Fractionated radiation alters the extracellular matrix produced by muscle-invasive bladder cancer cells

Muscle-invasive bladder cancer (MIBC) is a prevalent disease that can be treated with radiotherapy, but has a poor prognosis. Radiation-induced extracellular matrix (ECM) remodelling and fibrosis can induce tumour resistance and recurrence, but has not been studied in MIBC. Here we aimed to characterise the impact of radiation on the ECM composition of MIBC. Materials and MethodsThree MIBC cell lines (T24, UMUC3, J82) were treated with fractionated radiation. We used proteomics to analyse the ECM composition produced by surviving cancer cells and immunofluorescence to investigate changes in the morphology and number of ECM fibres. We evaluated the RNA expression of identified ECM proteins (FN1, COL5A1, COL1A1, TNF6AIP6, FLG) in one cystectomy (TCGA-BLCA, n=397) and two radiotherapy (BC2001, n=313; BCON, n=151) cohorts. ResultsThere were 613 proteins affected by radiation (padj<0.05, fold change >2 or <-2), 68 of which were ECM-associated proteins. There was a general increase in proteases and protease regulators but heterogeneity across cell lines. Enrichment analysis showed ECM organisation was the primary pathway affected. Immunofluorescence confirmed radiation affected ECM structure, generally, reducing the number, length and width of fibres. High FN1, COL5A1, COL1A1, TNF6AIP6 and FLG mRNA levels were adverse prognostic markers in clinical cohorts. ConclusionRadiation alters the composition and structure of the ECM produced by MIBC. As a proof-of-concept, we showed the expression of radiation-affected ECM genes are prognostic markers in MIBC. Future studies should validate these radiation-induced ECM changes in clinical samples.