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Breast cancer heterogeneity is reflected within the tumour microenvironment (TME), which consists of diverse cell types that play key roles in tumour progression, metastasis, and response to therapy. Cancer associated fibroblasts (CAFs) are a central component of the TME that have been shown to have both pro- and anti-tumour functions and are distinct from normal fibroblasts. CAF heterogeneity has been revealed by single-cell RNA sequencing studies which identified distinct clusters of CAFs in different breast cancer subtypes, which have been linked to patient prognostic outcomes. Some groups have investigated the heterogeneity of mammary fibroblasts and CAFs in mouse models; however, profiles of fibroblasts during different stages of breast tumorigenesis including the hyperplastic mammary gland have not yet been resolved. This project aims to (1) investigate the phenotype and characteristics of fibroblast populations in normal and hyperplastic mammary glands and the TME of mammary tumours from distinct genetically engineered mouse models, and (2) to study whether mammary fibroblasts from a hyperplastic environment can contribute to the cancerous transformation of epithelial cells. To investigate these aims, this project will use different genetically engineered mouse models of breast cancer to represent the heterogeneity seen in human breast cancers. Primary mouse mammary epithelial and stromal cells will be isolated and fractionated using fluorescence-activated cell sorting, and then used for cell co-culture assays. Confocal immunofluorescence imaging will be used to provide spatial information on the different fibroblast populations. Existing unpublished single-cell RNA sequencing data produced by our laboratory will be used to identify key genes regulating transformation from normal to cancer-associated fibroblasts. These genes will be manipulated using CRISPR-Cas9 technology in primary mammary fibroblasts. This research will expand our understanding of the role of fibroblasts in breast cancer progression. |