Effects of hypoxia on the proteomic profile of extracellular proteins produced by bladder cancer cells

  • Conrado Guerrero Quiles

Student thesis: Phd

Abstract

Introduction: There are many methods for measuring tumour hypoxia-an adverse prognostic factor in cancer particularly following radiotherapy-but none progressed to routine clinical use. A plasma biomarker would be useful due to ease of assay and repeat measurements. This project was a first step in trying to derive a tumour hypoxia-associated protein signature measurable in plasma. The extracellular matrix (ECM) and secretome produced by cancer cells are affected by hypoxic stress and potential reservoirs of candidate biomarkers that intravasate into blood. Therefore, the project aimed to: (1) characterise changes in cell derived ECMs (CDMs) induced by hypoxia; (2) investigate effects of hypoxia (alone and with radiation) on the morphology of ECM fibres, adhesion and migration; (3) characterise effects on secretome produced by cancer cell lines; and (4) generate a candidate plasma protein signature. Materials and methods: Four bladder cancer cell lines (T24, UMUC3, J82, RT4) were grown in hypoxia (0.2% O2) or normoxia (20.9% O2) for seven days. Cell derived ECM and secretome samples were analysed using mass spectrometry (MS). Morphology was investigated using immunofluorescence (IF) for fibronectin (FN) and collagen (COL) 5 and 1. For attachment, migration and IF co-localisation, cells were seeded onto seven-day-old hypoxic or normoxic ECM. Cell attachment was measured using crystal violet. Migration was measured using a scratch assay over 24-48 h. Co-localisation of FN, COL5/1 and E-cadherin was assessed using IF. Pre-treatment plasma samples were collected from bladder cancer patients (n=23) and tumour hypoxia assessed using a 24-gene signature. Plasma samples for the cohort were concentrated using liposomes and analysed with MS with respect to tumour hypoxia status. In silico analyses were performed using RStudio. Results: (1) Hypoxia changed the composition of ECM proteins produced by bladder cancer cells in vitro with pathway analyses showing effects would increase remodelling towards a fibrotic phenotype. Enrichment analyses showed hypoxia affects ECM structural proteins and ECM organisation. (2) While there was variation between lines, IF analysis tended to show hypoxia increased FN and decreased COL fibre numbers. E-cadherin co-localised with FN and COL fibres, which was affected by radiation. Hypoxic ECM increased cell adhesion but impaired cell migration independent (UMUC3) or dependent (T24, J82) of irradiation stress. (3) Analysis of secretomes showed hypoxia was likely to affect ECM remodelling and neutrophil immunity. Analysis of extracellular proteins (ECM + secretome) produced by bladder cancer cells confirmed hypoxia affects ECM remodelling and fibrosis as well as neutrophil infiltration and activity. (4) Fifteen proteins were differentially expressed in plasma in relation to tumour hypoxia scores. However, only AHNAK2 was differentially expressed in relation to tumour hypoxia scores. Comparison of plasma composition of high and low tumour hypoxia groups identified a three-protein plasma hypoxia signature (VK3, IGHV, OR5V1) that grouped the bladder cancer cohort according to hypoxia status in unsupervised clustering analyses. Conclusions: Hypoxia affects the composition of the extracellular environment produced by bladder cancer cells in vitro. Changes were associated with ECM remodelling, fibrosis, neutrophil recruitment and activation. Hypoxic ECM promotes cancer cell attachment but impairs migration, which was affected by radiation stress and regulated by E-cadherin signalling. A candidate 3-protein plasma signature able to stratify bladder cancer patients according to hypoxia stress was identified, which should be further evaluated in a larger prospective cohort. This study provided key insight about hypoxia regulation of the extracellular environment in bladder cancer, as well as proposing for the first time a hypoxic plasma protein signature for bladder cancer.
Date of Award1 Aug 2023
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorAnthony Whetton (Supervisor), Jonathan Humphries (Supervisor), Catharine West (Supervisor) & Ananya Choudhury (Supervisor)

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