Abstract PR08: Biomechanical preconditioning of circulating tumor cell adhesion during metastasis

Abstract

Introduction: Mechanical forces may greatly affect the ability and propensity of cancer cells to metastasize. Tumor cells are subject to many mechanical forces within their microenvironment in vivo. These mechanical forces may play a role in cancer cell regulation in combination with biochemical signals. However, much less is known about the role of mechanical forces in regulation of cancer cells. We have recently developed a novel high throughput mechanofluidic platform for assaying cancer cell adhesion under flow in a 96-well format. This device functions like a cone and plate viscometer in each well by inducing shear stress on cells cultured in a standard 96-well plate. We have used this device to study how the mechanics of the tumor microenvironment or applied mechanical forces alter a cancer cells propensity to adhere during metastatic extravasation. Mechanical compliance of the microenvironment regulates circulating tumor cell adhesion. We first examined how the compliance of the tumor environment can alter the adhesion of cancer cells during metastasis. MDA-MB-231 breast cancer cells were cultured on hydrogels with stiffnesses ranging from 0.2-50 kPa for the Young's modulus. Using the high throughput flow system we conducted adhesion assays to endothelial cells under flow followed by a detachment assay in which we increased the applied shear stress to determine the strength of attachment. We found that the compliance of the substrate cancer cells were grown on did not alter the initial adhesion to activated endothelial cells but markedly altered the strength of adhesion. The cells grown on 0.2 kPa substrates remained adhered under high stress (20 dynes/cm2) five-fold more than cells grown on 50 kPa substrates. We also examined whether compliance of the microenvironment of the endothelial cells could regulate their ability to recruit circulated tumor cells. We grew endothelial cells on substrates of varying compliance and then examined the adhesion of cancer cells grown on standard culture plates. In endothelial cells grown on compliant substrates (0.2 and 0.5 kPa) we found that cancer cells adhered less than to endothelial cells on stiffer substrates. Applied mechanical forces regulate circulating tumor cell adhesion to endothelial cells and extracellular matrix (ECM). We next examined how application of a cyclic stretching force to cancer cells would alter their adhesion to endothelial cells (ECs) and ECM. We applied a cyclic physiological strain to cancer cells for 24 hours and then performed adhesion assays with breast cancer cells to activated ECs, non-activated ECs, and purified ECM. We found that cyclic strain caused more cancer cells to adhere to activated ECs than their non-strained counterparts, however the strained cancer cells did not adhere as strongly to the activated ECs. Strained cancer cells adhered more to collagen I, laminin, and vitronectin, while they adhered less to collagen II and fibronectin than non-strained cells. The strength of adhesion to ECM was increased for collagen II, fibronectin, and laminin, but not for collagen I or vitronectin. To determine which integrins were involved in the strain-induced change in adhesion, a small library of integrin inhibitors was used to treat cancer cells while applying strain for 24 hours. Cilengitide, P11, ATN-161, Bio 1211, and RGDS peptides reduced the adhesion of cancer cells back to the level of non-strained cancer cells, indicating the role of αvβ3, αvβ5, α5β1, and α4β1 integrins in cancer cell sensing and reaction to the applied cyclic strain. Conclusion: Our studies have shown that application of mechanical forces such as the stiffness of the substrate that cancer cells and endothelial cells are cultured on or the application of cyclic strain can alter the adhesiveness and strength of adhesion of cancer cells to an endothelial monolayer or ECM under shear stress. Citation Format: Adrianne Spencer, Zhiying Zhu, Katerina Lee, Jason Lee, Chris Spruell, Chad Williams, Peter Voyvodic, Ashwin Ramaswami, Ning Jiang, Aaron Baker. Biomechanical preconditioning of circulating tumor cell adhesion during metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR08.