Proteomic Profiling of Hypoxia-Induced Changes in Cell-Derived Extracellular Matrix From Bladder Cancer Cell Lines.

Abstract

PURPOSE/OBJECTIVE(S)\nThe extracellular matrix (ECM) is an important component of the tumor microenvironment with key roles in cancer development, metastasis, inflammation and treatment resistance. There are synergies between a cancerous ECM and hypoxia. Hypoxia is a predominant feature of advance bladder cancer. It influences ECM remodeling, and a cancerous ECM can also promote hypoxia signaling through HIF1. We hypothesized that proteomic profiling of the hypoxic-induced ECM will identify potential new biomarkers and therapeutic targets. Our first objective was to identify differentially expressed proteins in the ECM from cells cultured in hypoxia vs normoxia and compare with proteins found in plasma from patients undergoing radiotherapy.\n\n\nMATERIALS/METHODS\nUMUC3, J82, RT4 and T24 bladder cancer cell lines were cultured under normoxic (21% O2) and hypoxic condition (0.2% O2). Cell cultures were decellularized with NH4OH, and the cell-derived ECM (CDM) proteins recovered. CDM samples were then in-gel trypsin digested and tandem mass spectrometry performed. Experiments were performed in biological triplicate. Hypoxia-induced cellular and CDM changes were validated by western blotting. In vitro potential biomarkers were validated comparing with LC-SWATH-MS data of longitudinal plasma samples from 10 bladder cancer patients undergoing radiotherapy.\n\n\nRESULTS\nOur results showed a strong influence of hypoxia on bladder CDM. The abundance of 66 out of 186 detected ECM proteins changed significantly (P < 0.05) in response to hypoxia in at least one of the cells lines. The results also highlighted diversity of CDM composition in response to hypoxia as only one protein (angiopoetin-4) was up-regulated across all four cell lines. The 66 proteins segregated samples into hypoxic and normoxic groups when analyzed with PCA and heatmap clustering. Gene ontological analysis indicated that the hypoxia-induced changes in CDM proteins have functions related to ECM structure, cell adhesion binding, integrin binding and grow factor activity. LC-SWATH analysis showed that several of the CDM proteins e.g., TGFB1, ANGPTL4, GDF5 were detectable in plasma from bladder cancer patients.\n\n\nCONCLUSION\nThe CDM composition changes in response to hypoxia. The 66 proteins identified stratified bladder cells lines according to oxygenation status and several were identified in plasma samples from bladder cancer patients. This newly identified bladder hypoxic matrisome signature will be evaluated further in plasma samples from a bladder cancer cohort as a potential biomarker for patient stratification to identify those likely to benefit from hypoxia-modifying treatments.