Fig 2: TNF-α-induced epithelial-to-mesenchymal transition regulates expression of immune checkpoint molecules in Hep3B cells. Reverse transcription-quantitative PCR analysis demonstrated (A) higher expression of PD-L1, PD-L2, CD73 and B7-H3, and (B) lower expression of VTCN1 and VISTA upon treatment with 20 ng/ml TNF-α for 72 h. n=3. *P<0.05, **P<0.01, ****P<0.001. (C) Western blot analysis demonstrated upregulation of PD-L1 and CD73 in Hep3B cells upon treatment with 20 ng/ml TNF-α for 72 h. n=3. *P<0.05, ****P<0.001 vs. control. GAPDH was used as the loading control. TNF-α, tumor necrosis factor-α; VTCN1, V-set domain-containing T-cell activation inhibitor 1; VISTA, V-domain immunoglobulin suppressor of T-cell activation; PD-L, programmed death receptor ligand.

Fig 3: Association of TNF-α in combination with immune checkpoints and survival in patients with HCC. Kaplan-Meier survival curves for the analysis of (A) gene expression of PD-L1/TNF-α and overall survival, and (B) gene expression of PD-L2/TNF-α and recurrence free survival in samples from patients with HCC. Red curve represents the high-risk group, while green curve represents the low-risk group (8). Markers (+) represent censoring samples. TNF-α, tumor necrosis factor-α; PD-L, programmed death receptor ligand; HR, hazard ratio; HCC, hepatocellular carcinoma.

Fig 4: Reversal of EMT returns immune checkpoint expression to control levels in Hep3B cells. Fluorescence microscopy showed (A) higher expression of PD-L1 upon EMT induction and lower expression of PD-L1 following reversal assay and (B) higher expression of CD73 upon EMT induction and lower expression of CD73 following reversal assay in Hep3B cells. Scale bar, 100 µm. Magnification, ×40. (C) Western blot analysis demonstrated higher expression of PD-L1, CD73, B7-H3 and PD-L2 upon EMT induction and lower expression following reversal assay in Hep3B cells. n=3. *P<0.05, **P<0.01. GAPDH was used as the loading control. EMT, epithelial-to-mesenchymal transition; TNF-α, tumor necrosis factor-α; MET, mesenchymal-to-epithelial transition; PD-L, programmed death receptor ligand.

Fig 5: A heat map presenting data on 20 immune checkpoints from a TMA containing 124 GC patient samples.The abscissa represents the patient number of 124 patients in the TMA, and the ordinate represents the expression of LSECtin, VISTA, PD-L1, IDO, TIM-3, TNFRSF14, B7M4, CEACAM1, NECTIN2, CD44, CD155, ADENOSINE, TLR4, HMGB1, SIGLEC6, FAS-L, SIGLEC15, CD73, Galectin-9 and PD-L2.