The role of PD-L1 in the radiation response and prognosis for esophageal squamous cell carcinoma related to IL-6 and T-cell immunosuppression

Abstract

The aim of this study was to assess the significance of programmed cell death 1 ligand 1 (PD-L1) in esophageal squamous cell carcinoma (ESCC) and its association with IL-6 and radiation response. Weretrospectively enrolled 162 patients with ESCC, and examined the correlation between PD-L1 levels and clinical outcomes in esophageal cancer patients. Furthermore, the human esophageal SCC cell line CE81T and TE2 were selected for cellular experiments to investigate the role of PD-L1 in T cell functions and radiation response. Here we demonstrated that PD-L1 expression was significantly higher in esophageal cancer specimens than in non-malignant epithelium. In clinical outcome analysis, this staining of PD-L1 was positively linked to the clinical T4 stage (p=0.004), development of LN metastasis (p=0.012) and higher loco-regional failure rate (p=0.0001). In addition, the frequency of PD-L1 immunoreactivity was significantly higher in IL-6-positive esophageal cancer specimens. When IL-6 signaling was inhibited in vitro, the level of PD-L1 is significantly down-regulated. PD-L1 is a significant predictor for poor treatment response and shorter survival.As demonstrated through in vitro experiments, Irradiation increased PD-L1 expression in human esophageal cancer cells. The inhibition of T cell functions including proliferation and cytotoxicity against tumor cells might be the mechanisms responsible to the role of PD-L1 in radiation response. In conclusion, PD-L1 is important in determining the radiation response and could predict the prognosis of patients with esophageal SCC. Therefore, we suggest inhibition of PD-L1 as a potential strategy for the treatment of esophageal SCC.

Keywords: CD8+ T cell; IL-6; PD-L1; esophageal SCC.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors confirm that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.

Figures

Figure 1. PD-L1 levels in esophageal SCC

a. Representative images of IHC staining with an anti-PD-L1 antibody of esophageal cancer and adjacent non-malignant epithelium from TMA blocks. b. IHC staining with an anti-PD-L1 antibody of human esophageal cancer specimens. Images of representative slides are shown at magnifications of ×100 (left panel) and ×200 (right panel).

Figure 2. Correlation between PD-L1 and IL-6 levels

a. IL-6 levels correlate positively withPD-L1 levels in human esophageal cancer specimens (p<0.001). Representative images of positive IL-6 and PD-L1 staining on slides from a selected tumor specimen, and representative negative staining for IL-6 andPD-L1 on slides from another tumor specimen, are shown. b. IL-6 levels measured using ELISA in plasma samples obtained from cancer patients with IL-6 negative staining (n = 27) and PD-L1 positive staing (n = 29) disease. The lines indicate the mean values (p<0.001). c. IL-6 levels measured using ELISA in plasma samples obtained from cancer patients with PD-L1 negative staining (n = 25) and PD-L1 positive staing (n = 31) disease. The lines indicate the mean values (p < 0.001).

Figure 3. Role of IL-6 signaling on PD-L1 expression in human esophageal cancer

The levels of PD-L1 were evaluated by a. FACS with PD-L1 antibody. Each column is shown as the means of 3 separate experiments; bars, SD.*, P<0.05; and b. IF staining for human esophageal cancer cells at 48h after IL-6 regulation in vitro (Blue, DAPI; Green, PD-L1). c. Effect of inhibited IL-6 signaling on PD-L1 protein levels as determined by immunoblotting (W, proteins were extracted from cells under control condition; IL-6Ab, proteins were extracted from cells incubated in the presence of 5 μg/ml IL-6 neutralizing antibodies for 48h; AG, proteins were extracted from cells incubated in the presence of 50 μM AG490 for 48 h; LY, proteins were extracted from cells incubated in the presence of 50 μM LY294002 for 48h).

Figure 4. Correlation between irradiation, PD-L1 in cancer cells, and the function of cytotoxic T cells

The levels of PD-L1 were evaluated by a. Western blot analysis and b. FACS with PD-L1 antibody for human esophageal cancer cells at indicated time after 6Gy irradiation or 48h after RT with 0, 3, 6, 9Gy in vitro. Representative slides are shown. Each column is shown as the means of 3 separate experiments; bars, SD.*, P<0.05. c. The effect of PD-L1 blockade on the suppressing ability of tumor cells for T cells proliferation was evaluated by FACS. Representative images and quantitative data are shown. Each column is shown as the means of 3 separate experiments; bars, SD.*, P<0.05; d. The effect of PD-L1 blockade on the CD8+ T cells cytotoxicity against cancer cells was evaluated by FACS. Representative images and quantitative data are shown.

Figure 4. Correlation between irradiation, PD-L1 in cancer cells, and the function of cytotoxic T cells

The levels of PD-L1 were evaluated by a. Western blot analysis and b. FACS with PD-L1 antibody for human esophageal cancer cells at indicated time after 6Gy irradiation or 48h after RT with 0, 3, 6, 9Gy in vitro. Representative slides are shown. Each column is shown as the means of 3 separate experiments; bars, SD.*, P<0.05. c. The effect of PD-L1 blockade on the suppressing ability of tumor cells for T cells proliferation was evaluated by FACS. Representative images and quantitative data are shown. Each column is shown as the means of 3 separate experiments; bars, SD.*, P<0.05; d. The effect of PD-L1 blockade on the CD8+ T cells cytotoxicity against cancer cells was evaluated by FACS. Representative images and quantitative data are shown.

Figure 5. Correlation between PD-L1 level and clinical outcome

Survival differences according to a. the positive staining of PD-L1; b. patients who underwent surgery or not; and c. the response to CCRT for all 162 patients. The PD-L1-positive group exhibited shorter survival than the PD-L1-negative group.