PROJECT SUMMARY High-grade serous ovarian carcinoma (HGSOC) is the most lethal cancer of the female reproductive system with less than 50% of patients surviving 5 years after diagnosis. A woman’s total lifetime number of ovulations is a key risk factor for developing ovarian cancer, and factors that repress ovulation including use of oral contraceptives significantly reduce HGSOC incidence. Despite the association with ovulation, HGSOC typically presents as late-stage, metastatic cancer in post-menopausal women who are no longer ovulating. Therefore, a major question in the field is: how do ovulation during a woman’s reproductive prime and the aging ovary post- menopause contribute to ovarian cancer development. Here, I propose two distinct aspects of the aging ovarian microenvironment that promote HGSOC initiation and pathogenesis. First, follicular fluid, which becomes fibro- inflammatory with advanced reproductive age, triggers tumor initiation in the fallopian tube epithelium (FTE). Second, changes to the composition and biomechanics of the aging ovarian extracellular matrix (ECM) allow for enhanced tumor cell adhesion and invasion. Recent data suggest that HGSOC tumors arise from the FTE, and exposure to follicular fluid results in DNA damage in FTE cells both in vivo and in vitro. However, the direct effect of reproductive aging on FTE tumorigenesis has not been examined. Work from our lab has identified a series of fibro-inflammatory cytokines that increase in human follicular fluid with advanced reproductive age, including VEGF which has roles in HGSOC metastasis and is involved in tumor initiation in other organ systems. In Aim 1 of this proposal, I will test the hypothesis that follicular fluid induces neoplastic transformation of the FTE in an age-dependent manner, through the action of candidate cytokine VEGF. I will treat FTE cell lines with human follicular fluid across an aging continuum or a dose-curve of recombinant-human VEGF to determine effects to cell proliferation, DNA damage, oxidative stress, and global gene expression. After tumor initiation in the FTE, tumor cells migrate to the ovary for expansion. Work from our lab has characterized significant changes to the composition of the ovarian ECM with advanced reproductive age including increased levels of collagen I and III, which increase tissue stiffness. HGSOC cells have been shown to favor a 3D collagen-rich matrix for expansion and stiffer matrices are known to increase cancer cell motility. In Aim 2, I will determine the mechanism by which the tissue environment of the aging ovarian ECM promotes HGSOC pathogenesis by investigating the independent functions of ECM composition and ovarian tissue stiffness on HGSOC cell adhesion and invasion. To this end, I will assess in vitro HGSOC cell adhesion and invasion on hydrogels that recapitulate the stiffness and collagen abundance of the aging ovary. Furthermore, I will evaluate HGSOC cell adhesion and invasion in reproductiv...