The relationship between expression of PD-L1 and HIF-1α in glioma cells under hypoxia

Abstract

Hypoxia inducible factor-1α (HIF-1α) up-regulates the expression of programmed death ligand-1 (PD-L1) in some extracranial malignancies. However, whether it could increase PD-L1 expression in intracranial tumor is still unknown. Here, we explored the relationship between HIF-1α and PD-L1 expression in glioma, and investigated their clinical significance. In glioma patients, HIF-1α and PD-L1 were overexpressed in high grade glioma tissues and were significantly associated with poor survival. In glioma cells, PD-L1 expression was induced under hypoxia condition, and the enhanced PD-L1 expression was abrogated by either HIF-1α knock-down or HIF-1α inhibitor treatment. Furthermore, ChIP-qPCR analysis showed the direct binding of HIF-1α to PD-L1 proximal promoter region, providing evidence that HIF-1α up-regulates PD-L1 in glioma. In glioma murine model, the combination treatment with HIF-1α inhibitor and anti-PD-L1 antibody caused a more pronounced suppressive effect on tumor growth compared to either monotherapy. Immunologically, the combination treatment improved both dendritic cell (DC) and CD8⁺ T cell activation. Overall, our results demonstrated that positive correlation between PD-L1 and HIF-1α in glioma, and provide an alternative strategy, inhibiting HIF-1α, as combination therapies with immunotherapies to advance glioma treatment.

Keywords: Glioma; HIF-1 α; Hypoxia; Immunotherapy; PD-L1.

Fig. 1

The relationship of PD-L1 and HIF-1α expression in tumor tissue of glioma patients and their impact on the overall survival. a Immunohistochemistry (IHC) analysis of HIF-1α and PD-L1 in tissue sections of glioma patients. Typical image of positive expressions of HIF-1α (≥ 1%) and PD-L1(≥ 5%) in tissue sections of one patient with grade IV glioma; Typical image of negative expressions of HIF-1α (< 1%) and PD-L1 (< 5%) in tissue sections of one patient with grade II glioma. b PD-L1 and HIF-1α expression in patients with different grades glioma. PD-L1 and HIF-1α expressions in high-grade glioma (HGG) group and low-grade glioma (LGG) group; PD-L1 and HIF-1α expressions in grade II to grade III groups; Correlation analysis of PD-L1 and HIF-1α expression (r = 0.412, P < 0.001) in all glioma patients in our cohort. For (A) to (B), the data were presented as mean ± SEM. *P < 0.05, ***P < 0.001. c The overall survival of glioma patients. Statistical significance was determined by log-rank (Mantel-Cox) test

Fig. 2

Hypoxia up-regulate PD-L1 expression via HIF-1α in glioma cell lines and combination treatment with HIF-1α inhibitor and anti–PD-L1 antibody can reduce tumor growth in murine model of glioma. a qPCR analysis of HIF-1α and PD-L1 mRNA expression in U251 and U343 lines with different treatments as indicated. The qPCR data were normalized to GAPDH. The data were presented as mean ± SEM. P values were calculated by unpaired two-tailed Student’s t tests. *P < 0.05, **P < 0.01. b Western blot analysis of U251 and U343 cells with different treatments using indicated antibodies. c Chromatin immunoprecipitation (ChIP) analysis of the PD-L1 promoter in U251 cells using anti-HIF-1α mAb. The experiments were performed in triplicates and repeated three times. d Immunofluorescence staining of HIF-1α and PD-L1 expression in tumor cells analyzed by confocal microscopy. Representative images are shown. Scale bars, 50 μm. e Mice bearing GL261 cells were divided into the indicated treatment groups. The tumor volumes of mice treated with control, anti–PD-L1 monoclonal antibody, HIF-1α inhibitor (PX-478), or combined anti–PD-L1 antibody and PX-478 were measured and plotted (n = 5). Tumor volume was measured twice weekly. Data are presented as mean ± SEM. and the statistical significance was determined by two-way ANOVA. f Survival from mice receiving the indicated treatments as described in e. Statistical significance was determined by log-rank (Mantel-Cox) test. For (e) to (f) *P < 0.05, **P < 0.01. g The HE staining of intracranial tumor and immunohistochemistry analysis of CD8⁺ T cells in intracranial tumor from mice receiving control, anti–PD-L1 antibody, PX-478, or anti–PD-L1 antibody and PX-478. h Representative flow cytometry analysis and quantification of CD4⁺ T, CD8⁺ T, CD11c⁺ DC and CD11b⁺ myeloid cells populations in GL261 tumors with the indicated treatments (n = 5). i Quantification flow cytometry analysis of the PD-L1 expression on CD45⁻, CD3⁺, CD11c⁺ and CD11b⁺ cells (n = 5). j Representative flow cytometry analysis and quantification of CD8⁺ INF- γ⁺ T cells in GL261 tumors and the MFI of INF- γ in CD8⁺ T cells in U261 tumors at day 14 after treatment (n = 5). For (h) to (j), data are presented as means ± SEM. P values were calculated by unpaired two-tailed Student’s t tests. *P < 0.05, **P < 0.01