Cells respond to physical stimuli, such as stiffness1, fluid shear stress2 and hydraulic pressure3,4. Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer5. However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology.

Fluorescence illumination can cause phototoxicity that negatively affects living samples. This study demonstrates that much of the phototoxicity and photobleaching experienced with live-cell fluorescence imaging occurs as a result of “illumination overhead” (IO). This occurs when a sample is illuminated but fluorescence emission is not being captured by the microscope camera. Several technological advancements have been developed, including fast-switching LED lamps and transistor-transistor logic (TTL) circuits, to diminish phototoxicity caused by IO. These advancements are not standard features on most microscopes and many biologists are unaware of their necessity for live-cell imaging. IO is particularly problematic when imaging rapid processes that require short exposure times. This study presents a workflow to optimize imaging conditions for measuring both slow and dynamic processes while minimizing phototoxicity on any standard microscope. The workflow includes a guide on how to (1) determine the maximum image exposure time for a dynamic process, (2) optimize excitation light intensity and (3) assess cell health with mitochondrial markers.

LIM domain containing proteins are important regulators of diverse cellular processes, and play pivotal roles in regulating the actin cytoskeleton. Lipoma Preferred Partner (LPP) is a member of the zyxin family of LIM proteins that has long been characterized as a promoter of mesenchymal/fibroblast cell migration. More recently, LPP has emerged as a critical inducer of tumor cell migration, invasion and metastasis. LPP is thought to contribute to these malignant phenotypes by virtue of its ability to shuttle into the nucleus, localize to adhesions and, most recently, to promote invadopodia formation. In this review, we will examine the mechanisms through which LPP regulates the functions of adhesions and invadopodia, and discuss potential roles of LPP in mediating cellular responses to mechanical cues within these mechanosensory structures.

Cell migration regulates diverse (patho)physiological processes, including cancer metastasis. According to the Osmotic Engine Model, polarization of NHE1 at the leading edge of confined cells facilitates water uptake, cell protrusion and motility. The physiological relevance of the Osmotic Engine Model and the identity of molecules mediating cell rear shrinkage remain elusive. Here, we demonstrate that NHE1 and SWELL1 preferentially polarize at the cell leading and trailing edges, respectively, mediate cell volume regulation, cell dissemination from spheroids and confined migration. SWELL1 polarization confers migration direction and efficiency, as predicted mathematically and determined experimentally via optogenetic spatiotemporal regulation. Optogenetic RhoA activation at the cell front triggers SWELL1 re-distribution and migration direction reversal in SWELL1-expressing, but not SWELL1-knockdown, cells. Efficient cell reversal also requires Cdc42, which controls NHE1 repolarization. Dual NHE1/SWELL1 knockdown inhibits breast cancer cell extravasation and metastasis in vivo, thereby illustrating the physiological significance of the Osmotic Engine Model.

Background: The p66ShcA redox protein is the longest isoform of the Shc1 gene and is variably expressed in breast cancers. In response to a variety of stress stimuli, p66ShcA becomes phosphorylated on serine 36, which allows it to translocate from the cytoplasm to the mitochondria where it stimulates the formation of reactive oxygen species (ROS). Conflicting studies suggest both pro-and anti-tumorigenic functions for p66ShcA, which prompted us to examine the contribution of tumor cell-intrinsic functions of p66ShcA during breast cancer metastasis. Methods: We tested whether p66ShcA impacts the lung-metastatic ability of breast cancer cells. Breast cancer cells characteristic of the ErbB2+/luminal (NIC) or basal (4T1) subtypes were engineered to overexpress p66ShcA. In addition, lung-metastatic 4T1 variants (4T1-537) were engineered to lack endogenous p66ShcA via Crispr/Cas9 genomic editing. p66ShcA null cells were then reconstituted with wild-type p66ShcA or a mutant (S36A) that cannot translocate to the mitochondria, thereby lacking the ability to stimulate mitochondrial-dependent ROS production. These cells were tested for their ability to form spontaneous metastases from the primary site or seed and colonize the lung in experimental (tail vein) metastasis assays. These cells were further characterized with respect to their migration rates, focal adhesion dynamics, and resistance to anoikis in vitro. Finally, their ability to survive in circulation and seed the lungs of mice was assessed in vivo. Results: We show that p66ShcA increases the lung-metastatic potential of breast cancer cells by augmenting their ability to navigate each stage of the metastatic cascade. A non-phosphorylatable p66ShcA-S36A mutant, which cannot translocate to the mitochondria, still potentiated breast cancer cell migration, lung colonization, and growth of secondary lung metastases. However, breast cancer cell survival in the circulation uniquely required an intact p66ShcA S36 phosphorylation site. Conclusion: This study provides the first evidence that both mitochondrial and non-mitochondrial p66ShcA pools collaborate in breast cancer cells to promote their maximal metastatic fitness.