Snippet: Antibodies used include α-CD47 (B6H12, ThermoFisher Scientific, 11–0479-42), α-Calreticulin (ThermoFisher Scientific, PA3-900), α-CD11b (Miltenyi Biotec, 130–110-554), α-CD14 (R&D Systems, FAB3832P-025), α-CD38 (R&D Systems, FAB2404P), α-CD68 (BioLegend, 333,819), α-CD80 (BioLegend, 305,220), α-CD163 (R&D Systems, FAB1607P-025), α-CD206 (R&D Systems, FAB25342P), α-CD209 (R&D Systems, FAB161P-025), Alexa Fluor 488 donkey α-rabbit IgG (Invitrogen, A21206).

Techniques: Expressing, In Vitro, Phagocytosis Assay, Labeling, Staining, Flow Cytometry, Comparison, Incubation, Control