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University of Technology Sydney. Faculty of Science. The tumour stroma, and in particular the extracellular matrix (ECM) in breast cancer plays a crucial role in shaping progression and metastasis. It is clear that the extracellular proteome - known as the matrisome - is a diverse and dynamic network, which fluctuates spatio-temporally over tumourigenesis. Whilst the matrisome has been linked to growth and metastasis, it has not been profiled during mammary tumourigenesis. This thesis explores the use of proteomic mass spectrometry in order to quantify the temporal profile of the matrisome during tumourigenesis as well as its spatial organisation. For temporal proteomic analysis, early (8-10 weeks), mid (11-13 weeks) and late (14-16 weeks) polyoma middle-T antigen (PyMT) mammary tumours and age-matched healthy controls were analysed using liquid chromatography tandem mass spectrometry (LC-MS/MS). This was coupled to downstream matrisome analysis. Spatial-proteomics was performed using matrix-assisted laser desorption-ionisation mass spectrometry imaging (MALDI-MSI). Analytical pipelines for exploratory MALDI-MSI were optimised using bovine lens, murine brain and late-stage PyMT tumour tissues, while sample processing for increased proteome coverage were optimised with murine brain and late-stage PyMT tumour. Temporal LC-MS/MS analysis of PyMT tumours and matched fatpads highlights increased matrisome diversity within the tumour and uncovers distinct temporal profiles during tumourigenesis. This exploratory approach revealed identified COL12A1 as a novel and uncharacterised breast cancer protein secreted by cancer-associated fibroblasts which regulates collagen I organisation and is functionally linked metastatic dissemination. To perform exploratory spatial-proteomics with MALDI-MSI, an R-based false-discovery rate-controlled peptide and protein annotation pipeline was developed called High-resolution Informatics Toolbox in MALDI-MSI Proteomics (HIT-MAP). HIT-MAP annotates peptides and proteins which localise to physiologically relevant compartments in the bovine lens and murine brain. Differential tissue-processing was assessed for its ability to increase the coverage of the proteome when implementing MALDI-MSI coupled to HIT-MAP analysis. Finally, MALDI-MSI analysis of a late-stage PyMT tumour sample highlighted distinct matrisome profiles and biological processes associated with epithelial and stroma-like compartments. Together these findings highlight the dynamic regulation of the matrisome in mammary tumourigenesis and spatial organisation in the tumour microenvironment. This thesis provides evidence of the functional role that the matrisome, and in particular collagen XII plays in mammary tumour progression and metastasis. This thesis also provides a novel tool for exploratory spatial-proteomics using MALDI-MSI, which will facilitate downstream spatial-proteomics analysis. Finally, this thesis argues that using proteomics to understand the spatio-temporal profile of the matrisome will uncover therapeutic vulnerabilities in breast cancer to improve patient well-being.

Figure S1. Similar response to arachidonic acid in patients with non-smoking related end-stage lung disease and patients who underwent lung resection for thoracic malignancies. (DOC 153 kb)

Figure S1. Similar response to arachidonic acid in patients with non-smoking related end-stage lung disease and patients who underwent lung resection for thoracic malignancies. (DOC 153 kb)

Breast cancer is the second most common cancer globally. Most deaths from breast cancer are due to metastatic disease which often follows long periods of clinical dormancy 1 . Understanding the mechanisms that disrupt the quiescence of dormant disseminated cancer cells (DCC) is crucial for addressing metastatic progression. Infection with respiratory viruses (e.g. influenza or SARS-CoV-2) is common and triggers an inflammatory response locally and systemically 2,3 . Here we show that influenza virus infection leads to loss of the pro-dormancy mesenchymal phenotype in breast DCC in the lung, causing DCC proliferation within days of infection, and a greater than 100-fold expansion of carcinoma cells into metastatic lesions within two weeks. Such DCC phenotypic change and expansion is interleukin-6 (IL-6)-dependent. We further show that CD4 T cells are required for the maintenance of pulmonary metastatic burden post-influenza virus infection, in part through attenuation of CD8 cell responses in the lungs. Single-cell RNA-seq analyses reveal DCC-dependent impairment of T-cell activation in the lungs of infected mice. SARS-CoV-2 infected mice also showed increased breast DCC expansion in lungs post-infection. Expanding our findings to human observational data, we observed that cancer survivors contracting a SARS-CoV-2 infection have substantially increased risks of lung metastatic progression and cancer-related death compared to cancer survivors who did not. These discoveries underscore the significant impact of respiratory viral infections on the resurgence of metastatic cancer, offering novel insights into the interconnection between infectious diseases and cancer metastasis., Competing Interests: Additional Declarations: Yes there is potential Competing Interest. HM has consulted Astra Zeneca relating to the use of monoclonal antibodies in the prevention and treatment of SARS-CoV-2 infection. J.A.A-G is a scientific co-founder, scientific advisory board member and equity owner in HiberCell and receives financial compensation as a consultant for HiberCell, a Mount Sinai spin-off company focused on the research and development of therapeutics that prevent or delay the recurrence of cancer. J.A.A-G is also a consultant for Astrin Biosciences, Inc and chief mission officer of Samuel Waxman Cancer Research Foundation.