Hypoxia Is Associated with Increased Immune Infiltrates and Both Anti-Tumour and Immune Suppressive Signalling in Muscle-Invasive Bladder Cancer

Abstract

Hypoxia and a suppressive tumour microenvironment (TME) are both independent negative prognostic factors for muscle-invasive bladder cancer (MIBC) that contribute to treatment resistance. Hypoxia has been shown to induce an immune suppressive TME by recruiting myeloid cells that inhibit anti-tumour T cell responses. Recent transcriptomic analyses show hypoxia increases suppressive and anti-tumour immune signalling and infiltrates in bladder cancer. This study sought to investigate the relationship between hypoxia-inducible factor (HIF)-1 and -2, hypoxia, and immune signalling and infiltrates in MIBC. ChIP-seq was performed to identify HIF1α, HIF2α, and HIF1β binding in the genome of the MIBC cell line T24 cultured in 1% and 0.1% oxygen for 24 h. Microarray data from four MIBC cell lines (T24, J82, UMUC3, and HT1376) cultured under 1%, 0.2%, and 0.1% oxygen for 24 h were used. Differences in the immune contexture between high- and low-hypoxia tumours were investigated using in silico analyses of two bladder cancer cohorts (BCON and TCGA) filtered to only include MIBC cases. GO and GSEA were used with the R packages “limma” and “fgsea”. Immune deconvolution was performed using ImSig and TIMER algorithms. RStudio was used for all analyses. Under hypoxia, HIF1α and HIF2α bound to ~11.5–13.5% and ~4.5–7.5% of immune-related genes, respectively (1–0.1% O2). HIF1α and HIF2α both bound to genes associated with T cell activation and differentiation signalling pathways. HIF1α and HIF2α had distinct roles in immune-related signalling. HIF1 was associated with interferon production specifically, whilst HIF2 was associated with generic cytokine signalling as well as humoral and toll-like receptor immune responses. Neutrophil and myeloid cell signalling was enriched under hypoxia, alongside hallmark pathways associated with Tregs and macrophages. High-hypoxia MIBC tumours had increased expression of both suppressive and anti-tumour immune gene signatures and were associated with increased immune infiltrates. Overall, hypoxia is associated with increased inflammation for both suppressive and anti-tumour-related immune signalling and immune infiltrates, as seen in vitro and in situ using MIBC patient tumours.

Emerging roles of MITF as a crucial regulator of immunity

“…Several studies have shown that HIF-1α induces DC-mediated inflammation and chemokine receptor type 7 (CCR7)-mediated migration of myeloid DCs (mDCs) 60 – 62 . In contrast, exposure to hypoxia has been shown to either inhibit or exert no effect on the proinflammatory function of DCs 63 , 64 . The HIF-1α level is also elevated in activated neutrophils 65 .…”