Background: A fundamental limitation in the development of new therapies to prevent metastatic cancer is a lack of in vitro systems that can accurately recapitulate the steps of cancer cell metastasis. Currently, most assays for examining the steps of metastasis fail to incorporate the biophysical forces experienced by tumor cells due to blood flow, or are low throughput and therefore not amenable to drug screening and high throughput experimentation. Methods: We have developed a novel high throughput mesofluidic platform for assaying 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 validated the fluid flow and alignment of the device and studied the adhesion of cultured leukocytic monocytes (THP-1 cells) and multiple cancer cell lines (HCT116, MDA-MB-231 and MCF-7 cells) to purified extracellular matrix molecules (ECM), endothelial cells and immobilized platelets. All assays were carried out under flow (0.5 dynes/cm2 of shear stress) and static conditions. Results: Our studies show that adhesion assays performed under flow yield markedly different results from static adhesion assays, and are better at identifying both aggressive cancer cells lines and known pathways for circulating cancer and immune cell adhesion. Treatment of breast cancer cells with a small library of integrin inhibitors demonstrated that these compounds had minimal effect on cancer cell adhesion to endothelial cells under static conditions, whereas under shear conditions many of these compounds reduced adhesion of cancer cells. In addition, a static adhesion assay of breast cancer cells to various types of ECM showed higher adhesion of the less aggressive MCF-7 cell line in comparison to the more aggressive MDA-MB-231 cell line. In contrast, flow incorporating assays showed increased adhesion of the MDA-MB-231 in comparison to the MCF-7 cell line. Finally, we performed a high throughput screening experiment using a kinase inhibitor library with 80 compounds and found that the shear based assay yielded notably different results from a similar screen under static conditions for cancer cell adhesion to endothelial cells, immune cell adhesion to endothelial cells and cancer cell adhesion to platelets. Conclusions: Our studies show that adhesion assays performed under flow yield markedly different results from static adhesion assays, and are better at identifying both aggressive cancer cells lines and known pathways for circulating cancer and immune cell adhesion. Thus, this high-throughput screening platform may enable the development of novel compounds to inhibit cancer metastasis and facilitate the study of the systems level behavior of cancer-endothelium adhesion. Citation Format: Adrianne Shearer, Chris Spruell, Victoria Le, Mar Creixell, Seema Nandi, Aaron B. Baker. Mesofluidic platform for high throughput screening for inhibitors of metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-008. doi:10.1158/1538-7445.AM2015-LB-008
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.
This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR08) of the Conference Proceedings. 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 B46.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.