(789b) ECM Stiffness and Modulus As Independent Controllers of Cancer Metastasis

Controllers of Cancer Metastasis 

Dannielle Ryman, Yuri Ebata, Max Nowak, Alfred J. Crosby, Shelly R. Peyton

University of Massachusetts Amherst, Amherst, MA, USA

Twenty to thirty percent of breast cancer cases will have metastases, which is the process by which individual cells leave the primary tumor site and colonize distant organs. Our objective is to study metastatic breast cancer at an early developmental stage, and to examine how the extracellular matrix (ECM) influences cell invasion at the primary tumor site. The ECM has been shown to affect cell motility via ligand-receptor interactions, and physical cues, such as matrix stiffness. Given that the primary tumor site considerably stiffens during disease progression, it seems likely that the ability of cells to sense and respond to these matrix features is relevant to cancer cell invasion.  It is now widely accepted that mechanical properties of the ECM can regulate cell migration. However, up to now, tissue modulus and stiffness have been used as interchangeable terms, and it is not clear if cell responses are sensitive to bulk tissue modulus, or, rather, tissue stiffness on the geometric length scale of the cell. We have adapted two experimental systems to quantify the independent effects of ECM stiffness and modulus on the migration of breast cancer cells.

First, we have a polymer microlens system that contains a PDMS base, which has a patterned array of cylindrical wells, coated with a thin, spuncoated film of polystyrene (PS). Cells only directly interact with the PS layer, but by controlling the geometry of both the PS and the PDMS, we can independently tune the stiffness and modulus that the cells experience. The inherent polymer architecture of the PDMS and PS controls the geometry-independent modulus, while the PDMS well diameter and thickness of the PS independently control the geometric stiffness. The completed microlenses consist of “compliant” regions of a thin film of PS (1.5N/m), and “stiff” regions where the PS film is supported by the PDMS (12 and 37N/m). Our second model system consists of poly(ethylene glycol) (PEG)-based hydrogels, wherein we can control the hydrogel thickness (on the order of tens of microns), independently of crosslinker concentration. The crosslinker content controls the hydrogel modulus, whereas the thickness of the gel overlaying a rigid glass coverslip controls the stiffness. Both the microlenses and the PEG hydrogels were coated with varying concentrations of fibronectin and collagen, and we quantified the migration of two metastatic breast cancer cell lines (MDA-MB-231 and BT549), and a non-metastatic control line (MCF-7). Thus far, our results show that the highly metastatic MDA-MB-231 are stiffness sensitive: cells achieve maximum cell speeds at an intermediate concentration of collagen I, on the lowest stiffness microlens regions. Interestingly, this stiffness dependence occurs on an incredibly high modulus substrate (PS modulus = 3GPa), suggesting that cell migration is regulated by substrate stiffness, rather than modulus. The less metastatic BT549 cells and non-metastatic MCF-7s do not exhibit any dependence on substrate stiffness. We are currently investigating what role integrin-binding proteins play in controlling mechanosensing across these geometric length scales, and if we can control mechanosensing via force transduction signaling pathways. By determining the appropriate length-scale by which mechanical properties regulate cancer metastasis, we hope to eventually uncover novel therapeutics to block cell invasion.