The effect of hypoxia on PD-L1 expression in bladder cancer

Abstract

Introduction Recent data has demonstrated that hypoxia drives an immunosuppressive tumour microenvironment (TME) via various mechanisms including hypoxia inducible factor (HIF)-dependent upregulation of programmed death ligand 1 (PD-L1). Both hypoxia and an immunosuppressive TME are targetable independent negative prognostic factors for bladder cancer. Therefore we sought to investigate whether hypoxia is associated with upregulation of PD-L1 in the disease. Materials and methods Three human muscle-invasive bladder cancer cell lines (T24, J82, UMUC3) were cultured in normoxia (20% oxygen) or hypoxia (1 and 0.1% oxygen) for 24 h. Differences in PD-L1 expression were measured using Western blotting, quantitative polymerase chain reaction (qPCR) and flow cytometry (≥3 independent experiments). Statistical tests performed were unpaired t tests and ANOVA. For in silico work an hypoxia signature was used to apply hypoxia scores to muscle-invasive bladder cancers from a clinical trial (BCON; n = 142) and TCGA (n = 404). Analyses were carried out using R and RStudio and statistical tests performed were linear models and one-way ANOVA. Results When T24 cells were seeded at < 70% confluence, there was decreased PD-L1 protein (p = 0.009) and mRNA (p < 0.001) expression after culture in 0.1% oxygen. PD-L1 protein expression decreased in both 0.1% oxygen and 1% oxygen in a panel of muscle-invasive bladder cancer cells: T24 (p = 0.009 and 0.001), J82 (p = 0.008 and 0.013) and UMUC3 (p = 0.003 and 0.289). Increasing seeding density decreased PD-L1 protein (p < 0.001) and mRNA (p = 0.001) expression in T24 cells grown in both 20 and 1% oxygen. Only when cells were 100% confluent, were PD-L1 protein and mRNA levels higher in 1% versus 20% oxygen (p = 0.056 and p = 0.037). In silico analyses showed a positive correlation between hypoxia signature scores and PD-L1 expression in both BCON (p = 0.003) and TCGA (p < 0.001) cohorts, and between hypoxia and IFNγ signature scores (p < 0.001 for both). Conclusion Tumour hypoxia correlates with increased PD-L1 expression in patient derived bladder cancer tumours. In vitro PD-L1 expression was affected by cell density and decreased PD-L1 expression was observed after culture in hypoxia in muscle-invasive bladder cancer cell lines. As cell density has such an important effect on PD-L1 expression, it should be considered when investigating PD-L1 expression in vitro.

HIF1α-dependent and independent pathways regulate the expression of PD-L1 in prostate cancer

“…This is in accordance with a previously reported study by Xu et al [24]. Similar results have been reported in other types of tumors, like bladder cancer and glioblastoma [25,26]. An important finding was also the association of HIF1α with low infiltration of PCs with TILs in accordance with previous studies in breast and head-neck cancer [27,28].…”

“…This is in accordance with a previously reported study by Xu et al [ 24 ]. Similar results have been reported in other types of tumors, like bladder cancer and glioblastoma [ 25 , 26 ].…”

Hypoxia-adapted Multiple Myeloma Stem Cells Resist γδ-T-Cell-mediated Killing by Modulating the Mevalonate Pathway

“…In this study, we found that the expression of PD-L1 on RPMI8226-HA cells was lower than that of RPMI8226-Normo cells (data not shown). Smith et al demonstrated that PD-L1 expression in bladder cancer was decreased under hypoxic conditions (31). These observations are in agreement with our data.…”

Negative Correlation Between 18F-RGD Uptake via PET and Tumoral PD-L1 Expression in Non-Small Cell Lung Cancer

“…However, only one (B16-F10 melanoma) of four murine cell lines studied showed an increase in the percentage of PD-L1 positive cells after culture in 0.1% oxygen. A study of the effect of hypoxia on PD-L1 expression in bladder cancer observed that hypoxia reduced PD-L1 expression and PD-L1 expression further decreased with increasing cell seeding density ( 24 ). It seems that the hypoxia-induced regulation of PD-L1 is tissue specific ( 12 , 22 , 24 ).…”

“…A study of the effect of hypoxia on PD-L1 expression in bladder cancer observed that hypoxia reduced PD-L1 expression and PD-L1 expression further decreased with increasing cell seeding density ( 24 ). It seems that the hypoxia-induced regulation of PD-L1 is tissue specific ( 12 , 22 , 24 ). E-cadherin is a tumor suppressor protein involved in cell-to-cell adhesion, and its suppression is a typical characteristic of epithelial-to-mesenchymal transition (EMT) induction ( 25 , 26 ).…”