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.

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.

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.

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.

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.

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.

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.

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