PROJECT SUMMARY In breast cancer, the median age of diagnose is 61 year old and the incidence then declines in elderly women. However, very few studies focus on the effect of age-related changes on tumorigenesis in the aged breast and almost all experimental models use young mice (3 months). Therefore there is a need to develop mouse models that best reflect the incidence of the disease in humans using aged rather than young or elderly mice. In order to begin characterizing the effect of aging in mammary tumorigenesis, we performed preliminary studies in an inducible mammary tumor model, in which all variables are identical except for age. After oncogene induction, we found that aged (18 months = 61 years in humans) mice have increased tumor incidence, compared to young (3 months) mice. In addition, tumors from aged mice express a unique genetic signature, compared to tumors from young mice. In contrast the increased tumor growth in vivo, aged mammary epithelial cells induced ex vivo formed smaller colonies compared to induced young mammary epithelial cells. Furthermore, our preliminary studies also suggest that the aged mammary gland is characterized by a decrease in collagen abundance and disorganized fiber orientation. Based on these findings, we propose to further analyze age-related alterations in both normal and tumor mammary cells, and microenvironment architecture (collagen abundance and orientation). In Aim 1, we will assess cell-intrinsic age-related changes in tumors arising from aged mice. We will perform transcriptome analysis of tumor cells arising from young and old females in two mouse mammary tumor models, MMTV-rtTA/TetO-ErbB2 and MMTV-rtTA/TetO-Ras. To identify potential novel targets in tumors arising from aged mice, we will compare the young and aged tumor signatures with young and old normal mammary epithelial cells. In Aim 2, we will investigate the cell-extrinsic effect of age-related changes in collagen composition on mammary tumor growth. First, we will further characterize the age-related changes in collagen abundance and orientation young and aged normal mammary glands using second harmonic generation imaging. We will also establish a 3D culture method using decellularized mammary glands from young and aged mice to study the effect of biological aging on tumor cell growth in vitro. We will confirm our studies in a series of swapping experiments using establish young and aged syngeneic cohorts. The purpose of the proposed studies is to improve our understanding of how age-related changes in the mammary gland contribute to breast cancer risk, and may identify new targets for breast cancer treatment in older women.