Fig 1: Knockdown of endosialin promotes the expression and secretion of CXCL9/10 in cancer-associated fibroblasts (CAFs) by activating INF-γ-STAT1 pathway. (A) Chemokine array to show the levels of different chemokines in IFN-γ-stimulated endosialin knockdown or control HFL1 cells. (B) RT-qPCR to show the mRNA level of CXCL9/10 in endosialin knockdown or control HFL1 cells. (C) ELISA to show the protein level of CXCL9/10 in the culture medium of endosialin knockdown or control HFL1 cells. (D) RT-qPCR to show the mRNA level of CXCL9/10 in endosialin overexpressing or control LX-2 cells. (E) ELISA to show the protein level of CXCL9/10 in the culture medium of endosialin overexpressing or control LX-2 cells. (F) ELISA to show the level of CXCL9/10 in subcutaneous Hepa1-6 tumor tissues of WT and ENKO mice (n=3). (G) Fluorescent imaging to show the migrated 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labeled T cells after co-culture with endosialin knockdown or control HFL1 cells in the presence of CXCL10 neutralizing antibody or control antibody (left) and quantification of the migrated T cells (right). Scale bar, 100 µm. (H) Western blot to detect the level of pSTAT1 in endosialin knockdown or control HFL1 cells. (I) IF staining to show the localization of STAT1 in endosialin knockdown or control HFL1 cells. Scale bar, 20 µm. (J) Western blot to detect the level of pSTAT1 in endosialin overexpressing or control LX-2 cells. (K) IF staining to show the localization of STAT1 in endosialin overexpressing or control LX-2 cells. Scale bar, 20 µm. Representative images are shown. Data are presented as the mean±SEM. *p<0.05; **p<0.01; ***p<0.001.
Fig 2: Combination therapy with endosialin antibody and PD-1 antibody shows synergistic antitumor effect. (A) Schematic image to show the process of combination therapy. (B) Picture of isolated tumors (left) and tumor weight (right) after mice were sacrificed after treatment (n=8). (C) Growth curve of the tumors in different groups after treatment. (D) Flow cytometry to show the percentage of CD4+ T cells and CD8+ T cells in the tumor tissues of different groups after treatment (left panel) and quantification of the flow cytometry data (right panel) (n=6). (E) Graphic diagram to describe the function of endosialin, which can inhibit the secretion of CXCL9/10 by inhibiting IFN-γ/JAK/STAT1 pathway in CAFs, thus inhibit the infiltration of CD8+ T cells into tumor tissues; and endosialin antibody can enhance the anti-tumor effect of PD1 antibody by increasing the infiltration of CD8+ T cells. Representative images are shown. Data are presented as the mean±SEM. *p<0.05; **p<0.01; ***p<0.001; ns, not significant. CAFs, cancer-associated fibroblasts.
Fig 3: High endosialin expression is correlated with low CD8+ T infiltration in clinical hepatocellular carcinoma (HCC) tissues. (A) Immunohistochemistry (IHC) staining of endosialin in HCC tissues and adjacent normal tissues (n=5). Scale bar, 100 µm (top panel), 20 µm (bottom panel). (B) Dual immunofluorescent (IF) staining of HCC tissues to show the correlation between endosialin expression and CD8+ T cell infiltration (n=4). Scale bar, 50 µm. (C) RT-qPCR to show the mRNA level of CXCL9/10 in endosialin knockdown or control primary cancer-associated fibroblasts (CAFs. (D) ELISA to show the protein level of CXCL9/10 in the culture medium of endosialin knockdown or control primary CAFs. (E) Fluorescent imaging to show the migrated 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labeled T cells after co-culture with endosialin knockdown or control primary CAFs at the presence of CXCL10 neutralizing antibody or control antibody (left) and quantification of the migrated T cells (right). Scale bar, 100 µm. (F) Western blot to detect the level of pSTAT1 in endosialin knockdown or control primary CAFs. (G) IF staining to show the localization of STAT1 in endosialin knockdown or control primary CAFs. Scale bar, 20 µm. Representative images are shown. Data are presented as the mean±SEM. *p<0.05; **p<0.01; ***p<0.001.
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