Impact of therapeutic ionising radiation on extracellular matrix proteins with application to breast tissue

Abstract

Breast cancer is the most common cancer for women around the world and advances in its treatment, such as in radiation therapy, have significantly improved prognosis and survival rates. Quality of life of survivors are, however, impacted by late side effects of treatment, like fibrosis, which is prevalent in breast radiotherapy patients. Current knowledge on the physiopathology of radiation-induced late side effects remains incomplete. Research had focused on the cellular impact of radiation while in contrast, the extracellular matrix (ECM) had been overlooked despite their importance in governing cellular behaviour and outcomes. To improve the understanding of roles that ECM proteins may play in mediating radiation-induced side effects, this PhD aimed to test the hypothesis that breast associated ECM proteins are profoundly altered by therapeutic doses of x-rays, using biomolecular techniques and a mass spectrometry-based analysis - peptide location fingerprinting (PLF). PLF is highly sensitive to the changes in proteolytic susceptibility of large ECM assemblies, allowing detection of regional structural changes within ECM proteins. Purified human collagen I and plasma fibronectin were first chosen as exemplar ECM proteins given their distinct composition and molecular structure. Collagen I and fibronectin responded differently to therapeutic x-rays; the primary structure of x-ray exposed collagen was fragmented with minimal impact to its triple helical structure, while irradiated fibronectin had structurally altered binding sites, resulting in increased binding to collagen. Many ECM components, including fibrillar collagens, exist as higher-order assemblies in tissue. To examine if the packing of fibrillar collagen I could impact its response to therapeutic x-rays, three increasingly complex forms of rat collagen were investigated: solubilised monomeric collagen, reconstituted collagen gel, and ex vivo tendon collagen. While solubilised collagen was fragmented by therapeutic x-rays, fibrillar collagen (gels and tendons) were not. However, proteolytic susceptibility (as analysed by PLF), and therefore structure, was altered in gel and tendon. The ultrastructure of collagen I appears to be crucial in determining its peptide fingerprint's response to therapeutic x-ray exposure. Finally, the impact of therapeutic x-ray doses in complex ECM-rich proteomes (in vitro fibroblast derived ECM (fECM) with ex vivo tissue ECM (tECM)) were analysed using PLF to screen for ECM proteins compromised by therapeutic x-rays. Mass spectrometry revealed similar collagen composition in both fECM and tECM. Collagen I was also found to be structurally similar in both matrices, but not for FN and basement membrane proteins. However, therapeutic x-rays was found to alter the proteolytic susceptibility of these ECM proteins regardless of their source, which may implicate their biological functions in vivo. In conclusion, this thesis successfully characterised the impact of therapeutic x-rays on crucial ECM proteins and demonstrated the capabilities of PLF as a multiscale proteomics tool. The work also identified a body of ECM proteins found in tissues, including the breast stroma, which are vulnerable to therapeutic x-rays. Further work will be needed to establish if these proteins are also affected by x-ray exposure in vivo and what the consequences might be for tissue physiology and the development of radiotherapy related pathologies.

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Michael Sherratt (Supervisor), Cliona Kirwan (Supervisor) & Sarah Cartmell (Supervisor)