A 3D tension bioreactor platform to study the interplay between ECM stiffness and tumor phenotype

Abstract

Extracellular matrix (ECM) structure, composition, and stiffness have profound effects on tissue development and pathologies such as cardiovascular disease and cancer. Accordingly, a variety of synthetic hydrogel systems have been designed to study the impact of ECM composition, density, mechanics, and topography on cell and tissue phenotype. However, these synthetic systems fail to accurately recapitulate the biological properties and structure of the native tissue ECM. Natural three dimensional (3D) ECM hydrogels, such as collagen or hyaluronic acid, feature many of the chemical and physical properties of tissue, yet, these systems have limitations including the inability to independently control biophysical properties such as stiffness and pore size. Here, we present a 3D tension bioreactor system that permits precise mechanical tuning of collagen hydrogel stiffness, while maintaining consistent composition and pore size. We achieve this by mechanically loading collagen hydrogels covalently-conjugated to a polydimethylsiloxane (PDMS) membrane to induce hydrogel stiffening. We validated the biological application of this system with oncogenically transformed mammary epithelial cell organoids embedded in a 3D collagen I hydrogel, either uniformly stiffened or calibrated to create a gradient of ECM stiffening, to visually demonstrate the impact of ECM stiffening on transformation and tumor cell invasion. As such, this bioreactor presents the first tunable 3D natural hydrogel system that is capable of independently assessing the role of ECM stiffness on tissue phenotype.

The physics of organoids: a biophysical approach to understanding organogenesis

“…Such attempts have been initiated and it was discovered, for example, that the differentiation of mammary cells in 3D depends on the stiffness of the environment (Alcaraz et al, 2008;Cassereau et al, 2015). The predilection of most organoids for Matrigel is a hurdle, but moving away from Matrigel to hydrogels, where the mechanical properties can be manipulated independently of biochemical components, will empower such approaches (Gjorevski et al, 2016;Greggio et al, 2013).…”

“…In this context, intestinal organoids embedded in bovine collagen gels or elastin domain-based engineered hydrogels with varying stiffness grow with various efficiencies (DiMarco et al, 2014(DiMarco et al, , 2015. Collagen gels loaded with polydimethylsiloxane (PDMS) further enable one to control stiffness independently of pore size (Cassereau et al, 2015).…”

Review: Mechanotransduction in ovarian cancer: Shearing into the unknown

“…81,82 For cancer cells, the stiffness of their surrounding ECM can influence metastasis, invasion, proliferation, and chemoresistance. [83][84][85][86] Numerous studies have proven that stiffer substrates enhance the metastatic phenotypes of cancer cells. [87][88][89][90][91] However, within the field of ovarian cancer, studies have resulted in contradictory findings.…”

Heralding a new paradigm in 3D tumor modeling

“…By using glutaraldehyde as a crosslinker to increase the stiffness of collagen gels independently from pore size or collagen concentration, Lang et al showed that 3D invasion is dependent on pore size; while increased matrix stiffness promotes 3D invasion in gels with large pores (small steric hindrance), increased matrix stiffness hinders cell invasion in gels with small pores (large steric hindrance) [79]. In another interesting study, the Weaver group reported the development of a 3D tension bioreactor platform to facilitate studies on ECM stiffness; by mechanically loading collagen gels to induce gel stiffening (via collagen strain hardening) while maintaining composition and pore size, increasing matrix stiffness was also found to enhance tumor cell invasion [80]. …”