Abstract
Tumor growth is regulated by a diverse set of extracellular influences, including paracrine crosstalk with stromal partners, and biophysical interactions with surrounding cells and tissues.Studies elucidating the role of physical force and the mechanical properties of the extracellular matrix (ECM) itself as regulators of tumor growth and invasion have been greatly catalyzed by the use of in vitro three-dimensional (3D) tumor models. These systems provide the ability to systematically isolate, manipulate, and evaluate impact of stromal components and extracellular mechanics in a platform that is both conducive to imaging and biologically relevant. However, recognizing that mechanoregulatory crosstalk is bi-directional and fully utilizing these models requires complementary methods for in situ measurements of the local mechanical environment. Here, in 3D tumor/fibroblast co-culture models of pancreatic cancer, a disease characterized by its prominent stromal involvement, we evaluate the use of particle-tracking microrheology to probe dynamic mechanical changes. Using videos of fluorescently labeled polystyrene microspheres embedded in collagen I ECM, we measure spatiotemporal changes in the Brownian motion of probes to report local ECM shear modulus and microheterogeneity. This approach reveals stiffening of collagen in fibroblast co-cultures relative to cultures with cancer cells only, which exhibit degraded ECM with heterogeneous microstructure. We further show that these effects are dependent on culture geometry with contrasting behavior for embedded and overlay cultures. In addition to potential application to screening stroma-targeted therapeutics, this work also provides insight into how the composition and plating geometry impact the mechanical properties of 3D cell cultures that are increasingly widely used in cancer biology.
Highlights
The tumor microenvironment encompasses a diverse set of growth-regulating signals that include biochemical and biophysical interactions with stromal cells and surrounding tissues.[1,2,3,4] A key component of this milieu is the extracellular matrix (ECM), a protein-based sca®olding that provides a support framework for healthy tissues, but which takes on altered composition and function as a regulator of tumor growth behavior
Inbroblast (þÞ and cell-free collagen ECM, probe trajectories are more nearly uniformly consistent with the latter description, re°ected in the small mean squared displacement (MSD) over all times probed (Fig. 3(a))
The geometry of 3D cultures as well as the presence or absence of stromal partners leads to signicant quantitative changes in both sample microheterogeneity as well as mean overall ECM rigidity
Summary
The tumor microenvironment encompasses a diverse set of growth-regulating signals that include biochemical and biophysical interactions with stromal cells and surrounding tissues.[1,2,3,4] A key component of this milieu is the extracellular matrix (ECM), a protein-based sca®olding that provides a support framework for healthy tissues, but which takes on altered composition and function as a regulator of tumor growth behavior. The study of mechanical regulation of tumor growth by stromal components has been facilitated in large part by the development of in vitro 3D tumor models, which have emerged as a powerful research platform in cancer biology in general.[14,15,16,17,18] The recreation of 3D tissue architecture in these model systems provides the ability to systematically isolate and evaluate roles of extracellular mechanical cues and tissue organization that are absent in traditional monolayer cell cultures yet more accessible to imaging than animal tumor models.19,20 3D tumor models utilizing physically customized sca®olds have already been used to gain insight into the e®ects of matrix composition and rheology impact on integrin and growth factor mediated signaling, and cell motility.[21,22]
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