Long Prehensile Protrusions Can Facilitate Cancer Cell Invasion through the Basement Membrane.

A basic process in cancer is the breaching of basement-membrane barriers to permit tissue invasion. Cancer cells can use proteases and physical mechanisms to produce initial holes in basement membranes, but how cells squeeze through this barrier into matrix environments is not well understood. We used a 3D invasion model consisting of cancer-cell spheroids encapsulated by a basement membrane and embedded in collagen to characterize the dynamic early steps in cancer-cell invasion across this barrier. We demonstrate that certain cancer cells extend exceptionally long (~30-100 µm) protrusions through basement membranes via actin and microtubule cytoskeletal function. These long protrusions use integrin adhesion and myosin II-based contractility to pull cells through the basement membrane for initial invasion. Concurrently, these long, organelle-rich protrusions pull surrounding collagen inward while propelling cancer cells outward through perforations in the basement-membrane barrier. These exceptionally long, contractile cellular protrusions can facilitate the breaching of the basement-membrane barrier as a first step in cancer metastasis.

Maintenance of pluripotency-like signature in the entire ectoderm leads to neural crest stem cell potential.

The ability of the pluripotent epiblast to contribute progeny to all three germ layers is thought to be lost after gastrulation. The later-forming neural crest (NC) rises from ectoderm and it remains poorly understood how its exceptionally high stem-cell potential to generate mesodermal- and endodermal-like derivatives is obtained. Here, we monitor transcriptional changes from gastrulation to neurulation using single-cell-Multiplex-Spatial-Transcriptomics (scMST) complemented with RNA-sequencing. We show maintenance of pluripotency-like signature (Nanog, Oct4/PouV, Klf4-positive) in undecided pan-ectodermal stem-cells spanning the entire ectoderm late during neurulation with ectodermal patterning completed only at the end of neurulation when the pluripotency-like signature becomes restricted to NC, challenging our understanding of gastrulation. Furthermore, broad ectodermal pluripotency-like signature is found at multiple axial levels unrelated to the NC lineage the cells later commit to, suggesting a general role in stemness enhancement and proposing a mechanism by which the NC acquires its ability to form derivatives beyond "ectodermal-capacity" in chick and mouse embryos.

A ubiquitin-based effector-to-inhibitor switch coordinates early brain, craniofacial, and skin development.

The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and neural crest-derived craniofacial structures are unclear. Here, biochemical disease-variant profiling reveals a posttranslational pathway that drives early ectodermal differentiation in the vertebrate head. The anteriorly expressed ubiquitin ligase CRL3-KLHL4 restricts signaling of the ubiquitous cytoskeletal regulator CDC42. This regulation relies on the CDC42-activating complex GIT1-ßPIX, which CRL3-KLHL4 exploits as a substrate-specific co-adaptor to recognize and monoubiquitylate PAK1. Surprisingly, we find that ubiquitylation converts the canonical CDC42 effector PAK1 into a CDC42 inhibitor. Loss of CRL3-KLHL4 or a disease-associated KLHL4 variant reduce PAK1 ubiquitylation causing overactivation of CDC42 signaling and defective ectodermal patterning and neurulation. Thus, tissue-specific restriction of CDC42 signaling by a ubiquitin-based effector-to-inhibitor is essential for early face, brain, and skin formation, revealing how cell-fate and morphometric changes are coordinated to ensure faithful organ development.

Biglycan regulates bone development and regeneration.

Endochondral bone development and regeneration relies on activation and proliferation of periosteum derived-cells (PDCs). Biglycan (Bgn), a small proteoglycan found in extracellular matrix, is known to be expressed in bone and cartilage, however little is known about its influence during bone development. Here we link biglycan with osteoblast maturation starting during embryonic development that later affects bone integrity and strength. Biglycan gene deletion reduced the inflammatory response after fracture, leading to impaired periosteal expansion and callus formation. Using a novel 3D scaffold with PDCs, we found that biglycan could be important for the cartilage phase preceding bone formation. The absence of biglycan led to accelerated bone development with high levels of osteopontin, which appeared to be detrimental to the structural integrity of the bone. Collectively, our study identifies biglycan as an influencing factor in PDCs activation during bone development and bone regeneration after fracture.

Maintenance of pluripotency in the entire ectoderm enables neural crest formation.

The ability of the pluripotent epiblast to contribute progeny to all three germ layers is thought to be lost after gastrulation. The later-forming neural crest (NC) rises from ectoderm and it remains poorly understood how its exceptionally high stem-cell potential to generate mesodermal- and endodermal-like cells is obtained. We monitored transcriptional changes from gastrulation to neurulation using single-cell-Multiplex-Spatial-Transcriptomics (scMST) complemented with RNA-sequencing. Unexpectedly, we find maintenance of undecided Nanog/Oct4-PouV/Klf4-positive pluripotent-like pan-ectodermal stem-cells spanning the entire ectoderm late in the neurulation process with ectodermal patterning completed only at the end of neurulation when pluripotency becomes restricted to NC, challenging our understanding of gastrulation. Furthermore, broad ectodermal pluripotency is found at all axial levels unrelated to the NC lineage the cells later commit to, suggesting a general role in stemness enhancement and proposing a mechanism by which the NC acquires its ability to form derivatives beyond "ectodermal-capacity" in chick and mouse embryos.

Mechanisms of Basement Membrane Micro-Perforation during Cancer Cell Invasion into a 3D Collagen Gel.

Cancer invasion through basement membranes represents the initial step of tumor dissemination and metastasis. However, little is known about how human cancer cells breach basement membranes. Here, we used a three-dimensional in vitro invasion model consisting of cancer spheroids encapsulated by a basement membrane and embedded in 3D collagen gels to visualize the early events of cancer invasion by confocal microscopy and live-cell imaging. Human breast cancer cells generated large numbers of basement membrane perforations, or holes, of varying sizes that expanded over time during cell invasion. We used a wide variety of small molecule inhibitors to probe the mechanisms of basement membrane perforation and hole expansion. Protease inhibitor treatment (BB94), led to a 63% decrease in perforation size. After myosin II inhibition (blebbistatin), the basement membrane perforation area decreased by only 15%. These treatments produced correspondingly decreased cellular breaching events. Interestingly, inhibition of actin polymerization dramatically decreased basement membrane perforation by 80% and blocked invasion. Our findings suggest that human cancer cells can primarily use proteolysis and actin polymerization to perforate the BM and to expand perforations for basement membrane breaching with a relatively small contribution from myosin II contractility.

Cell-3D matrix interactions: recent advances and opportunities.

Tissues consist of cells and their surrounding extracellular matrix (ECM). Cell-ECM interactions play crucial roles in embryonic development, differentiation, tissue remodeling, and diseases including fibrosis and cancer. Recent research advances in characterizing cell-matrix interactions include detailed descriptions of hundreds of ECM and associated molecules, their complex intermolecular interactions in development and disease, identification of distinctive modes of cell migration in different 3D ECMs, and new insights into mechanisms of organ formation. Exploring the roles of the physical features of different ECM microenvironments and the bidirectional regulation of cell signaling and matrix organization emphasize the dynamic nature of these interactions, which can include feedback loops that exacerbate disease. Understanding mechanisms of cell-matrix interactions can potentially lead to targeted therapeutic interventions.

Visualization of trigeminal ganglion sensory neuronal signaling regulated by Cdk5.

The mechanisms underlying facial pain are still incompletely understood, posing major therapeutic challenges. Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase involved in pain signaling. However, the regulatory roles of Cdk5 in facial pain signaling and the possibility of therapeutic intervention at the level of mouse trigeminal ganglion primary neurons remain elusive. In this study, we use optimized intravital imaging to directly compare trigeminal neuronal activities after mechanical, thermal, and chemical stimulation. We then test whether facial inflammatory pain in mice could be alleviated by the Cdk5 inhibitor peptide TFP5. We demonstrate regulation of total Ca2+ intensity by Cdk5 activity using transgenic and knockout mouse models. In mice with vibrissal pad inflammation, application of TFP5 specifically decreases total Ca2+ intensity in response to noxious stimuli. It also alleviates inflammation-induced allodynia by inhibiting activation of trigeminal peripheral sensory neurons. Cdk5 inhibitors may provide promising non-opioid candidates for pain treatment.

Atto 465 Derivative Is a Nuclear Stain with Unique Excitation and Emission Spectra Useful for Multiplex Immunofluorescence Histochemistry.

Multiplex immunofluorescence (mIF) is an effective technique for the maximal visualization of multiple target proteins in situ. This powerful tool is mainly limited by the spectral overlap of the currently available synthetic fluorescent dyes. The fluorescence excitation wavelengths ranging between 405 and 488 nm are rarely used in mIF imaging and serve as a logical additional slot for a fluorescent probe. In the present study, we demonstrate that the addition of 2,3,4,5,6-pentafluoroaniline to Atto 465 NHS ester, creating Atto 465-pentafluoroaniline (Atto 465-p), generates a bright nuclear stain in the violet-blue region of the visible spectrum. This allows the 405 nm excitation and emission, classically used for nuclear counterstains, to be used for the detection of another target protein. This increases the flexibility of the mIF panel and, with appropriate staining and microscopy, enables the quantitative analysis of at least six targets in one tissue section. (J Histochem Cytochem XX: XXX-XXX, XXXX).

Cell-extracellular matrix dynamics.

The sites of interaction between a cell and its surrounding microenvironment serve as dynamic signaling hubs that regulate cellular adaptations during developmental processes, immune functions, wound healing, cell migration, cancer invasion and metastasis, as well as in many other disease states. For most cell types, these interactions are established by integrin receptors binding directly to extracellular matrix proteins, such as the numerous collagens or fibronectin. For the cell, these points of contact provide vital cues by sampling environmental conditions, both chemical and physical. The overall regulation of this dynamic interaction involves both extracellular and intracellular components and can be highly variable. In this review, we highlight recent advances and hypotheses about the mechanisms and regulation of cell-ECM interactions, from the molecular to the tissue level, with a particular focus on cell migration. We then explore how cancer cell invasion and metastasis are deeply rooted in altered regulation of this vital interaction.

Fibrin is a critical regulator of neutrophil effector function at the oral mucosal barrier.

Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we report that the clotting factor fibrin is a critical regulator of neutrophil function at the oral mucosal barrier. We demonstrate that commensal microbiota trigger extravascular fibrin deposition in the oral mucosa. Fibrin engages neutrophils through the αMß2 integrin receptor and activates effector functions, including the production of reactive oxygen species and neutrophil extracellular trap formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis in mice and humans. Concordantly, genetic polymorphisms in PLG, encoding plasminogen, are associated with common forms of periodontal disease. Thus, fibrin is a critical regulator of neutrophil effector function, and fibrin-neutrophil engagement may be a pathogenic instigator for a prevalent mucosal disease.

3D mesenchymal cell migration is driven by anterior cellular contraction that generates an extracellular matrix prestrain.

We describe a cellular contractile mechanism employed by fibroblasts and mesenchymal cancer cells to migrate in 3D collagen gels. During 3D spreading, fibroblasts strongly deform the matrix. They protrude, polarize, and initiate migration in the direction of highest extracellular matrix (ECM) deformation (prestrain). This prestrain is maintained through anterior cellular contractions behind the leading edge prior to protrusion, coordinating a distinct 3D migration cycle that varies between cell types. Myosin IIA is required for strain polarization, generating anterior contractions, and maintaining prestrain for efficient directional cell migration. Local matrix severing disrupts the matrix prestrain, suppressing directional protrusion. We show that epithelial cancer and endothelial cells rarely demonstrate the sustained prestrain or anterior contractions. We propose that mesenchymal cells sense ECM stiffness in 3D and generate their own matrix prestrain. This requires myosin IIA to generate polarized periodic anterior contractions for maintaining a 3D migration cycle.

Direct comparison of five different 3D extracellular matrix model systems for characterization of cancer cell migration.

BACKGROUND: Three-dimensional (3D) in vitro model systems can bridge the gap between regular two-dimensional cell culture and whole-animal studies. Analyses of cancer cell migration and invasion increasingly use differing 3D systems, which may produce conflicting findings. AIMS: We directly compared different 3D extracellular matrix systems for studying cancer cell migration/invasion by analyzing cell morphologies and quantifying aspects of cell migration including speed and directional persistence using automated computer-based cell tracking. METHODS AND RESULTS: We performed direct comparisons of five different 3D extracellular matrix cell culture systems using both HT1080 fibrosarcoma and MDA-MB-231 breast carcinoma cell lines. The reconstituted 3D systems included two types of collagen hydrogel and tissue matrix gel (TMG) vs cell-derived matrices extracted from cultured primary human or cancer-associated fibroblasts. The fibrillar matrix architecture of these systems differed. 3D rat tail collagen and TMG matrices had short, randomly oriented collagen fibrils; bovine collagen had long, larger fibril bundles; and the cell-derived matrices were strongly oriented. HT1080 cells displayed rounded morphologies in all three reconstituted 3D matrices but became spindle shaped in the two cell-derived matrices. MDA-MB-231 cell morphologies were elongated in all matrices. Quantitative measures of cell migration parameters differed markedly between the different types of 3D matrix. Comparing the reconstituted matrices, cells migrated the most rapidly and furthest in TMG. Comparing TMG with cell-derived matrices, cells migrated more efficiently in the cell-derived matrices. The most notable differences were in directional persistence of migration, which was greatest in the two cell-derived matrices. CONCLUSION: The morphologies of matrix fibrils and cell shape, and particularly the efficiency and directionality of cell migration, differed substantially depending on the type of 3D matrix system. We suggest that it is important to employ the 3D model system that most closely resembles the matrix environment being studied for analyses of cancer cell migration and invasion.

Basement Membrane Regulates Fibronectin Organization Using Sliding Focal Adhesions Driven by a Contractile Winch.

We have discovered that basement membrane and its major components can induce rapid, strikingly robust fibronectin organization. In this new matrix assembly mechanism, α5ß1 integrin-based focal adhesions slide actively on the underlying matrix toward the ventral cell center through the dynamic shortening of myosin IIA-associated actin stress fibers to drive rapid fibronectin fibrillogenesis distal to the adhesion. This mechanism contrasts with classical fibronectin assembly based on stable or fixed-position focal adhesions containing αVß3 integrins plus α5ß1 integrin translocation into proximal fibrillar adhesions. On basement membrane components, these sliding focal adhesions contain standard focal adhesion constituents but completely lack classical αVß3 integrins. Instead, peripheral α3ß1 or α2ß1 adhesions mediate initial cell attachment but over time are switched to α5ß1 integrin-based sliding focal adhesions to assemble fibronectin matrix. This basement-membrane-triggered mechanism produces rapid fibronectin fibrillogenesis, providing a mechanistic explanation for the well-known widespread accumulation of fibronectin at many organ basement membranes.

Tissue Architectural Cues Drive Organ Targeting of Tumor Cells in Zebrafish.

Tumor cells encounter a myriad of physical cues upon arrest and extravasation in capillary beds. Here, we examined the role of physical factors in non-random organ colonization using a zebrafish xenograft model. We observed a two-step process by which mammalian mammary tumor cells showed non-random organ colonization. Initial homing was driven by vessel architecture, where greater numbers of cells became arrested in the topographically disordered blood vessels of the caudal vascular plexus (CVP) than in the linear vessels in the brain. Following arrest, bone-marrow- and brain-tropic clones exhibited organ-specific patterns of extravasation. Extravasation was mediated by ß1 integrin, where knockdown of ß1 integrin reduced extravasation in the CVP but did not affect extravasation of a brain-tropic clone in the brain. In contrast, silencing myosin 1B redirected early colonization from the brain to the CVP. Our results suggest that organ selectivity is driven by both vessel topography and cell-type-dependent extravasation.

Extracellular matrix dynamics in cell migration, invasion and tissue morphogenesis.

This review describes how direct visualization of the dynamic interactions of cells with different extracellular matrix microenvironments can provide novel insights into complex biological processes. Recent studies have moved characterization of cell migration and invasion from classical 2D culture systems into 1D and 3D model systems, revealing multiple differences in mechanisms of cell adhesion, migration and signalling-even though cells in 3D can still display prominent focal adhesions. Myosin II restrains cell migration speed in 2D culture but is often essential for effective 3D migration. 3D cell migration modes can switch between lamellipodial, lobopodial and/or amoeboid depending on the local matrix environment. For example, "nuclear piston" migration can be switched off by local proteolysis, and proteolytic invadopodia can be induced by a high density of fibrillar matrix. Particularly, complex remodelling of both extracellular matrix and tissues occurs during morphogenesis. Extracellular matrix supports self-assembly of embryonic tissues, but it must also be locally actively remodelled. For example, surprisingly focal remodelling of the basement membrane occurs during branching morphogenesis-numerous tiny perforations generated by proteolysis and actomyosin contractility produce a microscopically porous, flexible basement membrane meshwork for tissue expansion. Cells extend highly active blebs or protrusions towards the surrounding mesenchyme through these perforations. Concurrently, the entire basement membrane undergoes translocation in a direction opposite to bud expansion. Underlying this slowly moving 2D basement membrane translocation are highly dynamic individual cell movements. We conclude this review by describing a variety of exciting research opportunities for discovering novel insights into cell-matrix interactions.

Myosin II governs intracellular pressure and traction by distinct tropomyosin-dependent mechanisms.

Two-dimensional (2D) substrate rigidity promotes myosin II activity to increase traction force in a process negatively regulated by tropomyosin (Tpm) 2.1. We recently discovered that actomyosin contractility can increase intracellular pressure and switch tumor cells from low-pressure lamellipodia to high-pressure lobopodial protrusions during three-dimensional (3D) migration. However, it remains unclear whether these myosin II-generated cellular forces are produced simultaneously, and by the same molecular machinery. Here we identify Tpm 1.6 as a positive regulator of intracellular pressure and confirm that Tpm 2.1 is a negative regulator of traction force. We find that Tpm 1.6 and 2.1 can control intracellular pressure and traction independently, suggesting these myosin II-dependent forces are generated by distinct mechanisms. Further, these tropomyosin-regulated mechanisms can be integrated to control complex cell behaviors on 2D and in 3D environments.

Durotaxis by Human Cancer Cells.

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.

Extracellular Protein Fibulin-7 and Its C-Terminal Fragment Have In Vivo Antiangiogenic Activity.

Angiogenesis is crucial for tissue development and homeostasis; however, excessive angiogenesis can lead to diseases, including arthritis and cancer metastasis. Some antiangiogenic drugs are available, but side effects remain problematic. Thus, alternative angiogenesis inhibition strategies are needed. Fibulin-7 (Fbln7) is a newly discovered member of the fibulin protein family, a group of cell-secreted glycoproteins, that functions as a cell adhesion molecule and interacts with other extracellular matrix (ECM) proteins as well as cell receptors. We previously showed that a recombinant C-terminal Fbln7 fragment (Fbln7-C) inhibits tube formation by human umbilical vein endothelial cells (HUVECs) in vitro. In the present study, we examined the in vivo antiangiogenic activity of recombinant full-length Fbln7 (Fbln7-FL) and Fbln7-C proteins using a rat corneal angiogenesis model. We found that both Fbln7-FL and Fbln7-C inhibited neovascularization. Fbln7-C bound to vascular endothelial growth factor receptor 2 (VEGFR2), inhibiting VEGFR2 and ERK phosphorylation and resulting in reduced HUVEC motility. HUVEC attachment to Fbln7-C occurred through an interaction with integrin α5ß1 and regulated changes in cellular morphology. These results suggest that Fbln7-C action may target neovascularization by altering cell/ECM associations. Therefore, Fbln7-C could have potential as a therapeutic agent for diseases associated with angiogenesis.