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.

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.

Vinculin Regulates Directionality and Cell Polarity in 2D, 3D Matrix and 3D Microtrack Migration.

During metastasis, cells can use proteolytic activity to form tube-like "microtracks" within the extracellular matrix (ECM). Using these microtracks, cells can migrate unimpeded through the stroma. To investigate the molecular mechanisms of microtrack migration, we developed an in vitro 3D micromolded collagen platform. When in microtracks, cells tend to migrate unidirectionally. Since focal adhesions are the primary mechanism by which cells interact with the ECM, we examined the roles of several focal adhesion molecules in driving unidirectional motion. Vinculin knockdown results in the repeated reversal of migration direction compared with control cells. Tracking the position of the Golgi centroid relative to the position of the nucleus centroid reveals that vinculin knockdown disrupts cell polarity in microtracks. Vinculin also directs migration on 2D substrates and in 3D uniform collagen matrices, indicated by reduced speed, shorter net displacement and decreased directionality in vinculin-deficient cells. In addition, vinculin is necessary for Focal Adhesion Kinase (FAK) activation in 3D as vinculin knockdown results in reduced FAK activation in both 3D uniform collagen matrices and microtracks, but not on 2D substrates, and accordingly, FAK inhibition halts cell migration in 3D microtracks. Together, these data indicate that vinculin plays a key role in polarization during migration.

Microfabricated collagen tracks facilitate single cell metastatic invasion in 3D.

While the mechanisms employed by metastatic cancer cells to migrate remain poorly understood, it has been widely accepted that metastatic cancer cells can invade the tumor stroma by degrading the extracellular matrix (ECM) with matrix metalloproteinases (MMPs). Although MMP inhibitors showed early promise in preventing metastasis in animal models, they have largely failed clinically. Recently, studies have shown that some cancer cells can use proteolysis to mechanically rearrange their ECM to form tube-like "microtracks" which other cells can follow without using MMPs themselves. We speculate that this mode of migration in the secondary cells may be one example of migration which can occur without endogenous protease activity in the secondary cells. Here we present a technique to study this migration in a 3D, collagen-based environment which mimics the size and topography of the tracks produced by proteolytically active cancer cells. Using time-lapse phase-contrast microscopy, we find that these microtracks permit the rapid and persistent migration of noninvasive MCF10A mammary epithelial cells, which are unable to otherwise migrate in 3D collagen. Additionally, while highly metastatic MDAMB231 breast cancer cells are able to invade a 3D collagen matrix, seeding within the patterned microtracks induced significantly increased cell migration speed, which was not decreased by pharmacological MMP inhibition. Together, these data suggest that microtracks within a 3D ECM may facilitate the migration of cells in an MMP-independent fashion, and may reveal novel insight into the clinical challenges facing MMP inhibitors.

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.

A physical sciences network characterization of non-tumorigenic and metastatic cells.

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.

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.

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.

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.

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.

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.