Fig 2: Representative dual IF staining result to show the colocalization of Gal-9 with different cell markers. Dual-IF staining of Gal-9 and CD68 (A), Gal-9 and GPC3 (B), Gal-9 and CD163 (C), Gal-9 and CD206 (D). (blue: nuclei; red: Gal-9; green: CD68/GPC3/CD163/CD206; yellow: merge). Quantitation of the colocalization of Gal-9 and CD68/GPC3/CD163/CD206 is shown in (E–H). PCCs (above threshold) of Gal-9 and CD68/GPC3/CD163/CD206 were 0.83, -0.05, 0.84 and 0.38, respectively. Results showed that Gal-9 was mostly expressed on CD68+CD163+ KCs, but not HCC tumor cells or M2 macrophages. Scale bar, 50μm (left column) and 10μm (right column).

Fig 3: Expression of immune‐checkpoint molecule (ICM) ligands in head and neck squamous cell carcinoma (HNSCC) tissues. ICM ligands expression in the nest of case 11 is shown (A). Representative double immunostaining images with programmed cell death‐1 (PD‐1) in orange and cytokeratin in blue (upper right), galectin 9 in red and cytokeratin in blue (lower left) and CECAM‐1 in green and cytokeratin in blue (lower right) are shown in the same area with HE images (upper left). ICM ligand expression in the stroma is shown (B). Representative double immunostaining images with PD‐L1 in orange and CD204 in blue (upper right), CD68 in green and CD204 in blue (lower left) and galectin‐9 in red and CD204 in blue (lower right) are shown in the same area with the HE images (upper left). Distribution of programmed cell death receptor ligand 1 (PD‐L1) (orange) expressing macrophages along the periphery of the nest (cytokeratin in blue) was observed (C, upper panel) in an “immune‐excluded” case (case 14). In an “immune‐inflamed” case (case 24), high infiltration of PD‐L1 (orange)‐expressing macrophages into the nest (cytokeratin in blue) were observed (D, upper panel). These macrophages were positive for CD204 (C and D, lower panel). Upper right and lower right images of (C) and (D) indicate the expanded images of the white square left of the images. Magnifications are indicated in each image

Fig 4: Kaplan-Meier survival curve to show the association between Gal-9 expression and overall survival in HBV-associated HCC patients.

Fig 5: Survival of HCC patients with different Gal-9 mRNA levels from the GEPIA database. (http://gepia.cancer-pku.cn/; Cutoff-high (%) = 10; Cutoff-low (%) = 90). HCC patients with high Gal-9 mRNA level have significantly poor survival than those with low Gal-9 mRNA level (P=0.0012).