The key role of extracellular vesicles in the metastatic process

Extracellular vesicles (EVs), including exosomes, have a key role in the paracrine communication between organs and compartments. EVs shuttle virtually all types of biomolecules such as proteins, lipids, nucleic acids, metabolites and even pharmacological compounds. Their ability to transfer their biomolecular cargo into target cells enables EVs to play a key role in intercellular communication that can regulate cellular functions such as proliferation, apoptosis and migration. This has led to the emergence of EVs as a key player in tumor growth and metastasis through the formation of “tumor niches” in target organs. Recent data have also been shown that EVs may transform the microenvironment of primary tumors thus favoring the selection of cancer cells with a metastatic behavior. The release of EVs from resident non-malignant cells may contribute to the metastatic processes as well. However, cancer EVs may induce malignant transformation in resident mesenchymal stem cells, suggesting that the metastatic process is not exclusively due to circulating tumor cells. In this review, we outline and discuss evidence-based roles of EVs in actively regulating multiple steps of the metastatic process and how we can leverage EVs to impair metastasis.     

Apical P2XR contribute to [Ca2+]i signaling and Isc in mouse renal MCD

We examined P2X receptor expression and distribution in the mouse collecting duct (CD) and their functional role in Ca(2+) signaling. Both P2X(1) and P2X(4) were detected by RT-PCR and Western blot. Immunohistochemistry demonstrated apical P2X(1) and P2X(4) immunoreactivity in principal cells in the outer medullary CD (OMCD) and inner medullary CD (IMCD). Luminal ATP induced an increase in Ca(2+) signaling in native medullary CD (MCD) as measured by fluorescence imaging. ATP also induced an increase in Ca(2+) signaling in MCD cells grown in primary culture but not in the presence of P2XR antagonist PPNDS. Short circuit current (I(sc)) measurement with mouse IMCD cells showed that P2XR agonist BzATP induced a larger I(sc) than did P2YR agonist UTP in the apical membrane. Our data reveal for the first time that P2X(1) and P2X(4) are cell-specific with prominent immunoreactivity in the apical area of MCD cells. The finding that P2XR blockade inhibits ATP-induced Ca(2+) signaling suggests that activation of P2XR is a key step in Ca(2+)-dependent purinergic signaling. The result that activation of P2XR produces large I(sc) indicates the necessity of P2XR in renal CD ion transport.     

Proteomic analysis of necroptotic extracellular vesicles

Necroptosis is a regulated and inflammatory form of cell death. We, and others, have previously reported that necroptotic cells release extracellular vesicles (EVs). We have found that necroptotic EVs are loaded with proteins, including the phosphorylated form of the key necroptosis-executing factor, mixed lineage kinase domain-like kinase (MLKL). However, neither the exact protein composition, nor the impact, of necroptotic EVs have been delineated. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated and analyzed by mass spectrometry-based proteomics. A total of 3337 proteins were identified, sharing a high degree of similarity with exosome proteome databases, and clearly distinguishing necroptotic and control EVs. A total of 352 proteins were significantly upregulated in the necroptotic EVs. Among these were MLKL and caspase-8, as validated by immunoblot. Components of the ESCRTIII machinery and inflammatory signaling were also upregulated in the necroptotic EVs, as well as currently unreported components of vesicle formation and transport, and necroptotic signaling pathways. Moreover, we found that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we uncovered that necroptotic EVs contain tumor neoantigens, and are enriched with components of antigen processing and presentation. In summary, our study reveals a new layer of regulation during the early stage of necroptosis, mediated by the secretion of specific EVs that influences the microenvironment and may instigate innate and adaptive immune responses. This study sheds light on new potential players in necroptotic signaling and its related EVs, and uncovers the functional tasks accomplished by the cargo of these necroptotic EVs.Subject terms: Necroptosis, Cell death and immune response     

Extracellular Vesicles Secreted from Cancer Cell Lines Stimulate Secretion of MMP-9, IL-6, TGF-β1 and EMMPRIN

Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-β1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles.     

Expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells

A major function of macrophages is to engulf apoptotic cells to prevent them from releasing noxious materials as they die. Milk fat globule-EGF-factor 8 (MFG-E8) is a glycoprotein secreted by activated macrophages that works as a bridge between apoptotic cells and phagocytes by specifically recognizing phosphatidylserine exposed on apoptotic cells. In this study, we found that developmental endothelial locus-1 (Del-1), originally identified as an embryonic endothelial cell protein that binds alphavbeta3 integrin, is structurally and functionally homologous to MFG-E8. That is, both consist of a signal sequence, two epidermal growth factor domains and two factor VIII-homologous domains (C1 and C2). Del-1 bound to the apoptotic cells by recognizing phosphatidylserine via the factor VIII-homologous domains with an affinity similar to that of MFG-E8. The phagocytic activity of NIH 3T3 cells against apoptotic cells was enhanced by Del-1 through an interaction between the epidermal growth factor domain in Del-1 and alphavbeta3 integrin expressed in the NIH 3T3 cells. Screening of primary macrophages and macrophage cell lines for the expression of MFG-E8 and Del-1 indicated that MFG-E8 and Del-1 are expressed in different sets of macrophages. These results suggest the existence of macrophage subsets that use MFG-E8 or Del-1 differently to engulf apoptotic cells.     

Extracellular vesicles from thyroid cancer harbor a functional machinery involved in extracellular matrix remodeling

Extracellular vesicles (EVs) participate in cell-stroma crosstalk within the tumor microenvironment and fibroblasts (Fb) contribute to tumor promotion in thyroid cancer. However, the role of tumor-stroma derived EVs still needs to be deciphered. We hypothesized that the interaction of thyroid tumor cells with Fb would liberate EVs with a specific proteomic profile, which would have an impact on EV-functionality in thyroid tumor progression-related events. Tumor (TPC-1, 8505c) and non-tumor (NThyOri) thyroid cells were co-cultured with human Fb. EVs, obtained by ultracentrifugation of conditioned media, were characterized by nanoparticle tracking analysis and western blotting. EV-proteomic analysis was performed by mass-spectrometry, and metalloproteinases (MMPs) were studied by zymography. EV-exchange was evaluated using immunofluorescence, confocal microscopy and FACS. EVs expressed classical exosome markers, with EVs from thyroid tumor cell-Fb co-cultures showing a proteomic profile related to extracellular matrix (ECM) remodeling. Bidirectional crosstalk between Fb and TPC-1 cells produced significantly more EVs than their isolated cells, and potentiated EV-functionality. In line with this, Fb-TPC-1 derived EVs induced MMP2 activation in NThyOri supernatants, and MMP2 activity could be evidenced in Fb and TPC-1 contact-independent co-cultures. Besides, MMP2 interactors allowed us to discriminate between EVs from thyroid tumoral and non-tumoral milieus. Interestingly, Fb internalized more EVs from TPC-1 than from NThyOri producing cells. Fb and thyroid tumor cell crosstalk produces specialized EVs with an ECM remodeling proteomic profile, enabling activation of MMP2 and possibly facilitating ECM-degradation, which is potentially linked with thyroid tumor progression.     

Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance

Multidrug resistance (MDR) is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs) in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and function of MDR pump-rich EVs in cancer cells and their ability to confer multiple anticancer drug resistance.     

Extracellular vesicles in ovarian cancer chemoresistance,metastasis, and immune evasion

Chemoresistance and metastasis are the major challenges for the current ovarian cancer treatment. Understanding the mechanisms of ovarian cancer progression and metastasis is critically important for developing novel therapies. The advances in extracellular vesicles (EVs) research in recent years have attracted extensive attention. EVs contain a variety of proteins, RNAs, DNAs, and metabolites. Accumulating evidence indicates that ovarian cancer cells secrete a large amount of EVs, playing an important role in tumor progression and recurrence. In the microenvironment of ovarian tumor, EVs participate in the information transmission between stromal cells and immune cells, promoting the immune escape of ovarian cancer cells and facilitating cancer metastasis. Here, we review the recent advances of EVs in chemoresistance, mechanisms of metastasis, and immune evasion of ovarian cancer. Furthermore, we also discuss the challenges of EV research and future application of EVs as promising biomarker sources in response to therapy and in therapy-delivery approaches for ovarian cancer patients.Subject terms: Cancer microenvironment, Translational research     

Methylcholanthrene-Induced Sarcomas Develop Independently from NOX2-Derived ROS

Reactive oxygen species (ROS) produced by the inducible NADPH oxidase type 2 (NOX2) complex are essential for clearing certain infectious organisms but may also have a role in regulating inflammation and immune response. For example, ROS is involved in myeloid derived suppressor cell (MDSC)- and regulatory T cell (T_reg) mediated T- and NK-cell suppression. However, abundant ROS produced within the tumor microenvironment, or by the tumor itself may also yield oxidative stress, which can blunt anti-tumor immune responses as well as eventually leading to tumor toxicity. In this study we aimed to decipher the role of NOX2-derived ROS in a chemically (by methylcholanthrene (MCA)) induced sarcoma model. Superoxide production by NOX2 requires the p47^phox (NCF1) subunit to organize the formation of the NOX2 complex on the cell membrane. Homozygous mutant mice (NCF1*^/*) have a functional loss of their super oxide burst while heterozygous mice (NCF1*^/+) retain this key function. Mice harboring either a homo- or a heterozygous mutation were injected intramuscularly with MCA to induce sarcoma formation. We found that NOX2 functionality does not determine tumor incidence in the tested MCA model. Comprehensive immune monitoring in tumor bearing mice showed that infiltrating immune cells experienced an increase in their oxidative state regardless of the NOX2 functionality. While MCA-induced sarcomas where characterized by a T_reg and MDSC accumulation, no significant differences could be found between NCF1*^/* and NCF1*^/+ mice. Furthermore, infiltrating T cells showed an increase in effector-memory cell phenotype markers in both NCF1*^/* and NCF1*^/+ mice. Tumors established from both NCF1*^/* and NCF1*^/+ mice were tested for their in vitro proliferative capacity as well as their resistance to cisplatin and radiation therapy, with no differences being recorded. Overall our findings indicate that NOX2 activity does not play a key role in tumor development or immune cell infiltration in the chemically induced MCA sarcoma model.     

Effect of P2X purinergic receptors in tumor progression and as a potential target for anti-tumor therapy

The development of tumors is a complex pathological process involving multiple factors, multiple steps, and multiple genes. Their prevention and treatment have always been a difficult problem at present. A large number of studies have proved that the tumor microenvironment plays an important role in the progression of tumors. The tumor microenvironment is the place where tumor cells depend for survival, and it plays an important role in regulating the growth, proliferation, apoptosis, migration, and invasion of tumor cells. P2X purinergic receptors, which depend on the ATP ion channel, can be activated by ATP in the tumor microenvironment, and by mediating tumor cells and related cells (such as immune cells) in the tumor microenvironment. They play an important regulatory role on the effects of the skeleton, membrane fluidity, and intracellular molecular metabolism of tumor cells. Therefore, here, we outlined the biological characteristics of P2X purinergic receptors, described the effect of tumor microenvironment on tumor progression, and discussed the effect of ATP on tumor. Moreover, we explored the role of P2X purinergic receptors in the development of tumors and anti-tumor therapy. These data indicate that P2X purinergic receptors may be used as another potential pharmacological target for tumor prevention and treatment.

     

Extracellular nanovesicles‐transmitted circular RNA has_circ_0000190 suppresses osteosarcoma progression

Under the microenvironment, tumour progression is substantially affected by cell‐cell communication. In spite of the mediating effect of extracellular nanovesicles (EVs) on cell‐cell communication by packaging into circRNAs, the effect of EVs circRNA hsa_circ_0000190 (circ‐0000190) in osteosarcoma is still not clear. Circ‐0000190 expressions in tissues and EVs from plasma were compared between osteosarcoma patients and controls. Thereafter, receiver operating characteristic (ROC) curve was drawn and area under the curve was calculated to examine whether the diagnostic results were accurate, and the effect of EVs circ‐0000190 was dug out via the determination of cell phenotypes and animal assays. Results showed circ‐0000190 exhibited an obvious reduction in EVs and tissues of osteosarcoma patients (P < .05). It was also discovered that EVs encapsulated the majority of circ‐0000190, and EVs‐encapsulated circ‐0000190 could be applied to make a distinction between osteosarcoma patients and controls. Besides, EVs circ‐0000190 in osteosarcoma cells transported from normal cells weakened the capacities of osteosarcoma cells to migrate, proliferate and invade, so as to block their biological malignant behaviours (P < .05). In addition, under the action of EVs circ‐0000190, tumour growth was impeded and the expression of TET1 was inhibited via the competitive binding to miR‐767‐5p. In all, EVs circ‐0000190 has a good prospect as it can be regarded as a new biomarker for detecting osteosarcoma. EVs circ‐0000190 transported from normal cells to osteosarcoma cells impeded the in vitro and in vivo development of osteosarcoma, implying that EVs circ‐0000190 exerts an effect on communication between normal cells and osteosarcoma cells in the carcinogenesis process of osteosarcoma.     

Large oncosomes mediate intercellular transfer of functional microRNA

Prostate cancer cells release atypically large extracellular vesicles (EVs), termed large oncosomes, which may play a role in the tumor microenvironment by transporting bioactive molecules across tissue spaces and through the blood stream. In this study, we applied a novel method for selective isolation of large oncosomes applicable to human platelet-poor plasma, where the presence of caveolin-1-positive large oncosomes identified patients with metastatic disease. This procedure was also used to validate results of a miRNA array performed on heterogeneous populations of EVs isolated from tumorigenic RWPE-2 prostate cells and from isogenic non-tumorigenic RWPE-1 cells. The results showed that distinct classes of miRNAs are expressed at higher levels in EVs derived from the tumorigenic cells in comparison to their non-tumorigenic counterpart. Large oncosomes enhanced migration of cancer-associated fibroblasts (CAFs), an effect that was increased by miR-1227, a miRNA abundant in large oncosomes produced by RWPE-2 cells. Our findings suggest that large oncosomes in the circulation report metastatic disease in patients with prostate cancer, and that this class of EV harbors functional molecules that may play a role in conditioning the tumor microenvironment.     

Extracellular vesicles in pancreatic cancer progression and therapies

Pancreatic cancer (PC) is one of the leading causes of cancer-related death worldwide due to delayed diagnosis and limited treatments. More than 90% of all pancreatic cancers are pancreatic ductal adenocarcinoma (PDAC). Extensive communication between tumour cells and other cell types in the tumour microenvironment have been identified which regulate cancer hallmarks during pancreatic tumorigenesis via secretory factors and extracellular vesicles (EVs). The EV-capsuled factors not only facilitate tumour growth locally, but also enter circulation and reach distant organs to construct a pre-metastatic niche. In this review, we delineate the key factors in pancreatic ductal adenocarcinoma derived EVs that mediate different tumour processes. Also, we highlight the factors that are related to the crosstalk with cancer stem cells/cancer-initiating cells (CSC/CIC), the subpopulation of cancer cells that can efficiently metastasize and resist currently used chemotherapies. Lastly, we discuss the potential of EV-capsuled factors in early diagnosis and antitumour therapeutic strategies.Subject terms: Cancer microenvironment, Cancer stem cells     

Epstein–Barr virus noncoding RNAs from the extracellular vesicles of nasopharyngeal carcinoma (NPC) cells promote angiogenesis via TLR3/RIG-I-mediated VCAM-1 expression

Viral noncoding RNAs (Epstein–Barr virus-encoded RNAs, EBERs) are believed to play a critical role in the progression of lymphoma and nasopharyngeal carcinoma (NPC). However, the accurate mechanisms accounting for their oncogenic function have not been elucidated, especially in terms of interaction between tumor cells and mesenchymal cells. Here, we report that, in addition to NPC cells, EBERs are also found in endothelial cells in Epstein–Barr virus (EBV)-infected NPC parenchymal tissues, which implicates NPC-derived extracellular vesicles (EVs) in transmitting EBERs to endothelial cells. In support of this hypothesis, we first ascertained if EBERs could be transferred to endothelial cells via EVs isolated from NPC culture supernatant. Then, we clarified that EVs-derived EBERs could promote angiogenesis through stimulation of VCAM-1 expression. Finally, we explored the involvement of EBER recognition by TLR3 and RIG-I in NPC angiogenesis. Our observations collectively illustrate the significance and mechanism of EVs-derived EBERs in angiogenesis and underlie the interaction mechanisms between EBV-infected NPC cells and the tumor microenvironment.     

Extracellular vesicles and immunogenic stress in cancer

Tumor progression requires bidirectional cell-to-cell communication within a complex tumor microenvironment (TME). Extracellular vesicles (EVs) as carriers have the capacity to shuttle regulatory molecules, including nucleic acids, proteins, and lipids, between cancer cells and multiple stromal cells, inducing remarkable phenotypic alterations in the TME. Recently proposed the concept “immunogenic stress”, which means in some stressed microenvironment, cancer cells can release EVs containing specific immunoregulatory mediators, depending on the initiating stress-associated pathway, thereby provoking the changes of immune status in the TME. Considerable evidence has revealed that the intracellular mechanisms underlying the response to diverse stresses are mainly autophagy, endoplasmic reticulum (ER) stress reactions and the DNA damage response (DDR). In addition, the activation of immunogenic stress responses endows hosts with immune surveillance capacity; in contrast, several cargoes in EVs under immunogenic stress trigger a passive immune response by mediating the function of immune cells. This review discusses the current understanding of the immunogenic stress pathways in cancer and describes the interrelation between EVs and immunogenic stress to propose potential treatment strategies and biomarkers.Subject terms: Immunosurveillance, Autophagy, Cancer     

A novel 3D heterotypic spheroid model for studying extracellular vesicle-mediated tumour and immune cell communication

Cancer-derived extracellular vesicles (EVs) have emerged as important mediators of tumour-host interactions, and they have been shown to exert various functional effects in immune cells. In most of the studies on human immune cells, EVs have been isolated from cancer cell culture medium or patients' body fluids and added to the immune cell cultures. In such a setting, the physiological relevance of the chosen EV concentration is unknown and the EV isolation method and the timing of EV administration may bias the results. In the current study we aimed to develop an experimental cell culture model to study EV-mediated effects in human T and B cells at conditions mimicking the tumour microenvironment. We constructed a human prostate cancer cell line PC3 producing GFP-tagged EVs (PC3-CD63-GFP cells) and developed a 3D heterotypic spheroid model composed of PC3-CD63-GFP cells and human peripheral blood mononuclear cells (PBMCs). The transfer of GFP-tagged EVs from PC3-CD63-GFP cells to the lymphocytes was analysed by flow cytometry and fluorescence imaging. The endocytic pathway was investigated using three endocytosis inhibitors. Our results showed that GFP-tagged EVs interacted with a large fraction of B cells, however, the majority of EVs were not internalised by B cells but rather remained bound at the cell surface. T cell subsets differed in their ability to interact with the EVs - 15.7–24.1% of the total CD3⁺ T cell population interacted with GFP-tagged EVs, while only 0.3–5.8% of CD8⁺ T were GFP positive. Furthermore, a fraction of EVs were internalised in CD3⁺ T cells via macropinocytosis. Taken together, the heterotypic PC3-CD63-GFP and PBMC spheroid model provides the opportunity to study the interactions and functional effects of cancer-derived EVs in human immune cells at conditions mimicking the tumour microenvironment.     

M2 Macrophage Derived Extracellular Vesicle-Mediated Transfer of MiR-186-5p Promotes Colon Cancer Progression by Targeting DLC1

Colon cancer (CC) is one of the most common malignances in digestive tract. M2-polarized macrophages within the tumor microenvironment could facilitate CC cell growth by transferring molecules via extracellular vesicles, but the mechanisms are not fully elucidated. The current study aims to identify the possible effectors in M2 macrophage-derived extracellular vesicles (M2-EVs) and reveal related molecular mechanisms. In our study, we validated the promotion effects of M2-EVs on the proliferation and motility of CC cells, which was found to be dependent on the EVs enclosed molecules by a mild EVs digestion assay. Then we found that miR-186-5p was enriched in M2-EVs and was responsible for the tumor promoting functions of M2-EVs. Furthermore, mechanism investigation revealed M2-EVs transferring miR-186-5p inhibited DLC1 expression by targeting its 3''UTR, and restored DLC1 successfully neutralized the tumor-promoting effects of M2-EVs transferring miR-186-5p via inhibiting the β-catenin pathway. Our study revealed that M2-EVs facilitates the growth and motility of CC cells by delivering the enclosed miR-186-5p, which directly targets DLC1 mRNAs and facilitates their degradation, which could provide a potential biomarker and therapeutic target for CC.