HORMONAL AND MECHANICAL CUES ON DYNAMIC CELLULAR PROCESSES IN ENDOMETRIOSIS
Endometriosis is a chronic inflammatory and fibrotic condition in 1 in 10 women characterized by the abnormal growth of endometrial tissue outside of the uterus. Endometriotic tissues attach to the peritoneal mesothelium lining the pelvic cavity to establish harmful lesions and implants in ectopic sites, leading to pelvic pain, infertility, and reduced quality of life. Dysregulated behaviors such as cell migration, invasion, proliferation, and immune escape drive lesion formation. The endometriotic microenvironment contributing to these cell behaviors is complex, with notable components including high local levels of estradiol (E2), a potent estrogen molecule, fibrotic extracellular matrix stiffening, inflammatory markers, and diverse cellular interactions. However, dynamic cellular processes in endometriosis are still not well understood. Moreover, the field is understudied and would benefit from more accessible, diverse, and physiologically relevant in vitro models. This dissertation investigates how hormonal and mechanical microenvironmental cues regulate dynamic cellular processes during endometriosis progression using accessible in vitro models. Our first focus is on hormonal signaling, as E2 has been widely implicated in endometriosis pathogenesis. Whether commonly used cell culture models faithfully capture E2-driven migratory phenotypes, and how hormonal cues interact with the physical properties of the microenvironment, remain open questions. This work first examines the effects of E2 on actin organization, morphodynamic flexibility, and migratory behavior in an endometriotic epithelial cell line (12Z), with the goal of validating its hormone responsiveness. Unexpectedly, E2 sensitivity was found to be context-dependent, motivating a broader investigation into additional microenvironmental regulators of cell behavior. Our subsequent studies focus on the role of mechanical tissue stiffening during fibrosis, using polyacrylamide gels to model healthy and pathological matrices. We demonstrate that substrate stiffness regulates epithelial motility, cytoskeletal organization, and collective migration. We then develop an in vitro model of endometriotic implantation into the mesothelium and show that endometriotic spheroids preferentially disrupt mesothelial barriers on glass, while fibrotic stiffness produces graded effects. By characterizing hormone responses and integrating mechanobiology into co-culture in vitro systems, this dissertation advances our understanding of early lesion establishment. This work also informs future in vitro model design and provides tools for more physiologically informed endometriosis research.
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