Ovarian cancer is the most lethal cancer of the female reproductive system, with over 21,000 new ovarian cancer diagnoses and 14,000 deaths annually in the US. The total lifetime number of ovulations is a key risk factor for developing ovarian cancer. Factors that repress ovulation reduce the risk of ovarian cancer, such as oral contraceptives, pregnancy, lactation, and late menarche. The most common and deadly histotype of ovarian cancer, termed high grade serous cancer (HGSC), likely originates from the fallopian tube epithelial cells, and not the ovary. The frequent detection of tumors in the ovary, which resulted in the name “ovarian cancer”, suggests that the ovary provides a unique anatomical location for tumor migration and expansion. Since most research supports that the fallopian tube epithelium (FTE) is the source of ovarian cancer, it becomes critical to understand how ovulation contributes to tumor initiation in this site. Our team developed three-dimensional organotypic cultures supported in a state-of-the-art microfluidic platform that supports the ovary to produce dynamic hormone profiles that closely mimic the 28-day human reproductive menstrual cycle and ovulation on platform. This R01 renewal builds on this successful collaboration to expand our technology and models to elucidate the mechanisms whereby blocking ovulation prevents FTE carcinogenesis and ovarian colonization. We hypothesize that ovulation and the ovarian microenvironment contributes to the development FTE-derived high grade serous tumors and that ovarian secreted factors drive primary metastasis. Aim 1 leverages our ability to ovulate multiple ovaries in our MPS system called PREDICT-MOS to investigate mechanisms of transformation. Using a unique panel of isogenic cell lines that include non-tumorigenic cells, preneoplastic lesion models, and tumor models all derived from fallopian tube origin we will investigate how secreted factors from the ovary produced during ovulation impact proliferation, soft agar colony formation, and spheroids. We will determine if ovarian secretions increase DNA damage, replication stress, and copy number variation. Lastly, we test if exposure to ovulation drives tumor formation. Aim 2 will focus on the notion that ovulation generates a preneoplastic lesion and increased stemness through a secretory cell outgrowth using innovative iPSC-derived fallopian tube organoids. Organoids will be engineered to model early tumor lesions and monitored in terms of transformation in response to ovulation on platform. In Aim 3, we will investigate the mechanisms responsible for fallopian tube tumor cell colonization of the ovary. Proteomics of ovulatory secretions found both versican and IGF2 are higher after ovulation. We will investigate prevention of ovarian colonization by blocking versican and IGF2 signaling in cell lines, 3D bioprinted models, primary tissue, and in vivo. Overall, this grant will employ unique devices, primary human tissues, and...