Cancer Technologies
Peyton Clark (She/They)
Graduate Research Assistant
University of Alabama at Birmingham
Simpsonville, South Carolina, United States
MK Sewell-Loftin (she/her/hers)
Assistant Professor
The University of Alabama at Birmingham, United States
High grade serous ovarian carcinoma (HGSOC) accounts for 90% of deaths as a result of ovarian cancer (1). Additionally, in the U.S. the median age at diagnosis is 63 and most cases are in post-menopausal women (2). In contrast with the age of women diagnosed with HGSOC, most in vivo models of HGSOC use young animals. This discrepancy may add to the challenges of studying ovarian cancer, as aged animal models are expensive and require extensive care. An accurate in vitro model to study ovarian cancer progression is vital to understanding the mechanisms behind the disease in a physiologically relevant environment. To better understand differences in ovarian cancer progression in patients of different ages, we developed a 3D microtissue model of cell migration in response to mechanical and soluble stimuli. We hypothesize that media from senescent endothelial cells more closely mimics the stromal microenvironment and will promote more migration compared to media conditioned by nonsenescent endothelial cells.
To measure how age associated vascular senescence differences alter ovarian cancer cell migration, we utilized human SKOV3 cells tagged with a GFP reporter in a conditioned media study. The cells were seeded into the center chamber of a three-chamber microfluidic device, where the tissue chambers are connected in series with each other (3). Each chamber is an independent microtissue region and is connected to the other chambers with 20µm communication ports to allow transfer of nutrients and cell migration. The cells were suspended in a 9:1 fibrin-collagen matrix in the center chamber to mimic the natural tumor environment. The total protein concentration was 10 mg/ml. The side chambers were loaded with blank, cell-free gels. The cells were incubated for three days under conditions that did not allow flow of soluble factors between the adjacent chambers. On day 3, one side chamber of the device was treated with conditioned media from senescent human ovarian microvascular endothelial cells (HOMECs) and the other side chamber received conditioned media from non-senescent HOMECs. The center chamber was fed with control media. An inward flow direction was established that resulted in diffusion of soluble factors into the center chamber towards the SKOV3-GFP cells. Images were taken at the time of initial treatment and every 24hr for five days. The cells received fresh media every day. Migration into the side chambers was measured as percent area of fluorescence normalized to total area of the side chambers.
Our results show that there is increased cell migration towards the senescent media over a period of four days. The differences in migration began to even out on day five, but increased migration is still observed. The increased migration can also be observed in the immunofluorescence images taken each day. The chamber of the microtissue model that received the senescent conditioned media shows higher levels of cell migration than the chamber that received the non-senescent conditioned media. This trend continued through day five.
This pilot study demonstrated somewhat directed migration towards conditioned media from senescent endothelial cells compared to non-senescent, control cells. Our results suggest that secreted factors from senescent endothelial cells may promote migratory behavior of ovarian cancer cells. This may partially explain differences in ovarian cancer progression in patients of different ages. Also, the increase in cells in the chambers treated with senescent conditioned media may be partially due to enhanced proliferation. Future studies will test inhibiting proliferation to determine if increased cell counts are due to migration only or a combination of migration and proliferation. Additionally, it has been shown that there is a decrease in the production of hyaluronic acid (HA) in senescent cells (4). HA has been implicated in the progression of ovarian cancer, though its role is not yet fully understood. Future studies using this model will seek to further determine if the change in HA metabolism associated with aging and cellular senescence is contributing to the observed changes in migration behavior of ovarian cancer cells.
The authors would like to acknowledge funding for this project: R00-CA230202 (M.K.S.L), IMPACT Award, O’Neal Comprehensive Cancer Center (M.K.S.L). We would also like to thank Dr. Mythreye Karthikeyan and Dr. Asha Kumari for their contributions.