Fig 2: The diagram of MAGE-C3's promotion in cancer metastasis

Fig 3: MAGE-C3 enhances tumor cell mobility and promotes metastasis. (A) The ability of migration and invasion in MAGE-C3 overexpressing KYSE30 cells. Representative pictures are shown in (left panel, scale bar: 50µm.) and quantitative data (right panel) are shown. (B) Cell migration and invasion assay were performed on xCELLigence RTCA-DP system in MAGE-C3 overexpressing KYSE410 cells. (C, D) The ability of migration and invasion in MAGE-C3 knockdown KYSE30 cells (C) and KYSE140 cells (D). Representative pictures (left panel, scale bar: 50µm) and quantitative data (right panel). (E) The haematoxylin and eosin staining of lung metastatic nodules formed by MAGE-C3 overexpressing and control KYSE30 cells in mouse models (left panel). The numbers of lung metastatic nodules are shown in right panel. Scale bar: 400 µm. Three independent experiments were performed. *, P < 0.05; ***, P < 0.001

Fig 4: MAGE-C3 improves tumor cell invasion and migration via STAT3-mediated EMT. (A) Cell morphology of MAGE-C3 overexpressing KYSE30 and control cells. (B) Western blotting of metastatic markers (E-cadherin, N-cadherin, and vimentin), STAT3, and phosphorylated STAT3 (pSTAT3Tyr705) in MAGE-C3 overexpressing ESCC cells (KYSE30 and KYSE410) and control cells. (C) Western blotting of metastatic markers (E-cadherin, N-cadherin, and Vimentin), STAT3, and phosphorylated STAT3 (pSTAT3Tyr705) in MAGE-C3 shRNAs treated KYSE30 and KYSE140 cells (Shc3) and control shRNA treated cells (Shnc). (D) Western blotting of metastatic markers (E-cadherin, N-cadherin, and Vimentin), STAT3, and phosphorylated STAT3 (pSTAT3Tyr705) in MAGE-C3 overexpressing KYSE30 and KYSE410 cells with or without WP1066 treatment (5 µmol/L). (E, F) Transwell migration and invasion assays in MAGE-C3 overexpressing KYSE30 cells (E) and KYSE410 cells (F) with or without WP1066 treatment. Representative pictures (left panel, scale bar: 50 µm.) and quantitative data (right panel). Three independent experiments were performed. *, P < 0.05; ***, P < 0.001

Fig 5: MAGE-C3 promotes tumor progression through PD-L1-involved immunosuppression in vivo. (A) Upper: The exogenous MAGE-C3 in B16F10 cells. Under: The cellular proliferation of MAGE-C3 overexpressing B16F10 cells and control cells via RTCA-MP system. (B) The MAGE-C3 overexpressing B16F10 cells and control cells were bilaterally into the BALB/c nude mice. No significant effect of MAGE-C3 on tumor growth in nude mice was observed. (C) The MAGE-C3 overexpressing B16F10 cells and control cells were bilaterally into the C57BL/6 mice. The tumor weight and volume were increased in the MAGE-C3 overexpression group compared with those in the control group. (D) The survival of mice bearing MAGE-C3 overexpressing B16F10 and control tumor (n = 9). (E) The lung metastases of mice bearing MAGE-C3 overexpressing B16F10 were significantly increased than that in control tumor. (F) The CD8+ staining in lung metastasis loci of C57 BL/6 mice. (G) The percent of CD8+ T cell in all T lymphocytes of lung with metastasis detected by flow cytometry (n = 9). The infiltrated CD8+ T cell population was decreased in lung metastasis of MAGE-C3 overexpressing mice, whereas the percentage of PD-1+ CD8+ T cells was increased. Three independent experiments were performed. Scale bar: 40µm. * p < 0.05