Casey M. Kraning-Rush

Cellular Traction Stresses Increase with Increasing Metastatic Potential

Cancer cells exist in a mechanically and chemically heterogeneous microenvironment which undergoes dynamic changes throughout neoplastic progression. During metastasis, cells from a primary tumor acquire characteristics that enable them to escape from the primary tumor and migrate through the heterogeneous stromal environment to establish secondary tumors. Despite being linked to poor prognosis, there are no direct clinical tests available to diagnose the likelihood of metastasis. Moreover, the physical mechanisms employed by metastatic cancer cells to migrate are poorly understood. Because metastasis of most solid tumors requires cells to exert force to reorganize and navigate through dense stroma, we investigated differences in cellular force generation between metastatic and non-metastatic cells. Using traction force microscopy, we found that in human metastatic breast, prostate and lung cancer cell lines, traction stresses were significantly increased compared to non-metastatic counterparts. This trend was recapitulated in the isogenic MCF10AT series of breast cancer cells. Our data also indicate that increased matrix stiffness and collagen density promote increased traction forces, and that metastatic cells generate higher forces than non-metastatic cells across all matrix properties studied. Additionally, we found that cell spreading for these cell lines has a direct relationship with collagen density, but a biphasic relationship with substrate stiffness, indicating that cell area alone does not dictate the magnitude of traction stress generation. Together, these data suggest that cellular contractile force may play an important role in metastasis, and that the physical properties of the stromal environment may regulate cellular force generation. These findings are critical for understanding the physical mechanisms of metastasis and the role of the extracellular microenvironment in metastatic progression.

Biophysical control of invasive tumor cell behavior by extracellular matrix microarchitecture

Fibrillar collagen gels, which are used extensively in vitro to study tumor-microenvironment interactions, are composed of a cell-instructive network of interconnected fibers and pores whose organization is sensitive to polymerization conditions such as bulk concentration, pH, and temperature. Using confocal reflectance microscopy and image autocorrelation analysis to quantitatively assess gel microarchitecture, we show that additional polymerization parameters including culture media formulation and gel thickness significantly affect the dimensions and organization of fibers and pores in collagen gels. These findings enabled the development of a three-dimensional culture system in which cell-scale gel microarchitecture was decoupled from bulk gel collagen concentration. Interestingly, morphology and migration characteristics of embedded MDA-MB-231 cells were sensitive to gel microarchitecture independently of collagen gel concentration. Cells adopted a polarized, motile phenotype in gels with larger fibers and pores and a rounded or stellate, less motile phenotype in gels with small fibers and pores regardless of bulk gel density. Conversely, cell proliferation was sensitive to gel concentration but not microarchitecture. These results indicate that cell-scale gel microarchitecture may trump bulk-scale gel density in controlling specific cell behaviors, underscoring the biophysical role of gel microarchitecture in influencing cell behavior.

The role of the cytoskeleton in cellular force generation in 2D and 3D environments

To adhere and migrate, cells generate forces through the cytoskeleton that are transmitted to the surrounding matrix. While cellular force generation has been studied on 2D substrates, less is known about cytoskeletal-mediated traction forces of cells embedded in more in vivo-like 3D matrices. Recent studies have revealed important differences between the cytoskeletal structure, adhesion, and migration of cells in 2D and 3D. Because the cytoskeleton mediates force, we sought to directly compare the role of the cytoskeleton in modulating cell force in 2D and 3D. MDA-MB-231 cells were treated with agents that perturbed actin, microtubules, or myosin, and analyzed for changes in cytoskeletal organization and force generation in both 2D and 3D. To quantify traction stresses in 2D, traction force microscopy was used; in 3D, force was assessed based on single cell-mediated collagen fibril reorganization imaged using confocal reflectance microscopy. Interestingly, even though previous studies have observed differences in cell behaviors like migration in 2D and 3D, our data indicate that forces generated on 2D substrates correlate with forces within 3D matrices. Disruption of actin, myosin or microtubules in either 2D or 3D microenvironments disrupts cell-generated force. These data suggest that despite differences in cytoskeletal organization in 2D and 3D, actin, microtubules and myosin contribute to contractility and matrix reorganization similarly in both microenvironments.

Comparative mechanisms of cancer cell migration through 3D matrix and physiological microtracks

Tumor cell invasion through the stromal extracellular matrix (ECM) is a key feature of cancer metastasis, and understanding the cellular mechanisms of invasive migration is critical to the development of effective diagnostic and therapeutic strategies. Since cancer cell migration is highly adaptable to physiochemical properties of the ECM, it is critical to define these migration mechanisms in a context-specific manner. Although extensive work has characterized cancer cell migration in two- and three-dimensional (3D) matrix environments, the migration program employed by cells to move through native and cell-derived microtracks within the stromal ECM remains unclear. We previously reported the development of an in vitro model of patterned type I collagen microtracks that enable matrix metalloproteinase-independent microtrack migration. Here we show that collagen microtracks closely resemble channel-like gaps in native mammary stroma ECM and examine the extracellular and intracellular mechanisms underlying microtrack migration. Cell-matrix mechanocoupling, while critical for migration through 3D matrix, is not necessary for microtrack migration. Instead, cytoskeletal dynamics, including actin polymerization, cortical tension, and microtubule turnover, enable persistent, polarized migration through physiological microtracks. These results indicate that tumor cells employ context-specific mechanisms to migrate and suggest that selective targeting of cytoskeletal dynamics, but not adhesion, proteolysis, or cell traction forces, may effectively inhibit cancer cell migration through preformed matrix microtracks within the tumor stroma.

Controlling matrix stiffness and topography for the study of tumor cell migration

Cellular studies have long been performed on the bench top, within Petri dishes and flasks that expose cells to surroundings that differ greatly from their native environment. The complexity of a human tissue is such that to truly replicate a cell’s physiologic microenvironment in vitro is currently impossible. It is nevertheless important to determine how various factors of the microenvironment interact to drive cell behavior, particularly with regard to disease states, such as cancer. Here we focus on two key elements of the cellular microenvironment, matrix stiffness and architecture, in the context of tumor cell behavior. We discuss recent work focusing on the effects of these individual properties on cancer cell migration and describe one technique developed by our lab that could be applied to dissect the effects of specific structural and mechanical cues, and which may lead to useful insights into the potentially synergistic effects of these properties on tumor cell behavior.

Quantifying traction stresses in adherent cells.

Contractile force generation plays a critical role in cell adhesion, migration, and extracellular matrix reorganization in both 2D and 3D environments. Characterization of cellular forces has led to a greater understanding of cell migration, cellular mechanosensing, tissue formation, and disease progression. Methods to characterize cellular traction stresses now date back over 30 years, and they have matured from qualitative comparisons of cell-mediated substrate movements to high-resolution, highly quantitative measures of cellular force. Here, we will provide an overview of common methods used to measure forces in both 2D and 3D microenvironments. Specific focus will be placed on traction force microscopy, which measures the force exerted by cells on 2D planar substrates, and the use of confocal reflectance microscopy, which can be used to quantify collagen fibril compaction as a metric for 3D traction forces. In addition to providing experimental methods to analyze cellular forces, we discuss the application of these techniques to a large range of biomedical problems and some of the significant challenges that still remain in this field.

Substrate Stiffness Regulates PDGF-Induced Circular Dorsal Ruffle Formation Through MLCK

As atherosclerosis progresses, vascular smooth muscle cells (VSMCs) invade from the medial layer into the intimal layer and proliferate, contributing to atherosclerotic plaque formation. This migration is stimulated in part by platelet-derived growth factor (PDGF), which is released by endothelial cells and inflammatory cells, and vessel stiffening, which occurs with age and atherosclerosis progression. PDGF induces the formation of circular dorsal ruffles (CDRs), actin-based structures associated with increased cell motility. Here we show that mechanical changes in matrix stiffness enhance the formation of CDRs in VSMCs in response to PDGF stimulation. Our data indicate that matrix stiffness increases cellular contractility, and that intracellular pre-stress is necessary for robust CDR formation. When treated with agonists that promote contractility, cells increase CDR formation, whereas agonists that inhibit contractility lead to decreased CDR formation. Substrate stiffness promotes CDR formation in response to PDGF by upregulating Src activity through myosin light chain kinase. Together, these data indicate that vessel stiffening accompanying atherogenesis may exacerbate VSMC response to PDGF leading to CDR formation.

Vinculin regulates directionality and cell polarity in two- and three-dimensional matrix and three-dimensional microtrack migration

Cells can maneuver through 3D matrices by using tracks that exist in the matrix. The focal adhesion protein vinculin mediates the unidirectional movement through these tracks. Moreover, vinculin also promotes directional migration in 2D and 3D matrices. Vinculin’s role in migration is mediated by FAK activation.

Single cell-mediated collagen reorganization in 3D matrices

Cells use cytoskeletally-generated force to adhere, migrate and remodel their environment. While cellular forces generated by cells plated on 2D substrates is well-studied, much less is known about the forces generated by cells in 3D matrices, which more closely mimic the in vivo environment. Here, an approach to characterize cellular forces in 3D using confocal reflectance microscopy is presented. Remodeling of collagen fibrils due to the forces exerted by embedded cells was imaged in real-time as cells adhere to and contract the matrix. We implemented this approach in conjunction with 2D Traction Force Microscopy to compare cytoskeletally-mediated forces of cells in 3D collagen matrices to forces exerted by cells on 2D collagen-coated hydrogel substrates. Our results indicate that confocal reflectance microscopy of collagen fibrils can provide semi-quantitative information regarding cellular force in 3D matrices, and that the actin cytoskeleton plays a similar role in regulating cell contractility in both 2D and 3D microenvironments.

Molded Collagen Microchannels for the Study of Cancer Cell Invasion

Metastasis is the cause of 90% of cancer-related deaths and yet the precise mechanism of metastasis is poorly understood[1]. To metastasize, cells break free from the primary tumor, migrate through the surrounding tissue, and enter the vascular system to move to a secondary site. To migrate through the stroma, cell can both degrade the tissue and use physical force to move the tissue from its path. However, the relative roles of matrix degradation and cellular force are not well-understood. Previous work has shown that as cell move through the matrix, they create channels that other cells can then use to more easily escape from the primary tumor [2, 3]. To investigate the mechanisms by which metastatic cells move through 3D matrices, we fabricated microchannels from collagen that simulate the paths that are made and used by cells during metastasis. Here, we will present our method for molding channels in compliant collagen gels to investigate cell migration. The channels dimensions approximate the size of a cell and permit cell migration without the need for matrix degradation. We demonstrate that the channels cause persistent, unidirectional cell migration that is significantly faster than the migration observed in 3D matrices without channels. These channels provide a unique platform to probe 3D cellular migration and permit the dissection of the relative roles of matrix degradation and force generation in facilitating cell invasion.Copyright