In situ measurement of ECM rheology and microheterogeneity in embedded and overlaid 3D pancreatic tumor stroma co-cultures via passive particle tracking

Abstract

Tumor growth is regulated by a diverse set of extracellular in°uences, 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/¯broblast 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°uorescently 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 sti®ening of collagen in¯broblast co-cultures relative to cultures with cancer cells only, which exhibit degraded ECM with heterogeneous microstructure. We further show that these e®ects 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.

Photodestruction of Stromal Fibroblasts Enhances Tumor Response to PDT in 3D Pancreatic Cancer Coculture Models

“…Cultures were incubated at 37°C, and fresh cultures that supplemented with 2% Matrigel were substituted with the original medium every two days. The nodule growth was monitored every other day via dark-field microscopy with 5× objective to obtain high-contrast images (33). Longitudinal images were processed to obtain growth curves and size distribution data using analysis code that was previously described (38).…”

“…In this study, we adopted a heterotypic 3D coculture model, which allowed us to examine PDT response of PDAC cells in communication with stromal partners that recapitulate contrasting fibroblastic phenotypes (33). While sophisticated animal models have been developed to recapitulate PDAC stroma (34), the use of a custom in vitro model here allowed us to interrogate response to PDT when epithelial PDAC cells are allowed to interact with stromal cells exhibiting contrasting fibroblastic phenotypes in a controlled environment that restores key morphological and functional features of human disease (2,35).…”

Passive and Active Microrheology for Biomedical Systems

“…Understanding both directions of cell-environment interactions is essential in understanding the progression of pancreatic tumors. In a recent investigation into these mechanical relationships using VPT method, PANC-1 cells and MRC-5 (normal human fibroblast) cells were cultured in collagen I ECM to build pancreatic models: PANC-1 homoculture, PANC-1/MRC-5 co-culture, and cell-free control ( Jones et al, 2017 ). The resulting MSDs of the coculture and the control were similar, but the MSD of the PANC-1 homoculture revealed a high degree of structural heterogeneity.…”

Increased Stiffness Downregulates Focal Adhesion Kinase Expression in Pancreatic Cancer Cells Cultured in 3D Self-Assembling Peptide Scaffolds

“…Natural matrices, such as collagen, allow the modulation of stiffness by simply changing the concentration of the gel, but they also suffer degradation in vitro due to MMPs activity [ 33 , 34 ], which may induce changes in fiber orientation and thickness [ 33 ]. In fact, collagen degradation by cells can start as early as 6 h in culture on polymerized collagen substrate [ 35 ].…”