Breast cancer (BCa) is the second leading cause of cancer-related deaths in Australia. Despite recent improvements in early detection and adjuvant therapies, BCa incidence continues to rise, stressing the importance of identifying the factors that modulate BCa risk. While normal stroma protects against epithelial cell transformation, once a tumor has emerged, the stroma changes significantly to support cancer progression. Although the mechanisms responsible for these stromal changes are poorly understood, the importance of the stroma has been highlighted in recent work showing that stromal gene expression can predict clinical outcome in BCa patients. It is known that mammary gland involution is associated with an increased BCa risk, however it is not clear whether this is orchestrated by the stroma. Thus, we aim to isolate stromal fibroblasts, from the mammary glands of pubertal (active growth), adult (resting) and involuting (increased risk) mice, and determine their activation status. We will assess the fibroblasts for expression of known cancer associated fibroblast (activated) markers using IHC and western blot. We will correlate this with the proliferative ability of the fibroblasts in 2D assays, as well as their ability to modulate BCa growth in 3D assays (cell titre glo). To date IHC analysis has failed to accurately identify markers of activated fibroblasts, so we also aim to profile the stroma using FACS, to determine if this can more accurately estimate fibroblast activation levels. This study will define the fibroblast population present during times of increased BCa risk as well as during active growth and normal cycling.