Wnt/PCP signaling mediates breast cancer metastasis by promoting pro-invasive protrusion formation in collectively motile leader cells

As evidence supporting essential roles for collective cell migration in carcinoma metastasis continues to accumulate, a better understanding of the underlying cellular and molecular mechanisms will be critical to translating these findings to the treatment of advanced cancers. Here we report that Wnt/PCP, a non-canonical Wnt signaling pathway, mediates breast cancer collective migration and metastasis. We observe that mammary gland-specific knockout of Vangl2, a tetraspanin-like scaffolding protein required for Wnt5a-induced signaling and motility in cultured breast cancer cell lines, results in a striking decrease in metastatic efficiency but not primary tumor growth in the MMTV-NDL transgenic mouse model of HER2-positive breast cancer. We also observe that expression levels of core Wnt/PCP components Wnt5a, Vangl1 and Vangl2 are selectively elevated in K14-positive leader cells relative to follower cells within a collectively migrating cohort, and that Vangl2 expression selectively promotes RhoA activation in leading edge cells. Moreover, Vangl expression drives collective migration in three-dimensional ex vivo tumor organoids, and Vangl protein specifically accumulates within pro-migratory filamentous actin-rich protrusions of leader cells. Together, our observations point to a model whereby Wnt/PCP upregulation facilitates breast tumor collective cell motility by selectively augmenting the formation pro-migratory protrusions within leader cells.

Biophysical and biochemical attributes of hybrid epithelial/mesenchymal phenotypes

The Epithelial-Mesenchymal Transition (EMT) is a biological phenomenon associated with explicit phenotypic and molecular changes in cellular traits. Unlike the earlier-held popular belief of it being a binary process, EMT is now thought of as a landscape including diverse hybrid E/M phenotypes manifested by varying degrees of the transition. These hybrid cells can co-express both epithelial and mesenchymal markers and/or functional traits, and can possess the property of collective cell migration, enhanced tumor-initiating ability, and immune/targeted therapy-evasive features, all of which are often associated with worse patient outcomes. These characteristics of the hybrid E/M cells have led to a surge in studies that map their biophysical and biochemical hallmarks that can be helpful in exploiting their therapeutic vulnerabilities. This review discusses recent advances made in investigating hybrid E/M phenotype(s) from diverse biophysical and biochemical aspects by integrating live cell-imaging, cellular morphology quantification and mathematical modelling, and highlights a set of questions that remain unanswered about the dynamics of hybrid E/M states.

Label-free Cell Tracking Enables Collective Motion Phenotyping in Epithelial Monolayers

Collective cell migration is an umbrella term for a rich variety of cell behaviors, whose distinct character is essential for biological function, notably for cancer metastasis. One essential feature of collective behavior is the motion of cells relative to their immediate neighbors. We introduce an AI-based pipeline to segment and track cell nuclei from phase contrast images. Nuclei segmentation is based on a U‐Net convolutional neural network trained on images with nucleus staining. Tracking, based on the Crocker-Grier algorithm, quantifies nuclei movement and allows for robust downstream analysis of collective motion. Since the AI algorithm required no new training data, our approach promises to be applicable to and yield new insights for vast libraries of existing collective motion images. In a systematic analysis of a cell line panel with oncogenic mutations, we find that the collective rearrangement metric, D2min, which reflects non-affine motion, shows promise as an indicator of metastatic potential.

PTEN inhibits AMPK to control collective migration

PTEN is one of the most frequently mutated tumor suppressor gene in cancer. PTEN is generally altered in invasive cancers such as glioblastomas, but its function in collective cell migration and invasion is not fully characterized. Herein, we report that the loss of PTEN increases cell speed during collective migration of non-tumourous cells both in vitro and in vivo. We further show that loss of PTEN promotes LKB1-dependent phosphorylation and activation of the major metabolic regulator AMPK. In turn AMPK increases VASP phosphorylation, reduces VASP localization at cell-cell junctions and decreases the interjunctional transverse actin arcs at the leading front, provoking a weakening of cell-cell contacts and increasing migration speed. Targeting AMPK activity not only slows down PTEN-depleted cells, it also limits PTEN-null glioblastoma cell invasion, opening new opportunities to treat glioblastoma lethal invasiveness.

The role of the integrated stress response in keratinocyte migration

Background and Hypothesis: Cutaneous wound healing involves: hemostatic, inflammatory, proliferative, and tissue remodeling phases. Re-epithelialization can be modeled in vitro using human keratinocytes and artificial wounds. Previous work showed undifferentiated keratinocytes closing wounds in vitro using individual cell migration (ICM), whilst differentiated keratinocytes utilize collective cell migration (KCCM). Therefore, we hypothesize that ICM in vitro is equivalent to keratinocyte migration during squamous cell carcinoma metastasis in vivo and KCCM is a model for wound re-epithelialization. Furthermore, we hypothesize that the integrated stress response (ISR) is important in ICM and KCCM. The ISR is activated by environmental stresses that protein kinases (GCN2 and PERK) can detect and phosphorylate translation factor, eIF2a. Our goal is to define how the ISR, specifically GCN2 and PERK, influence keratinocyte migration. Methods: We will evaluate in vitro wound healing and kinetic variation in protein expression and cytoskeleton remodeling. We will utilize four keratinocyte cell lines, control human keratinocyte NTERTs, and CRISPR-derived gene knockouts of GCN2, PERK, and ISR effector gene ATF4. Quantitative analysis of wound healing is accomplished using an IncuCyte ZOOM instrument. Protein expression is measured via immunoblots following high density wounding. Cytoskeletal analyses was done by immunofluorescence. Results: Preliminary results show PERK-KO and GCN2-KO cells have reduced expression of F-actin. Immunoblots showed actin-binding protein, phospho-cofilin, at lower levels in PERK-KO and GCN2-KO cells than in NTERT cells. Wound healing assays showed differentiated keratinocytes healing faster than undifferentiated in all cells, except GCN2-KO. GCN2-KO cells healed significantly slower than other differentiated cells and undifferentiated GCN2-KO cells. Wound healing assays showed undifferentiated PERK-KO cells healing slower than other undifferentiated cell lines. Conclusion/Potential Impact: The results indicate PERK and GCN2 could be key components in ICM and CCM respectfully. GCN2 and PERK could thus be potential therapeutic targets to provide cost-effective therapeutics to enhance/inhibit keratinocyte migration.

Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration

Collective cell migration is essential for embryonic development and homeostatic processes. During zebrafish development, the posterior lateral line primordium (pLLP) navigates along the embryo flank by collective cell migration. The chemokine receptors, Cxcr4b and Cxcr7b, as well as their cognate ligand, Cxcl12a, are essential for this process. We corroborate that knockdown of the zebrafish cd9 tetraspanin orthologue, cd9b, results in mild pLL abnormalities. Through generation of CRISPR and TALEN mutants, we show that cd9a and cd9b function partially redundantly in pLLP migration, which is delayed in the cd9b single and cd9a; cd9b double mutants. This delay led to a transient reduction in neuromast numbers. Loss of both Cd9a and Cd9b sensitized embryos to reduced Cxcr4b and Cxcl12a levels. Together these results provide evidence that Cd9 modulates collective cell migration of the pLLP during zebrafish development. One interpretation of these observations is that Cd9 contributes to more effective chemokine signalling.

Supracellular organization confers directionality and mechanical potency to migrating pairs of cardiopharyngeal progenitor cells

Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that multicellular arrangements confer biochemical and biomechanical properties contributing to tissue scale organization. The Ciona cardiopharyngeal progenitors provide the simplest model of collective cell migration, with cohesive bilateral cell pairs polarized along the leader-trailer migration path while moving between the ventral epidermis and trunk endoderm. We use the Cellular Potts Model to computationally probe the distributions of forces consistent with shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we identify cardiopharyngeal progenitors as the simplest cell collective maintaining supracellular polarity with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy to align collective polarity with the direction of migration, as observed with three or more cells in silico and in vivo. Our approach reveals emerging properties of the migrating collective: cell pairs are more persistent, migrating longer distances, and presumably with higher accuracy. Simulations suggest that cell pairs can overcome mechanical resistance of the trunk endoderm more effectively when they are polarized collectively. We propose that polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis.

α4/α9 Integrins Coordinate Epithelial Cell Migration Through Local Suppression of MAP Kinase Signaling Pathways

Adhesion of basal keratinocytes to the underlying extracellular matrix (ECM) plays a key role in the control of skin homeostasis and response to injury. Integrin receptors indirectly link the ECM to the cell cytoskeleton through large protein complexes called focal adhesions (FA). FA also function as intracellular biochemical signaling platforms to enable cells to respond to changing extracellular cues. The α4β1 and α9β1 integrins are both expressed in basal keratinocytes, share some common ECM ligands, and have been shown to promote wound healing in vitro and in vivo. However, their roles in maintaining epidermal homeostasis and relative contributions to pathological processes in the skin remain unclear. We found that α4β1 and α9β1 occupied distinct regions in monolayers of a basal keratinocyte cell line (NEB-1). During collective cell migration (CCM), α4 and α9 integrins co-localized along the leading edge. Pharmacological inhibition of α4β1 and α9β1 integrins increased keratinocyte proliferation and induced a dramatic change in cytoskeletal remodeling and FA rearrangement, detrimentally affecting CCM. Further analysis revealed that α4β1/α9β1 integrins suppress extracellular signal-regulated kinase (ERK1/2) activity to control migration through the regulation of downstream kinases including Mitogen and Stress Activated Kinase 1 (MSK1). This work demonstrates the roles of α4β1 and α9β1 in regulating migration in response to damage cues.

Spontaneous rotations in epithelia as an interplay between cell polarity and RhoA activity at boundaries.

Directed flows of cells in vivo are essential in morphogenesis. They shape living matter in phenomena involving cell mechanics and regulations of the acto-myosin cytoskeleton. However the onset of coherent motion during collective cell migration is still poorly understood. Here we show that coherence is set by spontaneous alignments of cell polarity by designing cellular rings of controlled dimensions. A tug-of-war between opposite polarities dictates the onset of coherence, as assessed by tracking live cellular shapes and motions in various experimental conditions. In addition, we identify an internally driven constraint by cellular acto-myosin cables at boundaries as essential to ensure coherence and active force is generated as evaluated by the high RhoA activity. Its contribution is required to trigger coherence as shown by our numerical simulations based on a novel Vicsek-type model including free active boundaries. Altogether, spontaneous coherent motion results from basic interplay between cell orientations and active cables at boundaries.