Vascularized adipose tissue in a 3D microfluidic chip for the study of ovarian cancer intraperitoneal metastases

Abstract

Epithelial ovarian cancer has the highest mortality rate of any gynecologic malignancy in the US and worldwide', largely driven by late-stage diagnoses and high likelihoods of metastasis 2 . Ovarian cancer has a clear predilection to metastasize to the peritoneal cavity; 70% of patients present with intraperitoneal metastases at diagnostic surgery . Within the peritoneum, the adipose-rich omentum has been proposed as a preferential location for ovarian cancer metastasis owing to both its location and microenviroment containing adipose-derived cytokines'. Furthermore, intraperitoneal metastases in patients are highly associated with the formation of malignant ascites (the pathologic accumulation of fluid in the peritoneum) due to impaired fluid drainage and increased net filtration , increased intraperitoneal vascular permeability, and local tumor and activated-stromal secretion of vascular endothelial growth factor (VEGF) 5'6.

However, the relationship between ascites, vascular and mesothelial permeability, and ovarian cancer intraperitoneal metastases remains poorly understood. Modeling and understanding the mechanisms of tumor cell (TC) intraperitoneal dissemination and its effects on vascular barrier function is therefore crucial to the development of therapeutics targeting this step of the ovarian cancer metastatic cascade. While organotypic in vitro models aimed at replicating the human peritoneum and tumor microenvironment (TME) of ovarian cancer exist, none have been vascularized, preventing our ability to study transcoelomic metastases alongside the changes in vascular morphology and permeability implicated in the formation of malignant ascites. In this study, we have designed a vascularized ovarian cancer TME model of the human peritoneum in a 3D microfluidic system.

We employed this model to study vascular morphology and permeability, ovarian TC peritoneal attachment and invasion, as well as TC effects on microvascular barrier function. Our model incorporates a monolayer of mesothelium in parallel with on chip-differentiated primary omental adipocytes, as well as perfusable microvasculature assembled by vasculogenesis. The microvasculatures formed with primary adipocytes exhibited increased physiological relevance via decreased luminal diameter, increased branching, and increased expression of the adipose- and peritoneally-upregulated extracellular matrix (ECM) components collagen VI and fibronectin'- . Microvasculatures formed in parallel with mesothelial monolayers also exhibited increased fibronectin expression, and in contrast to adipocytes, increased vessel diameter and decreased branching.

The presence of omental adipocytes led to increased and more physiological values of vessel and mesothelial permeability, while microvessels cultured alongside a mesothelial monolayer exhibited increased endothelial barrier function. Using attachment and invasion assays, we found that mesothelial cells impeded TC attachment and invasion, while adipocyte and microvascular coculture both individually and synergistically increased TC attachment and invasion. The presence of TCs in our model resulted in mesothelial invasion into the gel channel as well as increased vascular permeability, highlighting the role of TC-signaling on vascular and mesothelial barrier function in the formation of malignant ascites. Our system provides a robust platform to elucidate TC-stromal cell interactions during epithelial ovarian cancer intraperitoneal metastases and presents the first in vitro vascularized model of the human peritoneum and ovarian cancer TME.