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Metastasis is a dynamic succession of events involving the dissemination of tumour cells to distant sites within the body, ultimately reducing the survival of patients with cancer. To colonize distant organs and, therefore, systemically disseminate within the organism, cancer cells and associated factors exploit several bodily fluid systems, which provide a natural transportation route. Indeed, the flow mechanics of the blood and lymphatic circulatory systems can be co-opted to improve the efficiency of cancer cell transit from the primary tumour, extravasation and metastatic seeding. Flow rates, vessel size and shear stress can all influence the survival of cancer cells in the circulation and control organotropic seeding patterns. Thus, in addition to using these fluids as a means to travel throughout the body, cancer cells exploit the underlying physical forces within these fluids to successfully seed distant metastases. In this Review, we describe how circulating tumour cells and tumour-associated factors leverage bodily fluids, their underlying forces and imposed stresses during metastasis. As the contribution of bodily fluids and their mechanics raises interesting questions about the biology of the metastatic cascade, an improved understanding of this process might provide a new avenue for targeting cancer cells in transit.This Review discusses the role of bodily fluids and their underlying forces and imposed stresses in metastasis, highlighting the contributions of fluid mechanics to tumour cell intravasation, intravascular arrest and extravasation as well as to dissemination of tumour-derived factors.

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.This Review describes the state of the art in imaging extracellular vesicles in animals to study their release, biodistribution and uptake, and covers labeling strategies, microscopy methods and discoveries made in model organisms.

Periodontitis is an inflammatory disease caused by bacterial infection. Recent studies have identified extracellular vesicles (EVs) as potential mediators of inflammation. This study aimed to evaluate the pro-inflammatory properties and miRNA content of EVs secreted in response to Porphyromonas gingivalis infection. Oral epithelial cells (ECs) were infected with P. gingivalis (MOI 100) for 24 h. EVs from infected (P.g.-ECs-EVs) and uninfected cells (ECs-EVs) were isolated, characterized, and tested on naive ECs. Cellular activity, inflammatory markers (TNF-α, IL-1β), and miRNA content of the secreted EVs were evaluated. Bioinformatic analysis was subsequently performed to identify potential targets of differentially expressed miRNAs. P. gingivalis infection increased EV production by 10 2 -fold. P.g.-ECs-EVs exhibited distinct properties compared to ECs-EVs, including higher metabolic activity and elevated TNF-α and IL-1β expression and secretion levels in exposed ECs. Several inflammation-related miRNAs were highly upregulated in P.g.-ECs-EVs (11- to 142-fold; p  < 0.001). P. gingivalis promotes the secretion of pro-inflammatory EVs by ECs, suggesting their role as key mediators in P. gingivalis -induced inflammation and periodontal tissue destruction.

The mechanisms regulating the extension of small unicellular tubes remain poorly defined. Here we identify several steps in Caenorhabditis elegans excretory canal growth, and propose a model for lumen extension. Our results suggest that the basal and apical excretory membranes grow sequentially: the former extends first like an axon growth cone; the latter extends next as a result of an osmoregulatory activity triggering peri-apical vesicles (a membrane reservoir) to fuse with the lumen. An apical cytoskeletal web including intermediate filaments and actin crosslinking proteins ensures straight regular lumen growth. Expression of several genes encoding proteins mediating excretory lumen extension, such as the osmoregulatory STE20-like kinase GCK-3 and the intermediate filament IFB-1, is regulated by ceh-26 (here referred to as pros-1 ), which we found essential for excretory canal formation. Interestingly, PROS-1 is homologous to vertebrate Prox1, a transcription factor controlling lymphatic vessel growth. Our findings have potential evolutionary implications for the origin of fluid-collecting organs, and provide a reference for lymphangiogenesis. Labouesse and colleagues examine the steps of excretory canal growth in nematodes. They delineate the importance of osmoregulated vesicle fusion with the lumen, and of a subapical cytoskeletal web to ensure straight lumen growth. They identify PROS-1 as a transcription factor essential for lumen growth through modulation of the osmosensitive kinase GCK-3 and intermediate filament protein IFB-1.

Among a plethora of functions, extracellular vesicles released by primary tumors spread in the organism and reach distant organs where they can induce the formation of a premetastatic niche. This constitutes a favorable microenvironment for circulating tumor cells which facilitates their seeding and colonization. In this review, we describe the journey of extracellular vesicles (EVs) from the primary tumor to the future metastatic organ, with a focus on the mechanisms used by EVs to target organs with a specific tropism (i.e., organotropism). We then highlight important tumor EV cargos in the context of premetastatic niche formation and summarize their known effects on extracellular matrix remodeling, angiogenesis, vessel permeabilization, resident cell activation, recruitment of foreign cells, and ultimately the formation of a pro‐inflammatory and immuno‐tolerant microenvironment. Finally, we discuss current experimental limitations and remaining opened questions in light of metastatic diagnosis and potential therapies targeting PMN formation.

Emerging evidence suggests that the function and position of organelles are pivotal for tumor cell dissemination. Among them, lysosomes stand out as they integrate metabolic sensing with gene regulation and secretion of proteases. Yet, how their function is linked to their position and how this controls metastasis remains elusive. Here, we analyze lysosome subcellular distribution in patient-derived melanoma cells and patient biopsies and show that lysosome spreading scales with melanoma aggressiveness. Peripheral lysosomes promote matrix degradation and cell invasion which is directly linked to the lysosomal and cell transcriptional programs. Using chemo-genetical control of lysosome positioning, we demonstrate that perinuclear clustering impairs lysosome secretion, matrix degradation and invasion. Impairing lysosome spreading significantly reduces invasive outgrowth in two in vivo models, mouse and zebrafish. Our study provides a direct demonstration that lysosome positioning controls cell invasion, illustrating the importance of organelle adaptation in carcinogenesis and suggesting its potential utility for diagnosis of metastatic melanoma. The function and position of organelles are pivotal for tumor cell dissemination. Here the authors use melanoma patient samples and animal models to show that peripheral localization of lysosomes promotes metastasis by favoring lysosome exocytosis and cell invasion.

A potential role for glycosphingolipids and lipid rafts in apical sorting was initially met with enthusiasm, but genetic analysis has since provided little support for it. A report now establishes that glycosphingolipids mediate apical sorting, and specifically help maintain apicobasal polarity in Caenorhabditis elegans .

In all organisms, cell polarity is fundamental for most aspects of cell physiology. In many species and cell types, it is controlled by the evolutionarily conserved PAR-3, PAR-6 and aPKC proteins, which are asymmetrically localized at the cell cortex where they define specific domains. While PAR proteins define the antero-posterior axis of the early C. elegans embryo, the mechanism controlling their asymmetric localization is not fully understood. Here we studied the role of endocytic regulators in embryonic polarization and asymmetric division. We found that depleting the early endosome regulator RAB-5 results in polarity-related phenotypes in the early embryo. Using Total Internal Reflection Fluorescence (TIRF) microscopy, we observed that PAR-6 is localized at the cell cortex in highly dynamic puncta and depleting RAB-5 decreased PAR-6 cortical dynamics during the polarity maintenance phase. Depletion of RAB-5 also increased PAR-6 association with clathrin heavy chain (CHC-1) and this increase depended on the presence of the GTPase dynamin, an upstream regulator of endocytosis. Interestingly, further analysis indicated that loss of RAB-5 leads to a disorganization of the actin cytoskeleton and that this occurs independently of dynamin activity. Our results indicate that RAB-5 promotes C. elegans embryonic polarity in both dynamin-dependent and -independent manners, by controlling PAR-6 localization and cortical dynamics through the regulation of its association with the cell cortex and the organization of the actin cytoskeleton.

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivo and are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146 dependent manner.

Background: Extracellular vesicles (EVs) contribute to tumour progression and metastasis by mediating the communication between tumour and stromal cells. Notably, tumour EVs have been shown to modify the microenvironment at distance from the primary tumour, creating a premetastatic niche where tumour cells can more easily settle. We have recently shown that two related GTPases, RalA and RalB contribute to EVs secretion in nematodes as well as in mouse mammary tumour cells (4T1 cells) (Hyenne et al., 2015). Methods: Here, we investigate the implication of the Ral-dependent EVs in tumour progression and metastasis formation in mice. Results: We first observed that both RalA and RalB are present in EVs secreted by 4T1 cells. 4T1 cells depleted for RalA or RalB have defects in late endosomal compartments (visualized by electron microscopy) and display distinct EV protein content (analysed by mass spectrometry), which suggest a role for RalA/B in EV biogenesis. To investigate the role of these GTPases in tumour progression and metastasis formation, we realized orthotopic injections in immuno-competent mice. We show that although cells depleted for RalA or RalB have different effects on primary mammary tumour growth, they both have reduced capacity of triggering lung metastasis. Preliminary observations suggest that neither RalA, nor RalB, affect tumour cell migration. Thus, to assess whether this phenotype could be related to differences in EV secretion or content, we investigated the functional properties of RalA/B dependent EVs. We observed that 4T1 EVs promote permeability of endothelial cells monolayer in vitro, and that this phenotype is impaired when 4T1 cells are depleted for RalA or RalB. We then used non-invasive whole-animal imaging to validate this observation in vivo where tumours depleted of RalA/B display a reduced vascular permeability. Summary/Conclusion: Altogether, our results show that RalA and RalB GTPases contribute to metastasis formation, possibly through the release of EVs, which promote local vascular permeability.