Pathological significance of abnormal recepteur d’origine nantais and programmed death ligand 1 expression in colorectal cancer

Figure 1 X-tile analysis to determine the optimal cut-off values for RON and PD-L1 expression and survival analysis in the GEO dataset. The optimal cut-off values are highlighted by black circles (left panels) and shown in histograms for the entire cohort (middle panels). Kaplan-Meier survival plots are shown in right panels. A: The optimal cut-off value for RON was 7.50 (χ² = 4.544, P = 0.033); B: The optimal cut-off value for PD-L1 was 4.30 (χ² = 4.078, P = 0.043); C: Effect of different RON and PD-L1 expression states on patient survival. ^aP < 0.05.

Figure 2 Four-color multiplex immunofluorescence staining of paraffin-embedded colorectal cancer tissues. A: High expression of RON and PD-L1 in tumor cells (TCs); B: High expression of RON in TCs and high expression of PD-L1 in tumor-infiltrating mononuclear cells (TIMCs); C: Lack of RON expression and high PD-L1 expression in TCs; D: Low expression of RON in TC and high PD-L1 expression in TIMCs; E: High RON expression in TCs and no PD-L1 expression in either TCs or TIMCs. Blue (DAPI) shows nuclei, green shows PD-L1, pink indicates RON, and red displays CK.

Figure 3 Immunohistochemical expression of RON and PD-L1 in colorectal cancer tissues. A: High (right) and negative (left) expression of RON in tumor cells (TCs); B: High (right) and negative (left) expression of PD-L1 in tumor-infiltrating mononuclear cells; C: High and negative expression of PD-L1 in TCs (original magnification, × 100 and × 200).

Figure 4 Kaplan-Meier analysis of overall survival of patients with colorectal cancer stratified by RON and/or PD-L1 expression. A: Overall survival (OS) according to RON expression in tumor cells (TCs); B: OS according to PD-L1 expression in TCs; C: OS according to PD-L1 expression in tumor-infiltrating mononuclear cells (TIMCs); D: OS according to RON and PD-L1 expression in TCs; E: OS according to RON expression in TCs and PD-L1 expression in TIMCs. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 5 Expression of RON and PD-L1 in HT29 cells after treatment with 2 nmol/L MSP, 2 nmol/L MSP + 2 μmol/L BMS-777607, or 2 μmol/L BMS-777607. A: Cellular immunofluorescence indicating the expression of RON and PD-L1 after treatment of HT29 cells with 2 nmol/L MSP, 2 nmol/L MSP + 2 μmol/L BMS-777607, or 2 μmol/L BMS-777607 for 24 h, respectively. DAPI indicates nuclei (blue color), FITC indicates PD-L1 (green color), and PE indicates RON (red color). Original magnification × 400 (all photomicrographs). B-C: HT29 cells were treated with 2 nmol/L MSP, 2 nmol/L MSP + 2 μmol/L BMS-777607, or 2 μmol/L BMS-777607 for 24 h, and the expression of RON and PD-L1 was detected by Western blot and quantified according to the immunoblots. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 6 Expression of RON and PD-L1, and activation of signaling pathways in HT29 cells. A: HT29 cells were treated with 2 nmol/L MSP, 2 nmol/L MSP + 2 μmol/L BMS-777607, or 2 μmol/L BMS-777607 for 1 h. The proteins analyzed include RON, phosphorylated RON, AKT, ERK1/2, phosphorylated AKT, and phosphorylated-ERK1/2. GAPDH was used as a loading control; B-D: The expression of p-RON, p-AKT, and p-ERK1/2 was detected by Western blot and quantified. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.