PhD Student University of Illinois Urbana-Champaign Urbana, Illinois, United States
Abstract Authors: Hannah S. Theriault1,6; Sarah Ibrahim2; Alison C. Nunes6; Sia Mittal3,6; Jenny Martinez1,6; Kathryn B.H. Clancy4,6; Romana A. Nowak2; Brendan A.C. Harley3,5,6
1. Department of Bioengineering, University of Illinois Urbana-Champaign (UIUC), Urbana, USA 2. Department of Animal Sciences, University of Illinois Urbana-Champaign (UIUC), Urbana, USA 3. Department of Chemical and Biomolecular Engineering, University of Illinois Urbana-Champaign (UIUC), Urbana, USA 4. Department of Anthropology, University of Illinois Urbana-Champaign (UIUC), Urbana, USA 5. Cancer Center at Illinois, University of Illinois Urbana-Champaign (UIUC), Urbana, USA 6. Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign (UIUC), Urbana, USA
Abstract Text: Endometriosis is a chronic inflammatory gynecological disease affecting 1 in 10 menstruators. It is identified laparoscopically by the presence of endometrial-like tissue around the pelvic cavity. These lesions cause dysmenorrhea, menorrhagia, fertility issues, and an increased risk of ovarian cancer (with ovarian endometriosis). Often, it takes a decade from symptom onset to diagnosis, thus, not much is known about the early stages of these lesions and their potential causes. Researchers believe that there is a positive correlation between phthalate exposure and endometriosis risk. Phthalates are known endocrine disruptors used in many everyday products to create flexible plastics. Di(2-ethylhexyl) phthalate (DEHP) is a particularly interesting research target for endometriosis as it is one of the most common phthalates and has been observed in animal models to alter estrogen synthesis. Previous in vitro work has examined the impact of DEHP on healthy endometrial cells and cancer-derived endometrial cells. Here we describe a 3D tissue engineering model to encapsulate endometriotic spheroids in physiologically relevant extracellular matrix to investigate the effects of DEHP on cell invasion, a critical early step in endometriotic lesion initiation. We create spheroids using cohorts of epithelial and stromal cells embedded into a 3D methacrylamide-functionalized gelatin (GelMA) hydrogel. Endometriotic cells are azurite-blue tagged human endometrial-endometriotic stromal cells (azb-iEC-ESCs; Fazleabas Lab, Michigan State University) and GFP-transduced human endometriotic epithelial cells (12z; ABM). Spheroids were formed from 5000 cells (1:3 mixture of GFP-12z:azb-iEC-ESCs), encapsulated in a GelMA precursor suspension, then UV polymerized. GelMA was synthesized utilizing a previously established method by the Harley Lab. GelMA was characterized with NMR (Agilent 600MHz NMR), and compressive testing (Instron 5943). 3D hydrogels were prepared with 5wt% or 7wt% GelMA, PBS, and 0.1% w/v lithium phenyl phosphinate photoinitiator. GelMA was photopolymerized under a UV light (λ = 365 nm, 7.14 mW cm–2, AccuCure Spot System ULM-3-365) for 30s. Spheroids were cultured in a 50/50 mixture of 12z complete culture medium (CCM) and iEC-ESC CCM. Media (0 μM DEHP, 1 μM DEHP, 0.1 μM DEHP, or 0.01 μM DEHP treatments) was changed every two days and spheroids were imaged on D0, D2, and D8. GelMA synthesized at 5wt% and UV polymerized for 30s fell within the stiffness of natural, healthy ovarian tissue (3.3 ± 2.5 kPa), with 7wt% hydrogels above this desired stiffness. We identified a ratio of epithelial and stromal endometriotic cells that mimic histological stains of lesions and supports outgrowth of spheroids within our system. We identified physiological doses of DEHP and observed differences in cell outgrowth patterns where control and 0.1 μM DEHP experienced the most outgrowth by D8. We observed visual differences in spheroid outgrowth of control vs. DEHP treatments, with reduced outgrowth in response to 1 μM DEHP and 0.01 μM DEHP treatments. Ongoing studies are extending the timeframe of studies of DEHP exposure and endometriotic cell outgrowth (epithelial vs. stromal). Ongoing efforts will also expand treatments to include MEHP (a primary metabolite of DEHP), increase the number of experimental replicates (n >6), and incorporate further analysis of gene expression and matrix remodeling in this model of endometriotic lesion initiation.
