Tumor metastasis is a complex phenomenon that poses significant challenges to current cancer therapeutics. While the biochemical signaling involved in promoting motile phenotypes is well understood, the role of biomechanical interactions has recently begun to be incorporated into models of tumor cell migration. Specifically, we propose the unjamming transition, adapted from physical paradigms describing the behavior of granular materials, to better discern the transition toward an invasive phenotype. In this review, we introduce the jamming transition broadly and narrow our discussion to the different modes of 3D tumor cell migration that arise. Then we discuss the mechanical interactions between tumor cells and their neighbors, along with the interactions between tumor cells and the surrounding extracellular matrix. We center our discussion on the interactions that induce a motile state or unjamming transition in these contexts. By considering the interplay between biochemical and biomechanical signaling in tumor cell migration, we can advance our understanding of biomechanical control in cancer metastasis.

To recapitulate prostate cancer metastasis, DU145 cells were cultured in a hyaluronic acid-based, bio-orthogonally constructed, protease-degradable hydrogels. In the presence of a covalently conjugated integrin-binding peptide (GRGDSP), DU145 cells formed tumoroids and exhibited small protrusions. Upon addition of soluble transforming growth factor beta 1 (TGFβ1), cells underwent morphological changes to form extended interconnected cellular networks. Contrarily, in RGD-free hydrogels, cells maintained spherical structures even in the presence of TGFβ1. In RGD-conjugated hydrogels, TGFβ1 induced nuclear localization of SMAD2/3, upregulating a wide range of TGFβ1 target genes and proteins. Prolonged exposure to TGFβ1 led to matrix remodeling and induced epithelial-to-mesenchymal transition in DU145 cells, with loss of epithelial markers and gain of mesenchymal markers. A pharmacological inhibitor of TGFβRI/ALK5, SB-431542, attenuated TGFβ1-induced morphological changes, abrogated nuclear localization of SMAD2/3, and restored the expression of key epithelial markers. Our findings highlight the cooperative role of TGFβ1 signaling and integrin-binding peptide in the acquisition of an aggressive phenotype and the promotion of tumor progression.

Collective motion is a phenomenon observed across length scales in nature - from bacterial swarming and tissue migration to the flocking of animals. The mechanisms underlying this behavior vary significantly depending on the biological system, ranging from hydrodynamic and chemical interactions in bacteria to mechanical forces in epithelial tissues and social alignment in animal groups. While collective motion often arises from the coordinated activity of independently motile agents, this work explores a novel context: the emergence of collective motion in systems of non-motile active agents. Inspired by the oscillatory shape dynamics observed in suspended cells such as neutrophils and fibroblasts, we model active dumbbells exhibiting limit-cycle oscillations in shape as a minimal representation of such systems. Through computational simulations, we demonstrate that hydrodynamic interactions between these dumbbells lead to three key phenomena - a density-dependent transition from sessile to collective motion, hydrodynamics-induced phase separation, and synchronization of oscillatory shape changes. We have explored the role of hydrodynamic interactions on these emergent properties of sessile active dumbbells. These results underscore the critical role of hydrodynamic coupling in enabling and organizing collective behaviors in systems lacking intrinsic motility. This study lays the groundwork for future investigations into the emergent behavior of active matter and its implications for understanding cell motility, tissue dynamics, and the development of bio-inspired materials.

Metastasis, the leading cause of cancer-related deaths, involves a complex cascade of events, including extravasation. Despite extensive research into metastasis, the mechanisms underlying extravasation remain unclear. Molecular targeted therapies have advanced cancer treatment, yet their efficacy is limited, prompting exploration into novel therapeutic targets. Here, we showed the association of polyploidy in MDA-MB-231 breast cancer cells and their extravasation, using microfluidic systems to reproduce the in vivo microvascular environment. We observed enhanced extravasation in polyploid cells alongside upregulated expression of genes involved in cell-substrate adhesion and cell mechanical dynamics. These findings offer insights into the relationship between polyploidy and extravasation, highlighting potential targets for cancer therapy.

Glioblastomas are aggressive brain tumors for which effective therapy is still lacking, resulting in dismal survival rates. These tumors display significant phenotypic plasticity, harboring diverse cell populations ranging from tumor core cells to dispersed, highly invasive cells. Neuron navigator 3 (NAV3), a microtubule-associated protein affecting microtubule growth and dynamics, is downregulated in various cancers, including glioblastoma, and has thus been considered a tumor suppressor. In this study, we challenge this designation and unveil distinct expression patterns of NAV3 across different invasion phenotypes. Using glioblastoma cell lines and patient-derived glioma stem-like cell cultures, we disclose an upregulation of NAV3 in invading glioblastoma cells, contrasting with its lower expression in cells residing in tumor spheroid cores. Furthermore, we establish an association between low and high NAV3 expression and the amoeboid and mesenchymal invasive phenotype, respectively, and demonstrate that overexpression of NAV3 directly stimulates glioblastoma invasive behavior in both 2D and 3D environments. Consistently, we observed increased NAV3 expression in cells migrating along blood vessels in mouse xenografts. Overall, our results shed light on the role of NAV3 in glioblastoma invasion, providing insights into this lethal aspect of glioblastoma behavior.

Cells under high confinement form highly polarized hydrostatic pressure-driven, stable leader blebs that enable efficient migration in low adhesion, environments. Here we investigated the basis of the polarized bleb morphology of metastatic melanoma cells migrating in non-adhesive confinement. Using high-resolution time-lapse imaging and specific molecular perturbations, we found that EGF signaling via PI3K stabilizes and maintains a polarized leader bleb. Protein activity biosensors revealed a unique EGFR/PI3K activity gradient decreasing from rear-to-front, promoting PIP3 and Rac1-GTP accumulation at the bleb rear, with its antagonists PIP2 and RhoA-GTP concentrated at the bleb tip, opposite to the front-to-rear organization of these signaling modules in integrin-mediated mesenchymal migration. Optogenetic experiments showed that disrupting this gradient caused bleb retraction, underscoring the role of this signaling gradient in bleb stability. Mathematical modeling and experiments identified a mechanism where, as the bleb initiates, CD44 and ERM proteins restrict EGFR mobility in a membrane-apposed cortical actin meshwork in the bleb rear, establishing a rear-to-front EGFR-PI3K-Rac activity gradient. Thus, our study reveals the biophysical and molecular underpinnings of cell polarity in bleb-based migration of metastatic cells in non-adhesive confinement, and underscores how alternative spatial arrangements of migration signaling modules can mediate different migration modes according to the local microenvironment.

Neuroblastoma is the most common extra-cranial solid tumour in children. Over half of all high-risk cases are expected to succumb to the disease even after chemotherapy, surgery, and immunotherapy. Although the importance of MYCN amplification in this disease is indisputable, the mechanistic details remain enigmatic. Here, we present a multicellular model of neuroblastoma comprising a continuous automaton, discrete cell agents, and a centre-based mechanical model, as well as the simulation results we obtained with it. The continuous automaton represents the tumour microenvironment as a grid-like structure, where each voxel is associated with continuous variables such as the oxygen level therein. Each discrete cell agent is defined by several attributes, including its cell cycle position, mutations, gene expression pattern, and more with behaviours such as cell cycling and cell death being stochastically dependent on these attributes. The centre-based mechanical model represents the properties of these agents as physical objects, describing how they repel each other as soft spheres. By implementing a stochastic simulation algorithm on modern GPUs, we simulated the dynamics of over one million neuroblastoma cells over a period of months. Specifically, we set up 1200 heterogeneous tumours and tracked the MYCN-amplified clone's dynamics in each, revealed the conditions that favour its growth, and tested its responses to 5000 drug combinations. Our results are in agreement with those reported in the literature and add new insights into how the MYCN-amplified clone's reproductive advantage in a tumour, its gene expression profile, the tumour's other clones (with different mutations), and the tumour's microenvironment are inter-related. Based on the results, we formulated a hypothesis, which argues that there are two distinct populations of neuroblastoma cells in the tumour; the p53 protein is pro-survival in one and pro-apoptosis in the other. It follows that alternating between inhibiting MDM2 to restore p53 activity and inhibiting ARF to attenuate p53 activity is a promising, if unorthodox, therapeutic strategy. The multicellular model has the advantages of modularity, high resolution, and scalability, making it a potential foundation for creating digital twins of neuroblastoma patients.

Aberrant Ras homologous (Rho) GTPase signalling is a major driver of cancer metastasis, and GTPase-activating proteins (GAPs), the negative regulators of RhoGTPases, are considered promising targets for suppressing metastasis, yet drug discovery efforts have remained elusive. Here, we report the identification and characterization of adhibin, a synthetic allosteric inhibitor of RhoGAP class-IX myosins that abrogates ATPase and motor function, suppressing RhoGTPase-mediated modes of cancer cell metastasis. In human and murine adenocarcinoma and melanoma cell models, including three-dimensional spheroid cultures, we reveal anti-migratory and anti-adhesive properties of adhibin that originate from local disturbances in RhoA/ROCK-regulated signalling, affecting actin-dynamics and actomyosin-based cell-contractility. Adhibin blocks membrane protrusion formation, disturbs remodelling of cell-matrix adhesions, affects contractile ring formation, and disrupts epithelial junction stability; processes severely impairing single/collective cell migration and cytokinesis. Combined with the non-toxic, non-pathological signatures of adhibin validated in organoids, mouse and Drosophila models, this mechanism of action provides the basis for developing anti-metastatic cancer therapies.

Gastric cancer (GC) is an aggressive and heterogeneous malignancy marked by cellular and molecular diversity. In GC, cancer cells invade locally in the stomach at stage I and can progress to metastasis in distant organs by stage IV, where it often becomes fatal.

We analyzed gene expression profiles from 719 stage I and stage IV GC patients across seven public datasets, conducting functional enrichment analysis to identify a gene signature linked to disease progression. Additionally, we developed an in vitro model of a simplified extracellular matrix (ECM) for cell-based assays.

Our analysis identified a progression-associated gene signature (APOD, COL1A2, FSTL1, GEM, LUM, and SPARC) that characterizes stage IV GC. This signature is associated with ECM organization and epithelial-to-mesenchymal transition (EMT), both of which influence the tumor microenvironment by promoting cell invasion and triggering EMT.

This gene signature may help identify stage I GC patients at higher risk, offering potential utility in early-stage patient management. Furthermore, our experimental ECM model may serve as a platform for investigating molecular mechanisms underlying metastatic spread in gastric cancer.

Breast cancer (BC) is the leading cause of death among women, primarily due to its potential for metastasis. As BC progresses, the extracellular matrix (ECM) produces more type-I collagen, resulting in increased stiffness. This alteration influences cellular behaviors such as migration, invasion, and metastasis. Specifically, cancer cells undergo changes in gene expression that initially promote an epithelial-to-mesenchymal transition (EMT) and subsequently, a transition from a mesenchymal to an amoeboid (MAT) migration mode. In this way, cancer cells can migrate more easily through the stiffer microenvironment. Despite their importance, understanding MATs remains challenging due to the difficulty of replicating in vitro the conditions for cell migration that are observed in vivo.

To address this challenge, we developed a three-dimensional (3D) growth system that replicates the different matrix properties observed during the progression of a breast tumor. We used this model to study the migration and invasion of the Triple-Negative BC (TNBC) cell line MDA-MB-231, which is particularly subject to metastasis.

Our results indicate that denser collagen matrices present a reduction in porosity, collagen fiber size, and collagen fiber orientation, which are associated with the transition of cells to a rounder morphology with bleb-like protrusions. We quantified how this transition is associated with a more persistent migration, an enhanced invasion capacity, and a reduced secretion of matrix metalloproteinases.

Our findings suggest that the proposed 3D growth conditions (especially those with high collagen concentrations) mimic key features of MATs, providing a new platform to study the physiology of migratory transitions and their role in BC progression.

Microfluidic chips are valuable tools for studying intricate cellular and cell-microenvironment interactions. Traditional in vitro cancer models lack accuracy in mimicking the complexities of in vivo tumor microenvironment. However, cancer-metastasis-on-a-chip (CMoC) models combine the advantages of 3D cultures and microfluidic technology, serving as powerful platforms for exploring cancer mechanisms and facilitating drug screening. These chips are able to compartmentalize the metastatic cascade, deepening the understanding of its underlying mechanisms. This article provides an overview of current CMoC models, focusing on distinctive models that simulate invasion, intravasation, circulation, extravasation, and colonization, and their applications in drug screening. Furthermore, challenges faced by CMoC and microfluidic technologies are discussed, while exploring promising future directions in cancer research. The ongoing development and integration of these models into cancer studies are expected to drive transformative advancements in the field.

Clinical outcome for patients suffering from HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. This is mostly due to highly invasive tumors that cause loco-regional relapses after initial therapeutic intervention and metastatic outgrowth. The molecular pathways governing the detrimental invasive growth modes in HNSCC remain however understudied. Here, we have established HNSCC patient derived organoid (PDO) models that recapitulate 3-dimensional invasion in vitro. Single cell mRNA sequencing was applied to study the differences between non-invasive and invasive conditions, and in a collective versus single cell invading PDO model. Differential expression analysis under invasive conditions in Collagen gels reveals an overall upregulation of a YAP-centered transcriptional program, irrespective of the invasion mode. However, we find that collectively invading HNSCC PDO cells show elevated levels of YAP transcription targets when compared to single cell invasion. Also, collectively invading cells are characterized by increased nuclear translocation of YAP within the invasive strands, which coincides with Collagen-I matrix alignment at the invasive front. Using gene set enrichment analysis, we identify immune cell-like migratory pathways in the single cell invading HNSCC PDO, while collective invasion is characterized by overt upregulation of adhesion and migratory pathways. Lastly, based on clinical head and neck cancer cohorts, we demonstrate that the identified collective invasion signature provides a candidate prognostic platform for survival in HNSCC. By uncoupling collective and single cell invasive programs, we have established invasion signatures that may guide new therapeutic options.

Despite recent experimental progress in characterizing cell migration mechanics, our understanding of the mechanisms governing rapid cell movement remains limited. To effectively limit tumor growth, antitumoral T cells need to rapidly migrate to find and kill cancer cells. To investigate the upper limits of cell speed, we developed a new hybrid stochastic-mean field model of bleb-based cell motility. We first examined the potential for adhesion-free bleb-based migration and show that cells migrate inefficiently in the absence of adhesion-based forces, i.e., cell swimming. While no cortical contractility oscillations are needed for cells to swim in viscoelastic media, high-to-low cortical contractility oscillations are necessary for cell swimming in viscous media. This involves a high cortical contractility phase with multiple bleb nucleation events, followed by an intracellular pressure buildup recovery phase at low cortical tensions, resulting in modest net cell motion. However, our model suggests that cells can employ a hybrid bleb- and adhesion-based migration mechanism for rapid cell motility and identifies conditions for optimality. The model provides a momentum-conserving mechanism underlying rapid single-cell migration and identifies factors as design criteria for engineering T cell therapies to improve movement in mechanically complex environments.

Cell migration requires the interplay among diverse migration patterns. The molecular basis of distinct migration programs is undoubtedly vital but not fully explored. Meanwhile, the lack of tools for investigating spontaneous migratory plasticity in a single living cell also adds to the hindrance. Here, we developed a micro/nanotechnology-enabled single-cell analytical platform to achieve coherent monitoring of spontaneous migratory pattern and signaling molecules. Via the platform, we unveiled a previously unappreciated STAT3 regionalization on the multifunctional regulations of migration. Specifically, nuclear STAT3 is associated with amoeboid migration, while cytoplasmic STAT3 promotes mesenchymal movement. Opposing effects of JAK2 multisite phosphorylation shape its response to STAT3 distribution in a dynamic and antagonistic manner, eventually triggering a reversible amoeboid-mesenchymal transition. Based on the above results, bioinformatics further revealed a possible downstream regulator of nucleocytoplasmic STAT3. Thus, our platform, as an exciting technological advance in single-cell migration research, can provide in-depth mechanism interpretations of tumor metastasis and progression.

Metastasis, the leading cause of cancer-related deaths, involves a complex cascade of events, including extravasation. Despite extensive research into metastasis, the mechanisms underlying extravasation remain unclear. Molecular targeted therapies have advanced cancer treatment, yet their efficacy is limited, prompting exploration into novel therapeutic targets. Here, we showed the association of polyploidy in MDA-MB-231 breast cancer cells and their extravasation, using microfluidic systems to reproduce the in vivo microvascular environment. We observed enhanced extravasation in polyploid cells alongside upregulated expression of genes involved in cell-substrate adhesion and cell mechanical dynamics. These findings offer insights into the relationship between polyploidy and extravasation, highlighting potential targets for cancer therapy.

The need for improved prediction of clinical response is driving the development of cancer models with enhanced physiological relevance. A new concept of 'precision biomaterials' is emerging, encompassing patient-mimetic biomaterial models that seek to accurately detect, treat and model cancer by faithfully recapitulating key microenvironmental characteristics. Despite recent advances allowing tissue-mimetic stiffness and molecular composition to be replicated in vitro, approaches for reproducing the 3D fibre architectures found in tumour extracellular matrix (ECM) remain relatively unexplored. Although the precise influences of patient-specific fibre architecture are unclear, we summarize the known roles of tumour fibre architecture, underlining their implications in cell-matrix interactions and ultimately clinical outcome. We then explore the challenges in reproducing tissue-specific 3D fibre architecture(s) in vitro, highlighting relevant biomaterial fabrication techniques and their benefits and limitations. Finally, we discuss imaging and image analysis techniques (focussing on collagen I-optimized approaches) that could hold the key to mapping tumour-specific ECM into high-fidelity biomaterial models. We anticipate that an interdisciplinary approach, combining materials science, cancer research and image analysis, will elucidate the role of 3D fibre architecture in tumour development, leading to the next generation of patient-mimetic models for mechanistic studies and drug discovery.

Metastasis accounts for the vast majority of breast cancer-related fatalities. Although the contribution of genetic and epigenetic modifications to breast cancer progression has been widely acknowledged, emerging evidence underscores the pivotal role of physical stimuli in driving breast cancer metastasis. In this review, we summarize the changes in the mechanics of the breast cancer microenvironment and describe the various forces that impact migrating and circulating tumor cells throughout the metastatic process. We also discuss the mechanosensing and mechanotransducing molecules responsible for promoting the malignant phenotype in breast cancer cells. Gaining a comprehensive understanding of the mechanobiology of breast cancer carries substantial potential to propel progress in prognosis, diagnosis, and patient treatment.

Deregulation of the relaxin family peptide system (RFPS) appears to increase the risk of range of cancers, including epithelial ovarian cancers (EOC). The present study examines the effect of relaxin family peptide receptor 1 (RXFP1) level on the biological properties of human epithelial ovarian adenocarcinoma cells (OVCAR4 and SKOV3). RXFP1 was downregulated (RXFP1↓) in the cells using the RXFP1 sgRNA CRISPR All-in-One Lentivirus set (pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro), and upregulated (RXFP1↑) using the RXFP1 CRISPRa sgRNA Lentivector (pLenti-U6-sgRNA-PGK-Neo) kit, which activates the RXFP1 gene when paired with dCas9-SAM. The changes taking place during adhesion to extracellular matrix (ECM) proteins were assessed in multi-well plates coated with collagen, fibronectin, laminin and gelatin. Cellular viability was monitored based on mitochondrial metabolic activity (MTT Assay, Alamar Blue Assay) and adenosine triphosphate production (ATP Assay). The rate of cell proliferation was determined based on the percentage of Ki67 immunoreactive cells and the numbers of cells in particular cell-cycle phases. The mesenchymal-like (Boyden Chamber Assay) and amoeboid-like movements (Wound Healing Assay) of ovarian cancer cells were also analyzed after transfection. RXFP1 downregulation decreased the adhesion properties of ovarian cancer cells and increased the tendency for apoptosis under stressful conditions. In contrast, RXFP1 upregulation had pro-proliferative, pro-survival and promigratory effects. Our findings confirm that the relaxin-2/RXFP1 signaling pathway plays a role in the promotion of growth and progression of ovarian cancer.

Prickle is an evolutionarily conserved family of proteins exclusively associated with planar cell polarity (PCP) signalling. This signalling pathway provides directional and positional cues to eukaryotic cells along the plane of an epithelial sheet, orthogonal to both apicobasal and left-right axes. Through studies in the fruit fly Drosophila, we have learned that PCP signalling is manifested by the spatial segregation of two protein complexes, namely Prickle/Vangl and Frizzled/Dishevelled. While Vangl, Frizzled, and Dishevelled proteins have been extensively studied, Prickle has been largely neglected. This is likely because its role in vertebrate development and pathologies is still being explored and is not yet fully understood. The current review aims to address this gap by summarizing our current knowledge on vertebrate Prickle proteins and to cover their broad versatility. Accumulating evidence suggests that Prickle is involved in many developmental events, contributes to homeostasis, and can cause diseases when its expression and signalling properties are deregulated. This review highlights the importance of Prickle in vertebrate development, discusses the implications of Prickle-dependent signalling in pathology, and points out the blind spots or potential links regarding Prickle, which could be studied further.

Many cells adhere to extracellular matrix for efficient cell migration. This adhesion is mediated by focal adhesions, a protein complex linking the extracellular matrix to the intracellular cytoskeleton. Focal adhesions have been studied extensively in mesenchymal cells, but recent research in physiological contexts and amoeboid cells suggest focal adhesion regulation differs from the mesenchymal focal adhesion paradigm. We used Dictyostelium discoideum to uncover new mechanisms of focal adhesion regulation, as Dictyostelium are amoeboid cells that form focal adhesion-like structures for migration. We show that PaxillinB, the Dictyostelium homologue of Paxillin, localizes to dynamic focal adhesion-like structures during Dictyostelium migration. Unexpectedly, reduced PaxillinB recruitment to these structures increases Dictyostelium cell migration. Quantitative analysis of focal adhesion size and dynamics show that lack of PaxillinB recruitment to focal adhesions does not alter focal adhesion size, but rather increases focal adhesion turnover. These findings are in direct contrast to Paxillin function at focal adhesions during mesenchymal migration, challenging the established focal adhesion model.

Cancer is still one of the leading causes of death, with an estimated 19.3 million new cases every year. Our paper presents the tumor-suppressing effect of Taenia crassiceps and Mesocestoides corti on B16F10 melanoma, the intraperitoneal application of which followed the experimental infection with these tapeworms, resulting in varying degrees of effectiveness in two strains of mice. In the case of M. corti-infected ICR mice, a strong tumor growth suppression occurred, which was accompanied by a significant reduction in the formation of distant metastases in the liver and lung. Tapeworm-infected C57BL/6J mice also showed a suppression of tumor growth and, in addition, the overall survival of infected C57BL/6J mice was significantly improved. Experiments with potential cross-reaction of melanoma and tapeworm antigens with respective specific antibodies, restimulation of spleen T cells, or the direct effect of tapeworm excretory-secretory products on melanoma cells in vitro could not explain the phenomenon. However, infections with T. crassiceps and M. corti increased the number of leukocytes possibly involved in anti-tumor immunity in the peritoneal cavity of both ICR and C57BL/6J mice. This study unveils the complex interplay between tapeworm infections, immune responses, and melanoma progression, emphasizing the need for further exploration of the mechanisms driving observed tumor-suppressive effects.

The ability of CD8+ T cells to infiltrate solid tumors and reach cancer cells is associated with improved patient survival and responses to immunotherapy. Thus, identifying the factors controlling T cell migration in tumors is critical, so that strategies to intervene on these targets can be developed. Although interstitial motility is a highly energy-demanding process, the metabolic requirements of CD8+ T cells migrating in a 3D environment remain unclear. Here, we demonstrate that the tricarboxylic acid (TCA) cycle is the main metabolic pathway sustaining human CD8+ T cell motility in 3D collagen gels and tumor slices while glycolysis plays a more minor role. Using pharmacological and genetic approaches, we report that CD8+ T cell migration depends on the mitochondrial oxidation of glucose and glutamine, but not fatty acids, and both ATP and ROS produced by mitochondria are required for T cells to migrate. Pharmacological interventions to increase mitochondrial activity improve CD8+ T cell intratumoral migration and CAR T cell recruitment into tumor islets leading to better control of tumor growth in human xenograft models. Our study highlights the rationale of targeting mitochondrial metabolism to enhance the migration and antitumor efficacy of CAR T cells in treating solid tumors.

Epithelial Mesenchymal Transition (EMT) is a dedifferentiation process implicated in many physio-pathological conditions including tumor transformation. EMT is regulated by several extracellular mediators and under certain conditions it can be reversible. Autophagy is a conserved catabolic process in which intracellular components such as protein/DNA aggregates and abnormal organelles are degraded in specific lysosomes. In cancer, autophagy plays a controversial role, acting in different conditions as both a tumor suppressor and a tumor-promoting mechanism. Experimental evidence shows that deep interrelations exist between EMT and autophagy-related pathways. Although this interplay has already been analyzed in previous studies, understanding mechanisms and the translational implications of autophagy/EMT need further study. The role of autophagy in EMT is not limited to morphological changes, but activation of autophagy could be important to DNA repair/damage system, cell adhesion molecules, and cell proliferation and differentiation processes. Based on this, both autophagy and EMT and related pathways are now considered as targets for cancer therapy. In this review article, the contribution of autophagy to EMT and progression of cancer is discussed. This article also describes the multiple connections between EMT and autophagy and their implication in cancer treatment.

Cell migration is a highly coordinated cellular event that determines diverse physiological and pathological processes in which the continuous interaction of a migrating cell with neighboring cells or the extracellular matrix is regulated by the physical setting of the extracellular microenvironment. In confined spaces, cell migration occurs differently compared to unconfined open spaces owing to the additional forces that limit cell motility, which create a driving bias for cells to invade the confined space, resulting in a distinct cell motility process compared to what is expected in open spaces. Moreover, cells in confined environments can be subjected to elevated mechanical compression, which causes physical stimuli and activates the damage repair cycle in the cell, including the DNA in the nucleus. Although cells have a self-restoring system to repair damage from the cell membrane to the genetic components of the nucleus, this process may result in genetic and/or epigenetic alterations that can increase the risk of the progression of diverse diseases, such as cancer and immune disorders. Furthermore, there has been a shift in the paradigm of bioengineering from the development of new biomaterials to controlling biophysical cues and fine-tuning cell behaviors to cure damaged/diseased tissues. The external physical cues perceived by cells are transduced along the mechanosensitive machinery, which is further channeled into the nucleus through subcellular molecular linkages of the nucleoskeleton and cytoskeleton or the biochemical translocation of transcription factors. Thus, external cues can directly or indirectly regulate genetic transcriptional processes and nuclear mechanics, ultimately determining cell fate. In this review, we discuss the importance of the biophysical cues, response mechanisms, and mechanical models of cell migration in confined environments. We also discuss the effect of force-dependent deformation of subcellular components, specifically focusing on subnuclear organelles, such as nuclear membranes and chromosomal organization. This review will provide a biophysical perspective on cancer progression and metastasis as well as abnormal cellular proliferation.

The TGFβ family member NODAL, repeatedly required during embryonic development, has also been associated with tumour progression. Our aim was to clarify the controversy surrounding its involvement in melanoma tumour progression. We found that the deletion of the NODAL exon 2 in a metastatic melanoma cell line impairs its ability to form tumours and colonize distant tissues. However, we show that this phenotype does not result from the absence of NODAL, but from a defect in the expression of a natural antisense transcript of NODAL, here called LADON. We show that LADON expression is specifically activated in metastatic melanoma cell lines, that its transcript is packaged in exosomes secreted by melanoma cells, and that, via its differential impact on the expression of oncogenes and tumour suppressors, it promotes the mesenchymal to amoeboid transition that is critical for melanoma cell invasiveness. LADON is, therefore, a new player in the regulatory network governing tumour progression in melanoma and possibly in other types of cancer.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, are able to invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis and here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the β1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. Spatially, sEV were secreted via filopodia-like cellular protrusions at the leading edge of migrating cells. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI. In vitro assays identified collagen VI as playing the key role in cell adhesion and invasion. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate migration and invasion by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction forces to enable VSMC movement within the complex vascular ECM network.

Solid cancer cells escape the primary tumor mass by transitioning from an epithelial-like state to an invasive migratory state. As they escape, metastatic cancer cells employ interchangeable modes of invasion, transitioning between fibroblast-like mesenchymal movement to amoeboid migration, where cells display a rounded morphology and navigate the extracellular matrix in a protease-independent manner. However, the gene transcripts that orchestrate the switch between epithelial, mesenchymal, and amoeboid states remain incompletely mapped, mainly due to a lack of methodologies that allow the direct comparison of the transcriptomes of spontaneously invasive cancer cells in distinct migratory states. Here, we report a novel single-cell isolation technique that provides detailed three-dimensional data on melanoma growth and invasion, and enables the isolation of live, spontaneously invasive cancer cells with distinct morphologies and invasion parameters. Via the expression of a photoconvertible fluorescent protein, compact epithelial-like cells at the periphery of a melanoma mass, elongated cells in the process of leaving the mass, and rounded amoeboid cells invading away from the mass were tagged, isolated, and subjected to single-cell RNA sequencing. A total of 462 differentially expressed genes were identified, from which two candidate proteins were selected for further pharmacologic perturbation, yielding striking effects on tumor escape and invasion, in line with the predictions from the transcriptomics data. This work describes a novel, adaptable, and readily implementable method for the analysis of the earliest phases of tumor escape and metastasis, and its application to the identification of genes underpinning the invasiveness of malignant melanoma.

This work describes a readily implementable method that allows for the isolation of individual live tumor cells of interest for downstream analyses, and provides the single-cell transcriptomes of melanoma cells at distinct invasive states, both of which open avenues for in-depth investigations into the transcriptional regulation of the earliest phases of metastasis.

Cancer incidence is increasing annually, and the invasion of cancer into the stroma significantly affects cancer metastasis. The stroma primarily comprises an abundant extracellular matrix (ECM) that interacts closely with cancer cells. Cancer cells use the ECM as a scaffold to migrate from a tumor via mechanical actions such as pushing and pulling the fibers. The purpose of this study is to clarify the effects of elastic modulus differences on cell migration behavior based on the same ECM fiber structure. We observe temporal changes in the morphology of cancer cells and the surrounding ECM to elucidate the relationship between changes in the mechanical properties of the ECM and the invasive behavior of cancer cells. We analyze the shape and migration distance of cancer cells and the displacement field of the ECM by varying the fiber elastic modulus but fixing the ECM density. Increasing the elastic modulus results in a protruding cell shape, which indicates the maximum displacement of the ECM around the cell. Additionally, differences in cell migration speed and dispersion based on the elastic modulus are observed. The behavior of cells with increasing elasticity is classified via cluster analysis. Owing to the chemical cross-linking of the fibers, some cells cannot deform the surrounding tissue. This is attributable to the gel state of the ECM and microscopic fluctuations in the fiber density around the cells. We successfully assessed the effect of changes in the ECM modulus on cell mortality and morphology to reveal the mechanism of cancer invasion.

The peritumoral stroma is a complex 3D tissue that provides cells with myriad biophysical and biochemical cues. Histologic observations suggest that during metastatic spread of carcinomas, these cues influence transformed epithelial cells, prompting a diversity of migration modes spanning single cell and multicellular phenotypes. Purported consequences of these variations in tumor escape strategies include differential metastatic capability and therapy resistance. Therefore, understanding how cues from the peritumoral stromal microenvironment regulate migration mode has both prognostic and therapeutic value. Here, we utilize a synthetic stromal mimetic in which matrix fiber density and bulk hydrogel mechanics can be orthogonally tuned to investigate the contribution of these two key matrix attributes on MCF10A migration mode phenotypes, epithelial-mesenchymal transition (EMT), and invasive potential. We develop an automated computational image analysis framework to extract migratory phenotypes from fluorescent images and determine 3D migration metrics relevant to metastatic spread. Using this analysis, we find that matrix fiber density and bulk hydrogel mechanics distinctly contribute to a variety of MCF10A migration modes including amoeboid, single mesenchymal, clusters, and strands. We identify combinations of physical and soluble cues that induce a variety of migration modes originating from the same MCF10A spheroid and use these settings to examine a functional consequence of migration mode -resistance to apoptosis. We find that cells migrating as strands are more resistant to staurosporine-induced apoptosis than either disconnected clusters or individual invading cells. Improved models of the peritumoral stromal microenvironment and understanding of the relationships between matrix attributes and cell migration mode can aid ongoing efforts to identify effective cancer therapeutics that address cell plasticity-based therapy resistances. STATEMENT OF SIGNIFICANCE: Stromal extracellular matrix structure dictates both cell homeostasis and activation towards migratory phenotypes. However decoupling the effects of myriad biophysical cues has been difficult to achieve. Here, we encapsulate electrospun fiber segments within an amorphous hydrogel to create a fiber-reinforced hydrogel composite in which fiber density and hydrogel stiffness can be orthogonally tuned. Quantification of 3D cell migration reveal these two parameters uniquely contribute to a diversity of migration phenotypes spanning amoeboid, single mesenchymal, multicellular cluster, and collective strand. By tuning biophysical and biochemical cues to elicit heterogeneous migration phenotypes, we find that collective strands best resist apoptosis. This work establishes a composite approach to modulate fibrous topography and bulk hydrogel mechanics and identified biomaterial parameters to direct distinct 3D cell migration phenotypes.

Solid tumor metastases cause most cancer-related deaths. The prevention of their occurrence misses suitable anti-metastases medicines newly labeled as migrastatics. The first indication of migrastatics potential is based on an inhibition of in vitro enhanced migration of tumor cell lines. Therefore, we decided to develop a rapid test for qualifying the expected migrastatic potential of some drugs for repurposing. The chosen Q-PHASE holographic microscope provides reliable multifield time-lapse recording and simultaneous analysis of the cell morphology, migration, and growth. The results of the pilot assessment of the migrastatic potential exerted by the chosen medicines on selected cell lines are presented.

The invasion of nerves by cancer cells, or perineural invasion (PNI), is potentiated by the nerve microenvironment and is associated with adverse clinical outcomes. However, the cancer cell characteristics that enable PNI are poorly defined. Here, we generated cell lines enriched for a rapid neuroinvasive phenotype by serially passaging pancreatic cancer cells in a murine sciatic nerve model of PNI. Cancer cells isolated from the leading edge of nerve invasion showed a progressively increasing nerve invasion velocity with higher passage number. Transcriptome analysis revealed an upregulation of proteins involving the plasma membrane, cell leading edge, and cell movement in the leading neuroinvasive cells. Leading cells progressively became round and blebbed, lost focal adhesions and filipodia, and transitioned from a mesenchymal to amoeboid phenotype. Leading cells acquired an increased ability to migrate through microchannel constrictions and associated more with dorsal root ganglia than nonleading cells. ROCK inhibition reverted leading cells from an amoeboid to mesenchymal phenotype, reduced migration through microchannel constrictions, reduced neurite association, and reduced PNI in a murine sciatic nerve model. Cancer cells with rapid PNI exhibit an amoeboid phenotype, highlighting the plasticity of cancer migration mode in enabling rapid nerve invasion.

Malignant melanoma is the most lethal form of skin cancer. Y-box binding protein 1 (YB-1) plays a prominent role in mediating metastatic behavior by promoting epithelial-to-mesenchymal transition (EMT). Migratory melanoma cells exhibit two major migration modes: elongated mesenchymal or rounded amoeboid. Using A375 melanoma cell line and the YB-1 knock-out model, we aimed to elucidate biochemical and biomechanical changes in migration signaling pathways in the context of melanoma metastases. We subjected A375 YB-1 knock-out and parental cells to atomic force microscopy (stiffness determination), immunolabelling, and proteome analysis. We found that YB-1 expressing cells were significantly stiffer compared to the corresponding YB-1 knock-out cell line. Our study demonstrated that the constitutive expression of YB-1 in A375 melanoma cell line appears to be closely related to known biomarkers of epithelial-to-mesenchymal transition, nestin, and vimentin, resulting in a stiffer phenotype, as well as a wide array of proteins involved in RNA, ribosomes, and spliceosomes. YB-1 knock-out resulted in nestin depletion and significantly lower vimentin expression, as well as global upregulation of proteins related to the cytoskeleton and migration. YB-1 knock-out cells demonstrated both morphological features and biochemical drivers of mesenchymal/ameboid migration. Melanoma is a highly plastic, adaptable, and aggressive tumor entity, capable of exhibiting characteristics of different migratory modes.

The ability of cells to switch between different invasive modes during metastasis, also known as invasion plasticity, is an important characteristic of tumor cells that makes them able to resist treatment targeted to a particular invasion mode. Due to the rapid changes in cell morphology during the transition between mesenchymal and amoeboid invasion, it is evident that this process requires remodeling of the cytoskeleton. Although the role of the actin cytoskeleton in cell invasion and plasticity is already quite well described, the contribution of microtubules is not yet fully clarified. It is not easy to infer whether destabilization of microtubules leads to higher invasiveness or the opposite since the complex microtubular network acts differently in diverse invasive modes. While mesenchymal migration typically requires microtubules at the leading edge of migrating cells to stabilize protrusions and form adhesive structures, amoeboid invasion is possible even in the absence of long, stable microtubules, albeit there are also cases of amoeboid cells where microtubules contribute to effective migration. Moreover, complex crosstalk of microtubules with other cytoskeletal networks participates in invasion regulation. Altogether, microtubules play an important role in tumor cell plasticity and can be therefore targeted to affect not only cell proliferation but also invasive properties of migrating cells.

In the past decades studies on anti-tumoral drugs inhibiting matrix metalloproteinase (MMPs) were disappointing. Recently, we demonstrated that mature endothelial cells (ECs) and endothelial colony forming cells (ECFCs) can switch between invasion modes to cope with challenging environments, performing the "amoeboid angiogenesis" in the absence of proteases activity.

We first set out to investigate by ELISA if the inhibitors of the main protease family involved in angiogenesis were differently expressed during breast cancer progression. We used Marimastat, a broad-spectrum MMP inhibitor, as a means of inducing amoeboid characteristics and studied VEGF role in amoeboid angiogenesis. Thus, we performed invasion and capillary morphogenesis assay, morphological, cell signaling and in vivo mouse studies.

Our data showed that TIMP1, TIMP2, alpha2-antiplasmin, PAI-1 and cystatin increase in breast cancer serum of patients with primary cancer and lymph node positive compared to healthy women. In vitro results revealed that the most high-powered protease inhibitors able to induce amoeboid invasion of ECFCs were TIMP1, 2 and 3. Surprisingly, Marimastat promotes ECFC invasion and tubular formation in vitro and in vivo, inducing amoeboid characteristics. We observed that the combination of Marimastat plus VEGF doesn't boost neither cell invasion nor vessel formation capacity. Moreover, inhibition of VEGF activity with Bevacizumab in the presence of Marimastat confirmed that amoeboid angiogenesis is independent from the stimulus of the main vascular growth factor, VEGF.

We underline the importance to consider the amoeboid mechanism of endothelial and cancer cell invasion, probably responsible for the failure of synthetic metalloproteinase inhibitors as cancer therapy and tumor resistance to VEGF-targeted therapies, to set-up new drugs to be used in cancer therapy.

Cell migration is essential in numerous living processes, including embryonic development, wound healing, immune responses, and cancer metastasis. From individual cells to collectively migrating epithelial sheets, the locomotion of cells is tightly regulated by multiple structural, chemical, and biological factors. However, the high complexity of this process limits the understanding of the influence of each factor. Recent advances in materials science, tissue engineering, and microtechnology have expanded the toolbox and allowed the development of biomimetic in vitro assays to investigate the mechanisms of cell migration. Particularly, three-dimensional (3D) hydrogels have demonstrated a superior ability to mimic the extracellular environment. They are therefore well suited to studying cell migration in a physiologically relevant and more straightforward manner than in vivo approaches. A myriad of synthetic and naturally derived hydrogels with heterogeneous characteristics and functional properties have been reported. The extensive portfolio of available hydrogels with different mechanical and biological properties can trigger distinct biological responses in cells affecting their locomotion dynamics in 3D. Herein, we describe the most relevant hydrogels and their associated physico-chemical characteristics typically employed to study cell migration, including established cell migration assays and tracking methods. We aim to give the reader insight into existing literature and practical details necessary for performing cell migration studies in 3D environments.

The tumor microenvironment consists of tumor cells, extracellular matrix, blood vessels, and non-tumor cells such as fibroblasts and immune cells. Crosstalk among components of this cellular ecosystem can transform non-malignant cells and promote tumor invasion and metastasis. Evidence is accumulating that the transcription factor STAT2, a downstream effector of type I interferon (IFN-I) signaling, can either inhibit or promote tumorigenesis depending on the unique environment presented by each type of cancer. STAT2 has long been associated with the canonical JAK/STAT pathway involved in various biological processes including reshaping of the tumor microenvironment and in antitumor immunity. This dichotomous tendency of STAT2 to both inhibit and worsen tumor formation makes the protein a curious, and yet relatively ill-defined player in many cancer pathways involving IFN-I. In this review, we discuss the role of STAT2 in contributing to either a tumorigenic or anti-tumorigenic microenvironment as well as chemoresistance.

Migration of single adherent cells is frequently observed in the developing and adult organism and has been the subject of many studies. Yet, while elegant work has elucidated molecular and mechanical cues affecting motion dynamics on a flat surface, it remains less clear how cells migrate in a 3D setting. In this review, we explore the changing parameters encountered by cells navigating through a 3D microenvironment compared to cells crawling on top of a 2D surface, and how these differences alter subcellular structures required for propulsion. We further discuss how such changes at the micro-scale impact motion pattern at the macro-scale.

Class I Myosins are a subfamily of motor proteins with ATPase activity and a characteristic structure conserved in all myosins: A N-Terminal Motor Domain, a central Neck and a C terminal Tail domain. Humans have eight genes for these myosins. Class I Myosins have different functions: regulate membrane tension, participate in endocytosis, exocytosis, intracellular trafficking and cell migration. Cell migration is influenced by many cellular components including motor proteins, like myosins. Recently has been reported that changes in myosin expression have an impact on the migration of cancer cells, the formation of infiltrates and metastasis. We propose that class I myosins might be potential markers for future diagnostic, prognostic or even as therapeutic targets in leukemia and other cancers.Abbreviations: Myo1g: Myosin 1g; ALL: Acute Lymphoblastic Leukemia, TH1: Tail Homology 1; TH2: Tail Homology 2; TH3: Tail Homology 3.

SALL2/Sall2 is a transcription factor associated with development, neuronal differentiation, and cancer. Interestingly, SALL2/Sall2 deficiency leads to failure of the optic fissure closure and neurite outgrowth, suggesting a positive role for SALL2/Sall2 in cell migration. However, in some cancer cells, SALL2 deficiency is associated with increased cell migration. To further investigate the role of Sall2 in the cell migration process, we used immortalized Sall2 knockout (Sall2 -/- ) and Sall2 wild-type (Sall2 +/+ ) mouse embryonic fibroblasts (iMEFs). Our results indicated that Sall2 positively regulates cell migration, promoting cell detachment and focal adhesions turnover. Sall2 deficiency decreased cell motility and altered focal adhesion dynamics. Accordingly, restoring Sall2 expression in the Sall2 -/- iMEFs by using a doxycycline-inducible Tet-On system recovered cell migratory capabilities and focal adhesion dynamics. In addition, Sall2 promoted the autophosphorylation of Focal Adhesion Kinase (FAK) at Y397 and increased integrin β1 mRNA and its protein expression at the cell surface. We demonstrated that SALL2 increases ITGB1 promoter activity and binds to conserved SALL2-binding sites at the proximal region of the ITGB1 promoter, validated by ChIP experiments. Furthermore, the overexpression of integrin β1 or its blockade generates a cell migration phenotype similar to that of Sall2 +/+ or Sall2 -/- cells, respectively. Altogether, our data showed that Sall2 promotes cell migration by modulating focal adhesion dynamics, and this phenotype is associated with SALL2/Sall2-transcriptional regulation of integrin β1 expression and FAK autophosphorylation. Since deregulation of cell migration promotes congenital abnormalities, tumor formation, and spread to other tissues, our findings suggest that the SALL2/Sall2-integrin β1 axis could be relevant for those processes.