Durotaxis by Human Cancer Cells.

Article Durotaxis by Human Cancer Cells Brian J. DuChez,1 Andrew D. Doyle,1 Emilios K. Dimitriadis,2 and Kenneth M. Yamada1,* 1 Cell Biology Section, Division of Intramural Research, National Institute of Dental and Craniofacial Research and 2Trans-NIH Shared Resource on Biomedical Engineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland ABSTRACT Durotaxis is a type of directed cell migration in which cells respond to a gradient of extracellular stiffness. Using automated tracking of positional data for large sample sizes of single migrating cells, we investigated 1) whether cancer cells can undergo durotaxis; 2) whether cell durotactic efficiency varies depending on the regional compliance of stiffness gradients; 3) whether a specific cell migration parameter such as speed or time of migration correlates with durotaxis; and 4) whether Arp2/3, previously implicated in leading edge dynamics and migration, contributes to cancer cell durotaxis. Although durotaxis has been characterized primarily in nonmalignant mesenchymal cells, little is known about its role in cancer cell migration. Diffusible factors are known to affect cancer cell migration and metastasis. However, because many tumor microenvironments gradually stiffen, we hypothesized that durotaxis might also govern migration of cancer cells. We evaluated the durotactic potential of multiple cancer cell lines by employing substrate stiffness gradients mirroring the physiological stiffness encountered by cells in a variety of tissues. Automated cell tracking permitted rapid acquisition of positional data and robust statistical analyses for migrating cells. These durotaxis assays demonstrated that all cancer cell lines tested (two glioblastoma, metastatic breast cancer, and fibrosarcoma) migrated directionally in response to changes in extracellular stiffness. Unexpectedly, all cancer cell lines tested, as well as noninvasive human fibroblasts, displayed the strongest durotactic migratory response when migrating on the softest regions of stiffness gradients (2–7 kPa), with decreased responsiveness on stiff regions of gradients. Focusing on glioblastoma cells, durotactic forward migration index and displacement rates were relatively stable over time. Correlation analyses showed the expected correlation with displacement along the gradient but much less with persistence and none with cell speed. Finally, we found that inhibition of Arp2/3, an actin-nucleating protein necessary for lamellipodial protrusion, impaired durotactic migration. INTRODUCTION Directional cell migration refers to the ability of a cell to polarize and move persistently in a specified direction, generally in response to an extracellular signal that biases the direction of movement. Signals in the extracellular space can take on many forms and can act to either attract or repel the cell. Chemotaxis, the most thoroughly studied and bestcharacterized mechanism of directed migration, involves a response to diffusible chemicals. Other factors—including substrate-bound gradients of extracellular proteins (haptotaxis), electric fields (galvanotaxis), contact guidance, and changes in substrate rigidity (durotaxis)—have also been shown to direct the movement of cells (for reviews, especially in cancer, see (1–10)). Directed migration can be contrasted with the chemokinetic, nondirectional migration that cells typically exhibit in homogeneous environments. Cells Submitted June 25, 2018, and accepted for publication January 7, 2019. *Correspondence: Editor: Margaret Gardel. https://doi.org/10.1016/j.bpj.2019.01.009 670 Biophysical Journal 116, 670–683, February 19, 2019 responding to a chemical stimulus can polarize and temporarily move directionally in an environment that lacks any gradient condition. However, the absence of a sufficiently strong external gradient to ‘‘bias’’ the direction of cell movement results in a population of cells that migrate in random directions. Durotaxis is a mechanism of directional migration in which a cell responds to an extracellular gradient of stiffness (6,11). Typically, durotactic migration involves cell movement toward regions of increasing stiffness across steps or up gradients of increasingly stiff substrates (6,11–21). Only durotactic migration toward increasing stiffness has been thoroughly documented; however, there is speculation that durotaxis toward increasingly soft substrates may occur (19,20). Mechanisms proposed to underlie durotaxis of fibroblastic (mesenchymal) cells include contractile mechanosensing, probing of the local substrate by filopodia, and focal adhesion signaling (15,17,21–24). Cancer cell migration is important for expanding tumor margins and initiating the metastatic cascade. Cells escape Durotaxis by Human Cancer Cells from the primary tumor through a variety of migratory mechanisms (25), and they enter the circulatory or lymphatic system. General hallmarks of malignant and normal cell migration on tissue culture substrates can be described as a cyclical series of sequential steps. These steps include 1) protrusion of the leading edge of the cell, 2) adhesion of the leading edge to the extracellular substrate, 3) forward translocation of the cell body, and 4) retraction of the trailing edge (26,27). The protrusive leading edge of a migrating cell is often characterized by a broad, sheet-like lamellipodium, which can also contain spike-like filopodia. These protrusions result from globular actin incorporation onto the barbed end of actin filaments during actin polymerization (28,29). Arp2/3 is a seven-protein complex responsible for initiating the growth of an extensive network of these actin filaments through increased actin branching. This polymerization against the plasma membrane helps push it forward during lamellipodia and filopodia formation (30–33), thereby playing a fundamental role in this crucial step of migration. Furthermore, Arp2/3 has been implicated in enabling cells to respond by directional migration to chemotactic (EGF but not PDGF) and haptotactic gradients (34,35). However, the role of Arp2/3 in durotaxis remains unknown. There is a strong correlation between stiffening of the tumor microenvironment and activation of epithelial-tomesenchymal transition pathways, tumor growth, and increased malignancy (e.g., see (36–39)). Consequently, cancer cells might employ durotaxis in the process of metastatic dissemination, but there has been little in vitro or in vivo evidence for the durotactic capacity of cancer cells to date. If they were capable of durotaxis, another unanswered question is whether cancer cells respond to a gradient of stiffness at a physiological range of stiffness associated with the compliance characteristics of different tissues throughout the body (40,41). In this study, we first developed software to automate cell tracking to analyze the migratory phenotypes o (...truncated)