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Cell invasion into the surrounding extracellular matrix or across tissue boundaries and endothelial barriers occurs in both physiological and pathological scenarios such as immune surveillance or cancer metastasis. Podosomes and invadopodia, collectively called ‘invadosomes’, are actin-based structures that drive the proteolytic invasion of cells, by forming highly regulated platforms for the localized release of lytic enzymes that degrade the matrix. Recent advances in high-resolution microscopy techniques, in vivo imaging and high-throughput analyses have led to considerable progress in understanding mechanisms of invadosomes, revealing the intricate inner architecture of these structures, as well as their growing repertoire of functions that extends well beyond matrix degradation. In this Review, we discuss the known functions, architecture and regulatory mechanisms of podosomes and invadopodia. In particular, we describe the molecular mechanisms of localized actin turnover and microtubule-based cargo delivery, with a special focus on matrix-lytic enzymes that enable proteolytic invasion. Finally, we point out topics that should become important in the invadosome field in the future.Podosomes and invadopodia, collectively called ‘invadosomes’, are actin-based structures that drive proteolytic invasion in various physiologically relevant cell types (including osteoclasts, immune cells, endothelial cells and fibroblasts) and in cancer cells. Recent work has expanded our understanding of the architecture and mechanisms of invadosomes, and has revealed their diverse functions beyond matrix degradation.

Metastases are initiated by disseminated tumor cells (DTCs) that colonize distant organs. Growing evidence suggests that the microenvironment of the primary tumor primes DTCs for dormant or proliferative fates. However, the manner in which this occurs remains poorly understood. Here, using the Window for High-Resolution Intravital Imaging of the Lung (WHRIL), we study the live lung longitudinally and follow the fate of individual DTCs that spontaneously disseminate from orthotopic breast tumors. We find that spontaneously DTCs have increased levels of retention, increased speed of extravasation, and greater survival after extravasation, compared to experimentally metastasized tumor cells. Detailed analysis reveals that a subset of macrophages within the primary tumor induces a pro-dissemination and pro-dormancy DTC phenotype. Our work provides insight into how specific primary tumor microenvironments prime a subpopulation of cells for expression of proteins associated with dissemination and dormancy. The understanding of the mechanisms underlying the ability of disseminated tumor cells (DTCs) to form metastasis is incomplete. Here, by using high-resolution intravital imaging of the murine lung to track the fate of breast-derived DTCs, the authors show that macrophages within the primary tumor induce a pro-dissemination and pro-dormancy phenotype in tumor cells, favouring their extravasation in the lung.

Navigation through the bulk tumour, entry into the blood vasculature, survival in the circulation, exit at distant sites and resumption of proliferation are all steps necessary for tumour cells to successfully metastasize. The ability of tumour cells to complete these steps is highly dependent on the timing and sequence of the interactions that these cells have with the tumour microenvironment (TME), including stromal cells, the extracellular matrix and soluble factors. The TME thus plays a major role in determining the overall metastatic phenotype of tumours. The complexity and cause-and-effect dynamics of the TME cannot currently be recapitulated in vitro or inferred from studies of fixed tissue, and are best studied in vivo, in real time and at single-cell resolution. Intravital imaging (IVI) offers these capabilities, and recent years have been a time of immense growth and innovation in the field. Here we review some of the recent advances in IVI of mammalian models of cancer and describe how IVI is being used to understand cancer progression and metastasis, and to develop novel treatments and therapies. We describe new techniques that allow access to a range of tissue and cancer types, novel fluorescent reporters and biosensors that allow fate mapping and the probing of functional and phenotypic states, and the clinical applications that have arisen from applying these techniques, reporters and biosensors to study cancer. We finish by presenting some of the challenges that remain in the field, how to address them and future perspectives.This Review covers recent advances in intravital imaging of mammalian models of cancer and describes how intravital imaging can help to understand the role of the tumour microenvironment in cancer progression and metastasis, and to develop novel treatments and therapies.

Key Points Chemotaxis is the phenomenon by which cell movement is directed in response to an extracellular chemical gradient. Factors that mediate chemotaxis are frequently mutated in cancer. Although most of the factors have dual roles in cell growth and survival, they also mediate cytoskeletal dynamics that results in chemotaxis, thus suggesting a potentially important role of chemotaxis in cancer. Tumour cells in vivo can move both randomly and directionally. However, invasion, migration and dissemination are most efficient when the cell is involved in directed migration. Different modes of directed migration have been described for tumour cells (amoeboid migration or mesenchymal migration for single cells and collective or streaming migration for groups of cells). The occurrence and frequency of these modes of migration in cancer is dependent on the type of cancer and the surrounding factors within the tumour microenvironment. Despite the various patterns of directed migration during tumour cell dissemination, the intracellular processes that direct the cell motility cycle in response to the chemoattractant are probably similar and are comprised of three steps: chemosensing, polarization and locomotion. First, polarized intracellular signals lead to asymmetric actin polymerization resulting in extension of the cell membrane in the direction of movement, thus creating the leading-edge protrusion. This is followed by integrin-mediated adhesion to the substrate on which the cell is moving, and then by detachment from the substrate and contraction of the trailing edge of the cell. In addition to cancer cells, directional migration to a chemokine source is observed in stromal cells, which frequently shape the tumour microenvironment to a more pro-metastatic state. A complex network of chemokines and growth factors is involved in the communication of tumour cells with stromal cells. This leads to several major events of cancer progression, such as immune evasion, angiogenesis, invasion and dissemination. Despite the strong experimental evidence for the involvement of chemotaxis signalling pathways in tumour cell dissemination, therapeutics under development are tested only for their ability to reduce the tumour size in patients with late-stage disease. A lack of relevant therapeutic end points in clinical practice, together with the current belief that dissemination occurs early in tumour progression, before clinical presentation, have brought scepticism to the development of anti-invasion and anti-dissemination drugs. We speculate that dissemination is not only a feasible but also a necessary therapeutic target if efficient long-term management of minimal residual disease is a goal in cancer treatment. The identification of therapeutic end points relevant to tumour cell dissemination will facilitate the development and appropriate use of therapeutics. Chemotaxis confers directionality to migrating tumour cells away from the tumour and recruits various cells to the tumour. This Review discusses the mechanisms of chemotaxis and how this process contributes to tumour progression. Chemotaxis of tumour cells and stromal cells in the surrounding microenvironment is an essential component of tumour dissemination during progression and metastasis. This Review summarizes how chemotaxis directs the different behaviours of tumour cells and stromal cells in vivo , how molecular pathways regulate chemotaxis in tumour cells and how chemotaxis choreographs cell behaviour to shape the tumour microenvironment and to determine metastatic spread. The central importance of chemotaxis in cancer progression is highlighted by discussion of the use of chemotaxis as a prognostic marker, a treatment end point and a target of therapeutic intervention.

Tumors often overcome the cytotoxic effects of chemotherapy through either acquired or environment-mediated drug resistance. In addition, signals from the microenvironment obfuscate the beneficial effects of chemotherapy and may facilitate progression and metastatic dissemination. Seminal mediators in chemotherapy-induced metastasis appear to be a wide range of hematopoietic, mesenchymal and immune progenitor cells, originating from the bone marrow. The actual purpose of these cells is to orchestrate the repair response to the cytotoxic damage of chemotherapy. However, these repair responses are exploited by tumor cells at every step of the metastatic cascade, ranging from tumor cell invasion, intravasation and hematogenous dissemination to extravasation and effective colonization at the metastatic site. A better understanding of the mechanistic underpinnings of chemotherapy-induced metastasis will allow us to better predict which patients are more likely to exhibit pro-metastatic responses to chemotherapy and will help develop new therapeutic strategies to neutralize chemotherapy-driven prometastatic changes.

Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. Here, we employ high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. Substantial enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent >60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch-Jagged signaling. In breast cancers from patients, the density of TMEM doorways correlates with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites. Intravital imaging reveals macrophage-driven de novo induction of cancer stem cells in vivo, and their dramatic enrichment on dissemination through TMEM doorways. These findings provide a mechanism for the validated ability of TMEM doorway density to be prognostic for distant recurrence of metastatic tumors in breast cancer patients.

Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA-Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT-dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell-cell junction formation, and regulation of cell migration, were enriched among EMT-associated alternatively splicing events. Our analysis suggested that most EMT-associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT-associated splicing pattern. Expression of EMT-associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT-dependent splicing changes occur commonly in human tumors. The functional significance of EMT-associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT-associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.

Entry of tumor cells into the blood stream is a critical step in cancer metastasis. Although significant progress has been made in visualizing tumor cell motility in vivo, the underlying mechanism of cancer cell intravasation remains largely unknown. We developed a microfluidic-based assay to recreate the tumor-vascular interface in three-dimensions, allowing for high resolution, real-time imaging, and precise quantification of endothelial barrier function. Studies are aimed at testing the hypothesis that carcinoma cell intravasation is regulated by biochemical factors from the interacting cells and cellular interactions with macrophages. We developed a method to measure spatially resolved endothelial permeability and show that signaling with macrophages via secretion of tumor necrosis factor alpha results in endothelial barrier impairment. Under these conditions intravasation rates were increased as validated with live imaging. To further investigate tumor-endothelial (TC-EC) signaling, we used highly invasive fibrosarcoma cells and quantified tumor cell migration dynamics and TC-EC interactions under control and perturbed (with tumor necrosis factor alpha) barrier conditions. We found that endothelial barrier impairment was associated with a higher number and faster dynamics of TC-EC interactions, in agreement with our carcinoma intravasation results. Taken together our results provide evidence that the endothelium poses a barrier to tumor cell intravasation that can be regulated by factors present in the tumor microenvironment.

While it has been established that a number of microenvironment components can affect the likelihood of metastasis, the link between microenvironment and tumor cell phenotypes is poorly understood. Here we have examined microenvironment control over two different tumor cell motility phenotypes required for metastasis. By high-resolution multiphoton microscopy of mammary carcinoma in mice, we detected two phenotypes of motile tumor cells, different in locomotion speed. Only slower tumor cells exhibited protrusions with molecular, morphological, and functional characteristics associated with invadopodia. Each region in the primary tumor exhibited either fast- or slow-locomotion. To understand how the tumor microenvironment controls invadopodium formation and tumor cell locomotion, we systematically analyzed components of the microenvironment previously associated with cell invasion and migration. No single microenvironmental property was able to predict the locations of tumor cell phenotypes in the tumor if used in isolation or combined linearly. To solve this, we utilized the support vector machine (SVM) algorithm to classify phenotypes in a nonlinear fashion. This approach identified conditions that promoted either motility phenotype. We then demonstrated that varying one of the conditions may change tumor cell behavior only in a context-dependent manner. In addition, to establish the link between phenotypes and cell fates, we photoconverted and monitored the fate of tumor cells in different microenvironments, finding that only tumor cells in the invadopodium-rich microenvironments degraded extracellular matrix (ECM) and disseminated. The number of invadopodia positively correlated with degradation, while the inhibiting metalloproteases eliminated degradation and lung metastasis, consistent with a direct link among invadopodia, ECM degradation, and metastasis. We have detected and characterized two phenotypes of motile tumor cells in vivo, which occurred in spatially distinct microenvironments of primary tumors. We show how machine-learning analysis can classify heterogeneous microenvironments in vivo to enable prediction of motility phenotypes and tumor cell fate. The ability to predict the locations of tumor cell behavior leading to metastasis in breast cancer models may lead towards understanding the heterogeneity of response to treatment.

Tumor-associated macrophages (TAMs) are a phenotypically diverse, highly plastic population of cells in the tumor microenvironment (TME) that have long been known to promote cancer progression. In this review, we summarize TAM ontogeny and polarization, and then explore how TAMs enhance tumor cell migration through the TME, thus facilitating metastasis. We also discuss how chemotherapy and host factors including diet, obesity, and race, impact TAM phenotype and cancer progression. In brief, TAMs induce epithelial-mesenchymal transition (EMT) in tumor cells, giving them a migratory phenotype. They promote extracellular matrix (ECM) remodeling, allowing tumor cells to migrate more easily. TAMs also provide chemotactic signals that promote tumor cell directional migration towards blood vessels, and then participate in the signaling cascade at the blood vessel that allows tumor cells to intravasate and disseminate throughout the body. Furthermore, while chemotherapy can repolarize TAMs to induce an anti-tumor response, these cytotoxic drugs can also lead to macrophage-mediated tumor relapse and metastasis. Patient response to chemotherapy may be dependent on patient-specific factors such as diet, obesity, and race, as these factors have been shown to alter macrophage phenotype and affect cancer-related outcomes. More research on how chemotherapy and patient-specific factors impact TAMs and cancer progression is needed to refine treatment strategies for cancer patients.