Abstract Ovarian cancer (OvCa) is the most fatal gynecologic malignancy and the fifth leading cause of overall cancer death among American women with a low (27%) 5-year survival rate, as 75% of women are diagnosed with disseminated intra-peritoneal (IP) metastasis. OvCa cells detach from the primary tumor and shed into the peritoneal cavity, adhere to the peritoneal mesothelial cell (MC) monolayer, intercalate within this layer, and invade into the submesothelial matrix, where they proliferate and form secondary lesions. More than one third of OvCa patients develop ascites that correlates with relapse and poor prognosis. The accumulated ascites can reach volumes up to 5 liters, causing a striking elevation in intraperitoneal pressure (IPP) from normally sub-atmospheric ~5 mmHg to as high as 27 mmHg, impacting the tumor microenvironment including host and tumor cells. The pressure created by accumulated ascites alters the force environment in the peritoneal cavity, causing strain and compressive forces on peritoneal structures. Yet, the mechanism through which ascites-induced IPP influences OvCa progression is poorly understood. The aim of this study is to understand how ascites-induced IPP promotes OvCa progression. To model the compressive force present in vivo due to accumulated tense ascites (~27 mmHg), we evaluated the effects of compressive force on primary human and murine MC monolayers and murine peritoneal explants using the Flexcell Compression System. Strikingly, compressed MC and peritoneal explants exhibited phenotypic alterations and formed abundant Tunneling Nanotubes (TNT), which are cell membrane extensions that modulate transfer of organelles between cells under stress. Furthermore, compression of OvCa cells adherent to murine peritoneal explants show intense formation of TNT between OvCa cells and peritoneal MC. Remarkably, TNT formed under compression displayed membrane distension and modulated the transfer of mitochondria from MC to OvCa cells. Moreover, collected peritoneal explants from OvCa murine model with intense ascites showed TNT formation and changes in the peritoneal sub-mesothelial collagen ultrastructure. This study reveals a novel response of OvCa cells and the tumor microenvironment to mechanical compression, uncovering a new mechanism by which OvCa metastatic success may be regulated. Note: This abstract was not presented at the meeting. Citation Format: Marwa Asem, Allison Young Young, Alejandro ClaureDeLaZerda, Matthew Ravosa, Sharon Stack. Regulation of ovarian tumor microenvironment dynamics by compressive stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1910.