Tumor-Derived Extracellular Vesicles: Multifunctional Entities in the Tumor Microenvironment.

Tumor cells release extracellular vesicles (EVs) that can function as mediators of intercellular communication in the tumor microenvironment. EVs contain a host of bioactive cargo, including membrane, cytosolic, and nuclear proteins, in addition to noncoding RNAs, other RNA types, and double-stranded DNA fragments. These shed vesicles may deposit paracrine information and can also be taken up by stromal cells, causing the recipient cells to undergo phenotypic changes that profoundly impact diverse facets of cancer progression. For example, this unique form of cellular cross talk helps condition the premetastatic niche, facilitates evasion of the immune response, and promotes invasive and metastatic activity. These findings, coupled with those demonstrating that the number and content of EVs produced by tumors can vary depending on their tumor of origin, disease stage, or response to therapy, have raised the exciting possibility that EVs can be used for risk stratification, diagnostic, and even prognostic purposes. We summarize recent developments and the current knowledge of EV cargoes, their impact on disease progression, and implementation of EV-based liquid biopsies as tumor biomarkers.

Recruitment of DNA to tumor-derived microvesicles.

The shedding of extracellular vesicles (EVs) represents an important but understudied means of cell-cell communication in cancer. Among the currently described classes of EVs, tumor-derived microvesicles (TMVs) comprise a class of vesicles released directly from the cell surface. TMVs contain abundant cargo, including functional proteins and miRNA, which can be transferred to and alter the behavior of recipient cells. Here, we document that a fraction of extracellular double-stranded DNA (dsDNA) is enclosed within TMVs and protected from nuclease degradation. dsDNA inclusion in TMVs is regulated by ARF6 cycling and occurs with the cytosolic DNA sensor, cGAS, but independent of amphisome or micronuclei components. Our studies suggest that dsDNA is trafficked to TMVs via a mechanism distinct from the multivesicular body-dependent secretion reported for the extracellular release of cytosolic DNA. Furthermore, TMV dsDNA can be transferred to recipient cells with consequences to recipient cell behavior, reinforcing its relevance in mediating cell-cell communication.

The ins and outs of microvesicles.

Microvesicles are a heterogeneous group of membrane-enclosed vesicles that are released from cells into the extracellular space by the outward budding and pinching of the plasma membrane. These vesicles are loaded with multiple selectively sorted proteins and nucleic acids. Although interest in the clinical potential of microvesicles is increasing, there is only limited understanding of different types of microvesicles and the mechanisms involved in their formation. Here, we describe what is presently known about this expanding and complex field of research focusing on the mechanism of biogenesis, cargo loading, and release of microvesicles.

Breaking Bad: Extracellular Vesicles Provoke Tumorigenic Responses Under Oxygen Deprivation.

Intercellular communication is vital to tumor progression. In this issue of Developmental Cell, Bertolini et al. (2020) describe how small extracellular vesicles released from hypoxic mammary tumor cells facilitate intercellular communication, leading to alterations in mitochondrial dynamics and acquisition of invasive phenotypes in normal epithelial cells.

Rab11b-mediated integrin recycling promotes brain metastatic adaptation and outgrowth.

Breast cancer brain metastases (BCBM) have a 5-20 year latency and account for 30% of mortality; however, mechanisms governing adaptation to the brain microenvironment remain poorly defined. We combine time-course RNA-sequencing of BCBM development with a Drosophila melanogaster genetic screen, and identify Rab11b as a functional mediator of metastatic adaptation. Proteomic analysis reveals that Rab11b controls the cell surface proteome, recycling proteins required for successful interaction with the microenvironment, including integrin ß1. Rab11b-mediated control of integrin ß1 surface expression allows efficient engagement with the brain ECM, activating mechanotransduction signaling to promote survival. Lipophilic statins prevent membrane association and activity of Rab11b, and we provide proof-of principle that these drugs prevent breast cancer adaptation to the brain microenvironment. Our results identify Rab11b-mediated recycling of integrin ß1 as regulating BCBM, and suggest that the recycleome, recycling-based control of the cell surface proteome, is a previously unknown driver of metastatic adaptation and outgrowth.

An ARF6-Exportin-5 axis delivers pre-miRNA cargo to tumour microvesicles.

Tumour-derived microvesicles (TMVs) comprise a class of extracellular vesicles released from tumour cells that are now understood to facilitate communication between the tumour and the surrounding microenvironment. Despite their significance, the regulatory mechanisms governing the trafficking of bioactive cargos to TMVs at the cell surface remain poorly defined. Here we describe a molecular pathway for the delivery of microRNA (miRNA) cargo to nascent TMVs involving the dissociation of a pre-miRNA/Exportin-5 complex from Ran-GTP following nuclear export and its subsequent transfer to a cytoplasmic shuttle comprised of ARF6-GTP and GRP1. As such, ARF6 activation increases the pre-miRNA cargo contained within TMVs through a process that requires the casein kinase 2-mediated phosphorylation of RanGAP1. Furthermore, TMVs were found to contain pre-miRNA processing machinery including Dicer and Argonaute-2, which allow for cell-free pre-miRNA processing within shed vesicles. These findings offer cellular targets to block the loading and processing of pre-miRNAs within TMVs.

Coordinated Regulation of Intracellular Fascin Distribution Governs Tumor Microvesicle Release and Invasive Cell Capacity.

Tumor cell invasion is one result of the bidirectional interactions occurring between tumor cells and the surrounding milieu. The ability of tumor cells to invade through the extracellular matrix is in part regulated by the formation of a class of protease-loaded extracellular vesicles, called tumor microvesicles (TMVs), which are released directly from the cell surface. Here we show that the actin bundling protein, fascin, redistributes to the cell periphery in a ternary complex with podocalyxin and ezrin, where it promotes TMV release. The peripheral localization of fascin is prompted by the loss of Rab35 signaling, which in turn unleashes ARF6 activation. The result is a mechanism through which Rab35 and ARF6 cooperatively and simultaneously regulate the distribution and localization of fascin and promote oncogenic signaling, which leads to TMV release while inhibiting invadopodium formation. These studies are clinically significant as fascin-loaded TMVs can be detected in bodily fluids and elevated fascin expression coupled with low Rab35 levels correlates with poor overall survival in some cancers.

Aberrant endocytosis leads to the loss of normal mitotic spindle orientation during epithelial glandular morphogenesis.

Epithelial cells form tissues with many functions, including secretion and environmental separation and protection. Glandular epithelial tissues comprise cysts and tubules that are formed from a polarized, single-epithelial cell layer surrounding a central, fluid-filled lumen. The pathways regulating key processes in epithelial tissue morphogenesis such as mitotic spindle formation are incompletely understood, but are important to investigate, as their dysregulation is a signature of epithelial tumors. Here, we describe a signaling axis that manifests in a defect in mitotic spindle orientation during epithelial growth and cystogenesis. We found that activation of the small GTPase ADP-ribosylation factor 6 (ARF6) results in the sustained internalization of cell-surface components such as the cMet receptor and the cell-adhesion molecule E-cadherin. The spindle orientation defect arising from elevated levels of ARF6-GTP required an increase in cMet endocytosis, but was independent of E-cadherin internalization or elevated extracellular signal-regulated kinase (ERK) activity resulting from internalized receptor signaling on endosomes. Misorientation of the mitotic spindle resulted in the development of epithelial cysts with structural abnormalities, the most conspicuous of which was the presence of multiple intercellular lumens. Abnormal mitotic spindle orientation was necessary but insufficient to disrupt glandular development, as blocking the strong prosurvival signal resulting from ERK hyperactivation yielded structurally normal cysts despite continued manifestation of spindle orientation defects. Our findings highlight a previously unknown link between ARF6 activation, cMet receptor internalization, and mitotic spindle orientation during epithelial glandular morphogenesis.

Extracellular microvesicles and invadopodia mediate non-overlapping modes of tumor cell invasion.

Tumor cell invasion requires the molecular and physical adaptation of both the cell and its microenvironment. Here we show that tumor cells are able to switch between the use of microvesicles and invadopodia to facilitate invasion through the extracellular matrix. Invadopodia formation accompanies the mesenchymal mode of migration on firm matrices and is facilitated by Rac1 activation. On the other hand, during invasion through compliant and deformable environments, tumor cells adopt an amoeboid phenotype and release microvesicles. Notably, firm matrices do not support microvesicle release, whereas compliant matrices are not conducive to invadopodia biogenesis. Furthermore, Rac1 activation is required for invadopodia function, while its inactivation promotes RhoA activation and actomyosin contractility required for microvesicle shedding. Suppression of RhoA signaling blocks microvesicle formation but enhances the formation of invadopodia. Finally, we describe Rho-mediated pathways involved in microvesicle biogenesis through the regulation of myosin light chain phosphatase. Our findings suggest that the ability of tumor cells to switch between the aforementioned qualitatively distinct modes of invasion may allow for dissemination across different microenvironments.

Tumor-derived microvesicles in the tumor microenvironment: How vesicle heterogeneity can shape the future of a rapidly expanding field.

Information transmission from tumor cells to non-tumor cells in the surrounding microenvironment via microvesicles is a more recently studied form of intercellular signaling that can have a marked impact on the tumor microenvironment. Tumor-derived microvesicles (TMVs) are packed with information including signaling proteins and nucleic acids, and can be taken up by target cells, enabling paracrine signaling. While previous research has focused on how vesicles released from pathologic cells differ from normal cells, the heterogeneity that exists within the TMV population itself is not fully characterized, and only beginning to be appreciated. In this review, we summarize current understanding of the biogenesis and roles of shed TMVs in the tumor microenvironment, and speculate on the consequences for tumor cell signaling in light of the hypothesis that there exists variance within the TMV population. The analysis of differential signaling upon cell-TMV interactions provides insights into potential mechanisms of intercellular communication.

Regulated delivery of molecular cargo to invasive tumour-derived microvesicles.

Cells release multiple, distinct forms of extracellular vesicles including structures known as microvesicles, which are known to alter the extracellular environment. Despite growing understanding of microvesicle biogenesis, function and contents, mechanisms regulating cargo delivery and enrichment remain largely unknown. Here we demonstrate that in amoeboid-like invasive tumour cell lines, the v-SNARE, VAMP3, regulates delivery of microvesicle cargo such as the membrane-type 1 matrix metalloprotease (MT1-MMP) to shedding microvesicles. MT1-MMP delivery to nascent microvesicles depends on the association of VAMP3 with the tetraspanin CD9 and facilitates the maintenance of amoeboid cell invasion. VAMP3-shRNA expression depletes shed vesicles of MT1-MMP and decreases cell invasiveness when embedded in cross-linked collagen matrices. Finally, we describe functionally similar microvesicles isolated from bodily fluids of ovarian cancer patients. Together these studies demonstrate the importance of microvesicle cargo sorting in matrix degradation and disease progression.

Tumor-derived microvesicles: shedding light on novel microenvironment modulators and prospective cancer biomarkers.

Recent advances in the study of tumor-derived microvesicles reveal new insights into the cellular basis of disease progression and the potential to translate this knowledge into innovative approaches for cancer diagnostics and personalized therapy. Tumor-derived microvesicles are heterogeneous membrane-bound sacs that are shed from the surfaces of tumor cells into the extracellular environment. They have been thought to deposit paracrine information and create paths of least resistance, as well as be taken up by cells in the tumor microenvironment to modulate the molecular makeup and behavior of recipient cells. The complexity of their bioactive cargo-which includes proteins, RNA, microRNA, and DNA-suggests multipronged mechanisms by which microvesicles can condition the extracellular milieu to facilitate disease progression. The formation of these shed vesicles likely involves both a redistribution of surface lipids and the vertical trafficking of cargo to sites of microvesicle biogenesis at the cell surface. Current research also suggests that molecular profiling of these structures could unleash their potential as circulating biomarkers as well as platforms for personalized medicine. Thus, new and improved strategies for microvesicle identification, isolation, and capture will have marked implications in point-of-care diagnostics for cancer patients.

Microvesicles: mediators of extracellular communication during cancer progression.

Microvesicles are generated by the outward budding and fission of membrane vesicles from the cell surface. Recent studies suggest that microvesicle shedding is a highly regulated process that occurs in a spectrum of cell types and, more frequently, in tumor cells. Microvesicles have been widely detected in various biological fluids including peripheral blood, urine and ascitic fluids, and their function and composition depend on the cells from which they originate. By facilitating the horizontal transfer of bioactive molecules such as proteins, RNAs and microRNAs, they are now thought to have vital roles in tumor invasion and metastases, inflammation, coagulation, and stem-cell renewal and expansion. This Commentary summarizes recent literature on the properties and biogenesis of microvesicles and their potential role in cancer progression.