In tissues, cells reside in confining microenvironments, which may mechanically restrict the ability of a cell to double in size as it prepares to divide. How confinement affects cell cycle progression remains unclear. We show that cells progressed through the cell cycle and proliferated when cultured in hydrogels exhibiting fast stress relaxation but were mostly arrested in the G0/G1 phase of the cell cycle when cultured in hydrogels that exhibit slow stress relaxation. In fast-relaxing gels, activity of stretch-activated channels (SACs), including TRPV4, promotes activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which in turn drives cytoplasmic localization of the cell cycle inhibitor p27Kip1, thereby allowing S phase entry and proliferation. Cell growth during G1 activated the TRPV4-PI3K/Akt-p27Kip1 signaling axis, but growth is inhibited in the confining slow-relaxing hydrogels. Thus, in confining microenvironments, cells sense when growth is sufficient for division to proceed through a growth-responsive signaling axis mediated by SACs.

No abstract

INTRODUCTION: Ovarian cancers have dynamic mechanical microenvironments. Ovarian cancer cells within the tumor microenvironment experience a range of shear and compressive stimuli from both internal and external sources including ascitic fluid flow, interstitial fluid flow, hydrostatic pressure, vascular blood flow, displacement of surrounding cells, and growth induced stress, all of which contribute to mechanotransduction. Ovarian cancer progression typically comes with the development of ascites within the peritoneal cavity, which create a unique mechanical microenvironment. External dynamic stimuli have been correlated with an increase in metastasis, cancer stem cell (CSC) marker expression, chemoresistance, and proliferation in a variety of cancers however, how these shear and compressive stimuli specifically contribute to ovarian cancer progression has been overlooked. To address this gap in knowledge, individual custom built bioreactors capable of applying tunable shear stress or compressive stimulus were constructed and characterized, to investigate the impact of physiologically relevant forces on ovarian cancer cells within a 3D culture environment. MATERIALS AND METHODS: A compression bioreactor capable of applying cyclic or static compression cycles, and a shear stress bioreactor equipped with tunable shear stress stimulus, were designed and fabricated in house. Both bioreactors utilize a 3D agarose-collagen type I interpenetrating network (IPN) hydrogel for mechanically supporting embedded cells. 20 kPa of pressure was applied for 24 hours to high grade serous OVCAR3 and OVSAHO ovarian cancer cells in either static or cyclic waveforms. Shear stress was applied to OVCAR3 and OVCAR8 cells for 24 hours via a peristaltic pump at shear stresses of 1, 5, or 11 dynes/cm2. Mechanical stimulus experienced by cells housed within the IPN gel composite was evaluated using finite element analysis (FEA). Histological stains were evaluated for morphological quantification and immunohistochemistry staining was used to evaluate proliferation and cell death. Changes in gene expression were monitored through qPCR analysis of experimental vs control groups. RESULTS AND DISCUSSION: The IPN hydrogel was characterized using SEM imaging and rheometric testing, and the viscoelastic modulus was determined to be 10.36±0.08 kPa. FEA of the bioreactors showed the distribution of compressive or shear forces throughout the cells within the hydrogel. Under compression, OVCAR3 cells were found to have a significant increase in cellular area, aspect ratio, and a significant decrease in roundness. The cells also displayed a two-fold increase in proliferation as quantified by Ki67 expression. Cell death was found to be significantly decreased under static compression conditions when compared to either cyclic or control conditions. qRTPCR analysis revealed a greater than 2-fold increase in the expression of COX-2, BCL2, E-Cadherin, C-SRC, and CDC42. Similar results were observed with shear stress stimulation. CONCLUSIONS: Innovative compression and shear stress bioreactors were designed and constructed to investigate the influence of physiologically relevant mechanical stimulus on ovarian cancer cells. Stimulated cells were found to exhibit cellular morphological changes, indicative of a motile phenotype. Ovarian cancer cells increased expression of a variety of genes tied to metastasis, mechanotransduction, cell cycle progression, and chemoresistance. The increased proliferation and decreased cell death indicate a cancer assistive mechanism from mechanical stimuli, and thus a potential treatment target within the activated mechanotransduction pathways. These dynamic in vitro 3D platform provide understanding of the influence of mechanical stimuli, and their influence on cellular response, a critical component of the mechanical environment in a variety of diseases and cell types. Citation Format: Caymen Novak, Eric Horst, Ciara Davis, Geeta Mehta. MECHANOTRANSDUCTION IN OVARIAN CANCERS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-080.

No abstract