Tumour hypoxia status provides prognostic information and predicts response to hypoxia-modifying treatments. a previous study by our group derived a 24-gene signature to assess hypoxia in bladder cancer. The objectives of the present study were to compare platforms for generating signature scores, identify cut-off values for prospective studies, assess intra-tumour heterogeneity and confirm hypoxia relevance. Briefly, RNA was extracted from prospectively collected diagnostic biopsies of muscle invasive bladder cancer (51 patients), and gene expression was measured using customised Taqman low density array (Tlda) cards, nanoString and clariom S arrays. cross-platform transferability of the gene signature was assessed using regression and concordance analysis. The cut-off values were the cohort median expression values. intra-and inter-tumour variability were determined in a retrospective patient cohort (n=51) with multiple blocks (2-18) from the same tumour. To demonstrate relevance, bladder cancer cell lines were exposed to hypoxia (0.1% oxygen, 24 h), and extracted rna was run on custom Tlda cards. Hypoxia scores (HS) values showed good agreement between platforms: clariom S vs. Tlda (r=0.72, P<0.0001; concordance 73%); clariom S vs. nanoString (r=0.84, P<0.0001; 78%); Tlda vs. nanoString (r=0.80, P<0.0001; 78%). cut-off values were 0.047 (Tlda), 7.328 (nanoString) and 6.667 (clariom S). intra-tumour heterogeneity in gene expression and HS (coefficient of variation 3.9%) was less than inter-tumour (7.9%) variability. HS values were higher in bladder cancer cells exposed to hypoxia compared with normoxia (P<0.02). in conclusion, the present study revealed that application of the 24-gene bladder cancer hypoxia signature was platform agnostic, cut-off values determined prospectively can be used in a clinical trial, intra-tumour heterogeneity was low and the signature was sensitive to changes in oxygen levels in vitro.
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.
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