Tumour-Derived Extracellular Vesicles (EVs): A Dangerous “Message in A Bottle” for Bone

Several studies have shown the importance of Extracellular Vesicles (EVs) in the intercellular communication between tumour and resident cells. Through EVs, tumour cells can trigger cell-signalling molecules and shuttle exogenous information to target cells, thus promoting spread of the disease. In fact, many processes are fuelled by EVs, such as tumour invasion and dormancy, drug-resistance, immune-surveillance escape, extravasation, extracellular matrix remodelling and metastasis. A key element is certainly the molecular profile of the shed cargo. Understanding the biochemical basis of EVs would help to predict the ability and propensity of cancer cells to metastasize a specific tissue, with the aim to target the release of EVs and to manipulate their content as a possible therapeutic approach. Moreover, EV profiling could help monitor the progression of cancer, providing a useful tool for more effective therapy. This review will focus on all the EV-mediated mentioned mechanisms in the context of both primary bone cancers and bone metastases.

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title: Ectosomes and exosomes: shedding the confusion between extracellular vesicles.
abstract: Long- and short-distance communication can take multiple forms. Among them are exosomes and ectosomes, extracellular vesicles (EVs) released from the cell to deliver signals to target cells. While most of our understanding of how these vesicles are assembled and work comes from mechanistic studies performed on exosomes, recent studies have begun to shift their focus to ectosomes. Unlike exosomes, which are released on the exocytosis of multivesicular bodies (MVBs), ectosomes are ubiquitous vesicles assembled at and released from the plasma membrane. Here we review the similarities and differences between these two classes of vesicle, suggesting that, despite their considerable differences, the functions of ectosomes may be largely analogous to those of exosomes. Both vesicles appear to be promising targets in the diagnosis and therapy of diseases, especially cancer.

title: Extracellular vesicles shuffling intercellular messages: for good or for bad.
abstract: The release of extracellular vesicles (EVs) is a highly conserved process exploited by diverse organisms as a mode of intercellular communication. Vesicles of sizes ranging from 30 to 1000nm, or even larger, are generated by blebbing of the plasma membrane (microvesicles) or formed in multivesicular endosomes (MVEs) to be secreted by exocytosis as exosomes. Exosomes, microvesicles and other EVs contain membrane and cytosolic components that include proteins, lipids and RNAs, a composition that differs related to their site of biogenesis. Several mechanisms are involved in vesicle formation at the plasma membrane or in endosomes, which is reflected in their heterogeneity, size and composition. EVs have significant promise for therapeutics and diagnostics and for understanding physiological and pathological processes all of which have boosted research to find modulators of their composition, secretion and targeting.

title: Ubiquitin-Dependent Sorting into the Multivesicular Body Pathway Requires the Function of a Conserved Endosomal Protein Sorting Complex, ESCRT-I
abstract: The multivesicular body (MVB) pathway is responsible for both the biosynthetic delivery of lysosomal hydrolases and the downregulation of numerous activated cell surface receptors which are degraded in the lysosome. We demonstrate that ubiquitination serves as a signal for sorting into the MVB pathway. In addition, we characterize a 350 kDa complex, ESCRT-I (composed of Vps23, Vps28, and Vps37), that recognizes ubiquitinated MVB cargo and whose function is required for sorting into MVB vesicles. This recognition event depends on a conserved UBC-like domain in Vps23. We propose that ESCRT-I represents a conserved component of the endosomal sorting machinery that functions in both yeast and mammalian cells to couple ubiquitin modification to protein sorting and receptor downregulation in the MVB pathway.

title: Extracellular vesicles: biology and emerging therapeutic opportunities
abstract: Within the past decade, extracellular vesicles have emerged as important mediators of intercellular communication, being involved in the transmission of biological signals between cells in both prokaryotes and higher eukaryotes to regulate a diverse range of biological processes. In addition, pathophysiological roles for extracellular vesicles are beginning to be recognized in diseases including cancer, infectious diseases and neurodegenerative disorders, highlighting potential novel targets for therapeutic intervention. Moreover, both unmodified and engineered extracellular vesicles are likely to have applications in macromolecular drug delivery. Here, we review recent progress in understanding extracellular vesicle biology and the role of extracellular vesicles in disease, discuss emerging therapeutic opportunities and consider the associated challenges.

title: Extracellular vesicles: masters of intercellular communication and potential clinical interventions.
abstract: Intercellular signaling via extracellular vesicles (EVs) is an underappreciated modality of cell-cell crosstalk that enables cells to convey packages of complex instructions to specific recipient cells. EVs transmit these instructions through their cargoes of multiple proteins, nucleic acids, and specialized lipids, which are derived from their cells of origin and allow for combinatorial effects upon recipient cells. This Review series brings together the recent progress in our understanding of EV signaling in physiological and pathophysiological conditions, highlighting how certain EVs, particularly exosomes, can promote or regulate infections, host immune responses, development, and various diseases - notably cancer. Given the diverse nature of EVs and their abilities to profoundly modulate host cells, this series puts particular emphasis on the clinical applications of EVs as therapeutics and as diagnostic biomarkers.

title: The biology and function of fibroblasts in cancer
abstract: Among all cells, fibroblasts could be considered the cockroaches of the human body. They survive severe stress that is usually lethal to all other cells, and they are the only normal cell type that can be live-cultured from post-mortem and decaying tissue. Their resilient adaptation may reside in their intrinsic survival programmes and cellular plasticity. Cancer is associated with fibroblasts at all stages of disease progression, including metastasis, and they are a considerable component of the general host response to tissue damage caused by cancer cells. Cancer-associated fibroblasts (CAFs) become synthetic machines that produce many different tumour components. CAFs have a role in creating extracellular matrix (ECM) structure and metabolic and immune reprogramming of the tumour microenvironment with an impact on adaptive resistance to chemotherapy. The pleiotropic actions of CAFs on tumour cells are probably reflective of them being a heterogeneous and plastic population with context-dependent influence on cancer.

title: Mesenchymal stroma: Role in osteosarcoma progression.
abstract: The initiation and progression of malignant tumors are supported by their microenvironment: cancer cells per se cannot explain growth and formation of the primary or metastasis, and a combination of proliferating tumor cells, cancer stem cells, immune cells mesenchymal stromal cells and/or cancer-associated fibroblasts all contribute to the tumor bulk. The interaction between these multiple players, under different microenvironmental conditions of biochemical and physical stimuli (i.e. oxygen tension, pH, matrix mechanics), regulates the production and biological activity of several soluble factors, extracellular matrix components, and extracellular vesicles that are needed for growth, maintenance, chemoresistance and metastatization of cancer. In osteosarcoma, a very aggressive cancer of young adults characterized by the extensive need for more effective therapies, this aspect has been only recently explored. In this view, we will discuss the role of stroma, with a particular focus on the mesenchymal stroma, contributing to osteosarcoma progression through inherent features for homing, neovascularization, paracrine cross-feeding, microvesicle secretion, and immune modulation, and also by responding to the changes of the microenvironment that are induced by tumor cells. The most recent advances in the molecular cues triggered by cytokines, soluble factors, and metabolites that are partially beginning to unravel the axis between stromal elements of mesenchymal origin and osteosarcoma cells, will be reviewed providing insights likely to be used for novel therapeutic approaches against sarcomas.

title: Mesenchymal Stem/Stromal Cells under Stress Increase Osteosarcoma Migration and Apoptosis Resistance via Extracellular Vesicle Mediated Communication
abstract: Studies have shown that mesenchymal stem/stromal cells (MSCs) from bone marrow are involved in the growth and metastasis of solid tumors but the mechanism remains unclear in osteosarcoma (OS). Previous studies have raised the possibility that OS cells may receive support from associated MSCs in the nutrient deprived core of the tumors through the release of supportive macromolecules and growth factors either in vesicular or non-vesicular forms. In the present study, we used stressed mesenchymal stem cells (SD-MSCs), control MSCs and OS cells to examine the hypothesis that tumor-associated MSCs in nutrient deprived core provide pro-proliferative, anti-apoptotic, and metastatic support to nearby tumor cells. Assays to study of the effects of SD-MSC conditioned media revealed that OS cells maintained proliferation when compared to OS cells grown under serum-starved conditions alone. Furthermore, OS cells in MSCs and SD-MSC conditioned media were significantly resistant to apoptosis and an increased wound healing rate was observed in cells exposed to either conditioned media or EVs from MSCs and SD-MSCs. RT-PCR assays of OS cells incubated with extracellular vesicles (EVs) from SD-MSCs revealed microRNAs that could potentially target metabolism and metastasis associated genes as predicted by in silico algorithms, including monocarboxylate transporters, bone morphogenic receptor type 2, fibroblast growth factor 7, matrix metalloproteinase-1, and focal adhesion kinase-1. Changes in the expression levels of focal adhesion kinase, STK11 were confirmed by quantitative PCR assays. Together, these data indicate a tumor supportive role of MSCs in osteosarcoma growth that is strongly associated with the miRNA content of the EVs released from MSCs under conditions that mimic the nutrient deprived core of solid tumors.

title: MicroRNAs in chromosomal translocation-associated solid tumors: learning from sarcomas.
abstract: Sarcomas are malignant solid tumors of mesenchymal origin which consist of 10-15% of all pediatric malignancies and associated with significantly high mortality rates despite current therapies. Oncogenic fusion genes, resulting from non-random chromosomal translocations, characterize a subset of sarcomas including rhabdomyosarcoma, the Ewing's sarcoma family of tumors, and synovial sarcoma. As investigators gain further insight into the role that fusion genes play in the development and progression of sarcomas, we are slowly uncovering novel molecules and pathways that are proving to be essential for the growth and maintenance of sarcomas and other malignancies. MicroRNAs (miRs) have been implicated in a diversity of human diseases including cancer. Only recently, has miR deregulation been shown to be an important component of sarcomagenesis. This review summarizes the recent discoveries tying miR deregulation to sarcoma biology and will discuss the potential and feasibility of miRs as novel therapeutic adjuncts to current therapies. The methodological approaches utilized in the study of miR biology and development of miR-based treatment regimens can serve as a paradigm for future investigations in other translocation-associated solid tumors.

title: Identification of Differentially Expressed MicroRNAs in Osteosarcoma
abstract: A limited number of reports have investigated the role of microRNAs in osteosarcoma. In this study, we performed miRNA expression profiling of osteosarcoma cell lines, tumor samples, and normal human osteoblasts. Twenty-two differentially expressed microRNAs were identified using high throughput real-time PCR analysis, and 4 (miR-135b, miR-150, miR-542-5p, and miR-652) were confirmed and validated in a different group of tumors. Both miR-135b and miR-150 have been previously shown to be important in cancer. We hypothesize that dysregulation of differentially expressed microRNAs may contribute to tumorigenesis. They might also represent molecular biomarkers or targets for drug development in osteosarcoma.

title: Blocking Tumor-Educated MSC Paracrine Activity Halts Osteosarcoma Progression
abstract: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets.  By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)–educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography.  We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients.  Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients.  23(14); 3721–33.

title: Ewing's sarcoma family of tumors: current management.
abstract: Ewing's sarcoma is the second most frequent primary bone cancer, with approximately 225 new cases diagnosed each year in patients less than 20 years of age in North America. It is one of the pediatric small round blue cell tumors, characterized by strong membrane expression of CD99 in a chain-mail pattern and negativity for lymphoid (CD45), rhabdomyosarcoma (myogenin, desmin, actin) and neuroblastoma (neurofilament protein) markers. Pathognomonic translocations involving the ews gene on chromosome 22 and an ets-type gene, most commonly the fli1 gene on chromosome 11, are implicated in the great majority of cases. Clinical presentation is usually dominated by local bone pain and a mass. Imaging reveals a technetium pyrophosphate avid lesion that, on plain radiograph, is destructive, diaphyseal and classically causes layered periosteal calcification. Magnetic resonance best defines the extent of the lesion. Biopsy should be undertaken by an experienced orthopedic oncologist. Approximately three quarters of patients have initially localized disease. About two thirds survive disease-free. Management, preferably at a specialist center with a multi-disciplinary team, includes both local control-either surgery, radiation or a combination-and systemic chemotherapy. Chemotherapy includes cyclic combinations, incorporating vincristine, doxorubicin, cyclophosphamide, etoposide, ifosfamide and occasionally actinomycin D. Topotecan in combination with cyclophosphamide has shown preliminary activity. Patients with initially metastatic disease fare less well, with about one quarter surviving. Studies incorporating intensive therapy followed by stem cell infusion show no clear benefit. New approaches include anti-angiogenic therapy, particularly since vascular endothelial growth factor is an apparent downstream target of the ews-fli1 oncogene.

title: Abnormal expression of neurofilament proteins in Ewing's sarcoma cell cultures.
abstract: A neural origin of Ewing's sarcoma (ES) has often been suggested and we have demonstrated neurofilament protein expression in ES cells. However, only the 200-kD subunit has been revealed in all of the ES cells analyzed. The 160- and 68-kD subunits were always absent. For these reasons, we have attempted to induce neural differentiation in 3 ES cell lines with different types of inducers: tetradecanoylphorbol-13-acetate (TPA) retinoic acid and nerve growth factor. When the cell lines were cultured for 7 days with TPA (10(-9) M) or retinoic acid (10(-7) M), only the 68-kD neurofilament subunit was slightly induced. No inducation was obtained when nerve growth factor was used, even at a 21-day culture. These results are in agreement with the putative neural origin of ES and may indicate an abnormal expression of neurofilament proteins in this tumor.

title: CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis.
abstract: Ewing sarcoma (EWS) is an aggressive bone tumor of uncertain cellular origin. CD99 is a membrane protein that is expressed in most cases of EWS, although its function in the disease is unknown. Here we have shown that endogenous CD99 expression modulates EWS tumor differentiation and malignancy. We determined that knocking down CD99 expression in human EWS cell lines reduced their ability to form tumors and bone metastases when xenografted into immunodeficient mice and diminished their tumorigenic characteristics in vitro. Further, reduction of CD99 expression resulted in neurite outgrowth and increased expression of beta-III tubulin and markers of neural differentiation. Analysis of a panel of human EWS cells revealed an inverse correlation between CD99 and H-neurofilament expression, as well as an inverse correlation between neural differentiation and oncogenic transformation. As knockdown of CD99 also led to an increase in phosphorylation of ERK1/2, we suggest that the CD99-mediated prevention of neural differentiation of EWS occurs through MAPK pathway modulation. Together, these data indicate a new role for CD99 in preventing neural differentiation of EWS cells and suggest that blockade of CD99 or its downstream molecular pathway may be a new therapeutic approach for EWS.

title: Chemotaxis in cancer
abstract: 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.

title: Exosome secretion promotes chemotaxis of cancer cells
abstract: Migration of cells toward chemical cues, or chemotaxis, is important for many biologic processes such as immune defense, wound healing and cancer metastasis. Although chemotaxis is thought to occur in cancer cells, it is less well characterized than chemotaxis of professional immune cells such as neutrophils. Here, we show that cancer cell chemotaxis relies on secretion of exosome-type extracellular vesicles. Migration of fibrosarcoma cells toward a gradient of exosome-depleted serum was diminished by knockdown of the exosome secretion regulator Rab27a. Rescue experiments in which chemotaxis chambers were coated with purified extracellular vesicles demonstrate that exosomes but not microvesicles affect both speed and directionality of migrating cells. Chamber coating with purified fibronectin and fibronectin-depleted exosomes demonstrates that the exosome cargo fibronectin promotes cell speed but cannot account for the role of exosomes in promoting directionality of fibrosarcoma cell movement during chemotaxis. These experiments indicate that exosomes contain multiple motility-promoting cargoes that contribute to different aspects of cell motility.

title: Progression of Osteosarcoma from a Non-Metastatic to a Metastatic Phenotype Is Causally Associated with Activation of an Autocrine and Paracrine uPA Axis
abstract: Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS) resulting in 10–20% long-term survival. The factors and pathways regulating these processes remain unclear, yet their identification is crucial in order to find new therapeutic targets. In this study we used a multi-omics approach to identify molecules in metastatic and non-metastatic OS cells that may contribute to OS metastasis, followed by validation in vitro and in vivo. We found elevated levels of the urokinase plasminogen activator (uPA) and of the uPA receptor (uPAR) exclusively in metastatic OS cells. uPA was secreted in soluble form and as part of the protein cargo of OS-secreted extracellular vesicles, including exosomes. In addition, in the tumour microenvironment, uPA was expressed and secreted by bone marrow cells (BMC), and OS- and BMC-derived uPA significantly and specifically stimulated migration of metastatic OS cells via uPA-dependent signaling pathways. Silencing of uPAR in metastatic OS cells abrogated the migratory response to uPA in vitro and decreased metastasis in vivo. Finally, a novel small-molecule inhibitor of uPA significantly (P = 0.0004) inhibited metastasis in an orthotopic mouse model of OS. Thus, we show for the first time that malignant conversion of OS cells to a metastatic phenotype is defined by activation of the uPA/uPAR axis in both an autocrine and paracrine fashion. Furthermore, metastasis is driven by changes in OS cells as well as in the microenvironment. Finally, our data show that pharmacological inhibition of the uPA/uPAR axis with a novel small-molecule inhibitor can prevent the emergence of metastatic foci.

title: Matrix metalloproteinases participate in osteosarcoma invasion.
abstract: Osteosarcoma (OS) is a highly malignant bone tumor and is the most frequent malignant bone tumor in children and adolescents. Metastases are the major cause of death, and patients with relapse have poor prognosis. Several solid tumors display enhanced expression of matrix metalloproteinases (MMPs), and recently MMP-inhibitors have entered clinical trials. A disturbance of the MMP system in favor of enhanced proteolytic activity may be suspected in OS because OS growth is accompanied by both enhanced local bone destruction and bone formation, two processes that are dependant on proteolytic enzymes. Thus, the aim of the present study was to evaluate the involvement of MMPs in a panel of human OS cell lines, xenografts and biopsies.



Expression of MMPs and their endogenous inhibitors were studied by zymography and Northern blot analyses. In vitro invasion of OS cell lines and effects of MMP-inhibitors (Marimastat and doxycycline) were assessed in the transwell chamber assay.



In vitro invasiveness was compared with gelatinase activity, and the most invasive cell line secreted the highest amounts of MMP-2 and MMP-9. Two different MMP-inhibitors significantly reduced OS cell invasion. The majority of the OS xenografts expressed both the inactive and active form of MMP-2 and in some cases also MMP-9. The biopsies from primary and metastatic OS also expressed MMP-2 mRNA. However, MMP-9 levels were higher in the biopsies than in the xenografts.



The obtained results support the hypothesis that MMPs and their endogenous inhibitors participate in the invasive process of human OS.

title: Increased expression of membrane-type matrix metalloproteinase-1 is correlated with poor prognosis in patients with osteosarcoma.
abstract: Numerous studies have demonstrated a correlation of matrix metalloproteinase (MMP) overexpression with the prognosis of various kinds of cancer. The current study investigated whether the expression of MMPs is correlated with the prognosis of osteosarcoma. Expression levels of MMP-2, -9, MT1-MMP and TIMP-2 were examined immunohistochemically in samples from 47 patients with osteosarcoma. Correlation of the positivity of staining with prognosis was analyzed with the Kaplan-Meier method, and statistically analyzed with log-rank test. Co-localization of MMP-2, MT1-MMP and TIMP-2 was determined by double staining with fluorescence-conjugated antibodies. Activities of gelatinases in representative tissues were examined with gelatin zymography. MMP-2 was expressed strongly in 60% of cases (28/47), and MMP-9, MT1- MMP and TIMP-2 was strongly positive in 61% (29/47), 45% (21/47), and 91% (43/47), respectively. Increased MT1-MMP expression was associated significantly with poor prognosis in overall survival (P=0.0480). In cases of overexpression for both MMP-2 and MT1-MMP, there was a tendency for poor prognosis (P=0.0969). In 36 cases who underwent neoadjuvant chemotherapy, wide resection of the tumors and post-operative adjuvant chemotherapy, increased expression of MT1-MMP resulted in a significant negative prognostic factor for disease-free survival (P=0.0143). Also, co-overexpression of MT1-MMP and MMP-2 showed a tendency to correlate to the reduced disease-free survival (P=0.0502). Increased gelatinase activity was observed in tissues of co-overexpression of MT1-MMP and MMP-2. The results of this study demonstrate the correlation of MT1-MMP expression and the oncological outcome of osteosarcoma patients, suggesting the prognostic significance of these proteins in osteosarcoma patients.

title: Secretion of active membrane type 1 matrix metalloproteinase (MMP‐14) into extracellular space in microvesicular exosomes
abstract: Membrane type 1 matrix metalloproteinase (MT1‐MMP, MMP14) is an efficient extracellular matrix (ECM) degrading enzyme that plays important roles in tissue homeostasis and cell invasion. Like a number of type I membrane proteins, MT1‐MMP can be internalized from the cell surface through early and recycling endosomes to late endosomes, and recycled to the plasma membrane. Late endosomes participate in the biogenesis of small (30–100 nm) vesicles, exosomes, which redirect plasma membrane proteins for extracellular secretion. We hypothesized that some of the endosomal MT1‐MMP could be directed to exosomes for extracellular release. Using cultured human fibrosarcoma (HT‐1080) and melanoma (G361) cells we provide evidence that both the full‐length 60 kDa and the proteolytically processed 43 kDa forms of MT1‐MMP are secreted in exosomes. The isolated exosomes were identified by their vesicular structure in electron microscopy and by exosomal marker proteins CD9 and tumor susceptibility gene (TSG101). Furthermore, exosomes contained β1‐integrin (CD29). The exosomes were able to activate pro‐MMP‐2 and degrade type 1 collagen and gelatin, suggesting that the exosomal MT1‐MMP was functionally active. The targeting of MT1‐MMP in exosomes represents a novel mechanism for cancer cells to secrete membrane type metalloproteolytic activity into the extracellular space.  2008.

title: Biotechnological approach for systemic delivery of membrane Receptor Activator of NF-κB Ligand (RANKL) active domain into the circulation
abstract: Deficiency of Receptor Activator of NF-κB Ligand (RANKL) prevents osteoclast formation causing osteopetrosis. RANKL is a membrane-bound protein cleaved into active soluble (s)RANKL by metalloproteinase 14 (MMP14). We created a bio-device that harbors primary osteoblasts, cultured on 3D hydroxyapatite scaffolds carrying immobilized MMP14 catalytic domain. Scaffolds were sealed in diffusion chambers and implanted in RANKL-deficient mice. Mice received 1 or 2 diffusion chambers, once or twice and were sacrificed after 1 or 2 months from implants. A progressive increase of body weight was observed in the implanted groups. Histological sections of tibias of non-implanted mice were negative for the osteoclast marker Tartrate-Resistant Acid Phosphatase (TRAcP), consistent with the lack of osteoclasts. In contrast, tibias excised from implanted mice showed TRAcP-positive cells in the bone marrow and on the bone surface, these latter morphologically similar to mature osteoclasts. In mice implanted with 4 diffusion chambers total, we noted the highest number and size of TRAcP-positive cells, with quantifiable eroded bone surface and significant reduction of trabecular bone volume. These data demonstrate that our bio-device delivers effective sRANKL, inducing osteoclastogenesis in RANKL-deficient mice, supporting the feasibility of an innovative experimental strategy to treat systemic cytokine deficiencies.

title: Extracellular Membrane Vesicles Derived from 143B Osteosarcoma Cells Contain Pro-Osteoclastogenic Cargo: A Novel Communication Mechanism in Osteosarcoma Bone Microenvironment1234
abstract: The bone microenvironment (BME) is the main hub of all skeletal related pathological events in osteosarcoma leading to tumor induced bone destruction, and decreasing overall bone quality and bone strength. The role of extra-cellular membrane vesicles (EMVs) as mediators of intercellular communication in modulating osteosarcoma-BME is unknown, and needs to be investigated. It is our hypothesis that osteosarcoma-EMVs contain pro-osteoclastogenic cargo which increases osteoclastic activity, and dysregulated bone remodeling in the osteosarcoma-BME. In this study, EMVs were isolated from the conditioned media of 143B and HOS human osteosarcoma cell cultures using differential ultracentrifugation. Nano-particle tracking analysis determined EMVs in the size range of 50-200 nm in diameter. The EMV yield from 143B cells was relatively higher compared to HOS cells. Transmission electron microscopy confirmed the ultrastructure of 143B-EMVs and detected multivesicular bodies. Biochemical characterization of 143B-EMVs detected the expression of bioactive pro-osteoclastic cargo including matrix metalloproteinases-1 and -13 (MMP-1, -13), transforming growth factor-β (TGF-β), CD-9, and receptor activator of nuclear factor kappa-β ligand (RANKL). Detection of a protein signature that is uniquely pro-osteoclastic in 143B-EMVs is a novel finding, and is significant as EMVs represent an interesting mechanism for potentially mediating bone destruction in the osteosarcoma-BME. This study further demonstrates that 143B cells actively mobilize calcium in the presence of ionomycin, and forskolin, and induce cytoskeleton rearrangements leading to vesicular biogenesis. In conclusion, this study demonstrates that 143B osteosarcoma cells generate EMVs mainly by mechanisms involving increased intracellular calcium or cAMP levels, and contain pro-osteoclastic cargo.

title: Matrix Metalloproteinase 1 promotes tumor formation and lung metastasis in an intratibial injection osteosarcoma mouse model.
abstract: Proteolytic degradation of the extracellular matrix (ECM) is an important process during tumor invasion. Matrix Metalloproteinase 1 (MMP-1) is one of the proteases that degrade collagen type I, a major component of bone ECM. In the present study, the biological relevance of MMP-1 in osteosarcoma (OS) tumor growth and metastasis was investigated in vitro and in vivo. Human OS cells in primary culture expressed MMP-1 encoding mRNA at considerably higher levels than normal human bone cells. In addition, MMP-1 mRNA and protein expression in the highly metastatic human osteosarcoma 143-B cell line was remarkably higher than in the non-metastatic parental HOS cell line. Stable shRNA-mediated downregulation of MMP-1 in 143-B cells impaired adhesion to collagen I and anchorage-independent growth, reflected by a reduced ability to grow in soft agar. Upon intratibial injection into SCID mice, 143-B cells with shRNA-downregulated MMP-1 expression formed smaller primary tumors and significantly lower numbers of lung micro- and macrometastases than control cells. Conversely, HOS cells stably overexpressing MMP-1 showed an enhanced adhesion capability to collagen I and accelerated anchorage-independent growth compared to empty vector-transduced control cells. Furthermore, and most importantly, individual MMP-1 overexpression in HOS cells enabled the formation of osteolytic primary tumors and lung metastasis while the HOS control cells did not develop any tumors or metastases after intratibial injection. The findings of the present study reveal an important role of MMP-1 in OS primary tumor and metastasis formation to the lung, the major organ of OS metastasis.

title: Role of MMP-9 and its tissue inhibitor TIMP-1 in human osteosarcomaFindings in 42 patients followed for 1–16 years
abstract: The activity of matrix metalloproteinases (MMPs) in degrading extracellular matrix is controlled by activation of proenzymes and inhibition of MMP tissue inhibitors (TIMPs). Patients and methods To assess the proteolytic cascade imbalance in malignancy progression, tissue expression and serum levels of MMP-2, MMP-9 and of their inhibitors TIMP-2 and TIMP-1 respectively were evaluated in 42 selected patients with high-grade osteosarcoma (OS). MMP-2, MMP-9, TIMP-2 and TIMP-1 were studied in biopsies by immunohistochemistry and in serum by ELISA test. Patients were subdivided into 3 groups according to their follow up: continuously disease-free, diagnosis of metastasis during follow-up, and metastasis at diagnosis. Immunohistochemistry demonstrated an imbalance between MMPs and TIMPs, with a more evident role for MMP-9 than for MMP-2 in tumor progression. TIMP-1 inhibitor in plasma was higher in patients with osteosarcoma than in a control group. This high value of TIMP-1 was particularly evident in the group of patients who later developed metastases and/or local recurrences, and in those with metastases at diagnosis. Our findings confirm the protective action of TIMP-1, as MMP inhibitor, but also show its activity as a growth factor underlining its multifunctional role in OS.

title: MicroRNA-143 regulates human osteosarcoma metastasis by regulating matrix metalloprotease-13 expression.
abstract: Pulmonary metastases are the main cause of death in patients with osteosarcoma, however, the molecular mechanisms of metastasis are not well understood. To detect lung metastasis-related microRNA (miRNA) in human osteosarcoma, we compared parental (HOS) and its subclone (143B) human osteosarcoma cell lines showing lung metastasis in a mouse model. miR-143 was the most downregulated miRNA (P < 0.01), and transfection of miR-143 into 143B significantly decreased its invasiveness, but not cell proliferation. Noninvasive optical imaging technologies revealed that intravenous injection of miR-143, but not negative control miRNA, significantly suppressed lung metastasis of 143B (P < 0.01). To search for miR-143 target mRNA in 143B, microarray analyses were performed using an independent RNA pool extracted by two different comprehensive miR-143-target mRNA collecting systems. Western blot analyses revealed that MMP-13 was mostly protein downregulated by miR-143. Immunohistochemistry using clinical samples clearly revealed MMP-13-positive cells in lung metastasis-positive cases, but not in at least three cases showing higher miR-143 expression in the no metastasis group. Taken together, these data indicated that the downregulation of miR-143 correlates with the lung metastasis of human osteosarcoma cells by promoting cellular invasion, probably via MMP-13 upregulation, suggesting that miRNA could be used to develop new molecular targets for osteosarcoma metastasis.

title: Exosomes from Osteosarcoma and normal osteoblast differ in proteomic cargo and immunomodulatory effects on T cells
abstract: Canine osteosarcoma (OSA) is the most common cancer of the appendicular skeleton and is associated with high metastatic rate to the lungs and poor prognosis. Recent studies have shown the impact of malignant‐derived exosomes on immune cells and the facilitation of immune evasion. In the current study, we have characterized the proteomic profile of exosomes derived from healthy osteoblasts and osteosarcoma cell lines. We investigated the direct impact of these exosomes on healthy T cells.  Proteomic cargo of the malignant exosomes was markedly different from osteoblastic exosomes and contained immunosuppressive proteins including TGF‐&bgr;, &agr; fetoprotein and heat shock proteins. OSA exosomes directly attenuated the rate of T cell proliferation, increased a regulatory (FoxP3+) CD4+ phenotype and diminished the expression of the activation marker CD25+ on CD8+ cells. Exosomes of osteoblasts also demonstrated a direct impact on T cells, but to a lesser degree.  Osteosarcoma‐derived exosomes compared to normal osteoblasts contain an immunomodulatory cargo, which reduced the rate of T cell proliferation and promoted T regulatory phenotype. Osteoblast‐derived exosomes can also reduce T cell activity, but to lesser degree compared to OSA exosomes and without promoting a T regulatory phenotype. HIGHLIGHTSProteomic profiles of osteoblasts and osteosarcoma exosomes have been characterized.Malignant exosomes have immunosuppressive phenotype.Osteosarcoma exosomes inhibit CD4+ and CD8+ T cell proliferation and activation.Osteosarcoma exosomes can upregulate the expression of FOXP3 and CD25+ in CD4+ T cells.

title: Proteomic Analysis of Exosomes and Exosome‐Free Conditioned Media From Human Osteosarcoma Cell Lines Reveals Secretion of Proteins Related to Tumor Progression
abstract: Osteosarcomas are the most prevalent bone tumors in pediatric patients, but can also occur later in life. Bone tumors have the potential to metastasize to lung and occasionally other vital organs. To understand how osteosarcoma cells interact with their micro‐environment to support bone tumor progression and metastasis, we analyzed secreted proteins and exosomes from three human osteosarcoma cell lines. Exosome isolation was validated by transmission electron microscopy (TEM) and immuno‐blotting for characteristic biomarkers (CD63, CD9, and CD81). Exosomal and soluble proteins (less than 100 kDa) were identified by mass spectrometry analysis using nanoLC‐MS/MS and classified by functional gene ontology clustering. We identified a secretome set of >3,000 proteins for both fractions, and detected proteins that are either common or unique among the three osteosarcoma cell lines. Protein ontology comparison of proteomes from exosomes and exosome‐free fractions revealed differences in the enrichment of functional categories associated with different biological processes, including those related to tumor progression (i.e., angiogenesis, cell adhesion, and cell migration). The secretome characteristics of osteosarcoma cells are consistent with the pathological properties of tumor cells with metastatic potential.  2017.

title: Serum miR-195 is a diagnostic and prognostic marker for osteosarcoma.
abstract: MicroRNAs have been proved to be successful diagnostic and prognostic markers in several cancers, including osteosarcoma. miR-195, as a tumor suppressor, has been reported to suppress osteosarcoma cell invasion and migration in vitro. However, its clinical value is still unclear. The aim of this study was to determine the expression pattern of miR-195 in osteosarcoma patients and to investigate its associations with tumor progression and prognosis.



miR-195 levels in sera from 166 osteosarcoma patients and 60 healthy controls were detected by real-time quantitative reverse transcription polymerase chain reaction. Then, the association of serum miR-195 level with clinicopathologic factors or survival of osteosarcoma patients was further evaluated.



miR-195 levels in sera from osteosarcoma patients were significantly lower than those in healthy controls (P < 0.001). Importantly, miR-195 could efficiently screen osteosarcoma patients from healthy controls (area under receiver operating characteristic curve = 0.892). Additionally, low miR-195 expression was more frequently occurred in osteosarcoma patients with advanced clinical stage (P = 0.002) and positive distant metastasis (P = 0.008). Univariate survival analysis revealed that osteosarcoma patients with low miR-195 expression had significantly shorter overall survival and disease-free survival than those with high miR-195 expression (both P < 0.001). Multivariate analysis further determined that low miR-195 expression was an independent and significant prognostic factor to predict poor overall survival (P = 0.002) and disease-free survival (P = 0.01).



Decreased expression of miR-195 in serum may be a novel biomarker for screening osteosarcoma and can predict poor prognosis. Detection of serum miR-195 expression may have potential applications for the diagnosis, prognosis, and treatment of osteosarcoma.

title: Circulating miR-148a is a significant diagnostic and prognostic biomarker for patients with osteosarcoma
abstract: The purpose of the present study was to detect the expression levels of circulating miR-148a in the peripheral blood of osteosarcoma patients and to further investigate the clinicopathological, diagnostic, and prognostic value of miR-148a. Eighty-nine patients with initially diagnosed osteosarcoma who successfully underwent surgical resection were enrolled in this prospective study. The expression levels of circulating miR-148a were detected by real-time quantitative RT-PCR. All statistical analyses were performed with SPSS 18.0 statistical software to determine the potential values of circulating miR-148a on the clinicopathological factors, diagnosis, and prognosis. The expression levels of circulating miR-148a in osteosarcoma patients were significantly higher than that in the healthy controls (P < 0.001), and miR-148a was capable of distinguishing osteosarcoma patients from healthy controls effectively (AUC = 0.783). In addition, miR-148a expression was significantly associated with tumor size (P = 0.049) and distant metastasis (P = 0.004). Univariate survival analysis demonstrated that patients with miR-148a high expression had significantly poor overall survival (P < 0.001) and disease-specific survival (P < 0.001) after 5 years’ operation. Multivariate survival analysis confirmed that miR-148a high expression was an independent predictor for unfavorable overall survival (P = 0.003) and disease-specific survival (P = 0.008), respectively. Our findings demonstrated that detection of circulating miR-148a expression in the peripheral blood have clinical potentials as an indicator of progressive phenotype, a novel diagnostic biomarker and a promising predictor to identify individuals with poor prognosis for osteosarcoma patients.

title: Clinical significance of circulating miR-25-3p as a novel diagnostic and prognostic biomarker in osteosarcoma
abstract: Emerging evidence has suggested that circulating microRNAs (miRNAs) in body fluids have novel diagnostic and prognostic significance for patients with malignant diseases. The lack of useful biomarkers is a crucial problem of bone and soft tissue sarcomas; therefore, we investigated the circulating miRNA signature and its clinical relevance in osteosarcoma.  Global miRNA profiling was performed using patient serum collected from a discovery cohort of osteosarcoma patients and controls and cell culture media. The secretion of the detected miRNAs from osteosarcoma cells and clinical relevance of serum miRNA levels were evaluated using in vitro and in vivo models and a validation patient cohort.  Discovery screening identified 236 serum miRNAs that were highly expressed in osteosarcoma patients compared with controls, and eight among these were also identified in the cell culture media. Upregulated expression levels of miR-17-5p and miR-25-3p were identified in osteosarcoma cells, and these were abundantly secreted into the culture media in tumor-derived exosomes. Serum miR-25-3p levels were significantly higher in osteosarcoma patients than in control individuals in the validation cohort, with favorable sensitivity and specificity compared with serum alkaline phosphatase. Furthermore, serum miR-25-3p levels at diagnosis were correlated with patient prognosis and reflected tumor burden in both in vivo models and patients; these associations were more sensitive than those of serum alkaline phosphatase.  Serum-based circulating miR-25-3p may serve as a non-invasive blood-based biomarker for tumor monitoring and prognostic prediction in osteosarcoma patients.

title: Increased expression of a set of genes enriched in oxygen binding function discloses a predisposition of breast cancer bone metastases to generate metastasis spread in multiple organs
abstract: Bone is the preferential site of distant metastasis in breast carcinoma (BrCa). Patients with metastasis restricted to bone (BO) usually show a longer overall survival compared to patients who rapidly develop multiple metastases also involving liver and lung. Hence, molecular predisposition to generate bone and visceral metastases (BV) represents a clear indication of poor clinical outcome. We performed microarray analysis with two different chip platforms, Affymetrix and Agilent, on bone metastasis samples from BO and BV patients. The unsupervised hierarchical clustering of the resulting transcriptomes correlated with the clinical progression, segregating the BO from the BV profiles. Matching the twofold significantly regulated genes from Affymetrix and Agilent chips resulted in a 15‐gene signature with 13 upregulated and two downregulated genes in BV versus BO bone metastasis samples. In order to validate the resulting signature, we isolated different MDA‐MB‐231 clonal subpopulations that metastasize only in the bone (MDA‐BO) or in bone and visceral tissues (MDA‐BV). Six of the signature genes were also significantly upregulated in MDA‐BV compared to MDA‐BO clones. A group of upregulated genes, including Hemoglobin B (HBB), were involved in oxygen metabolism, and in vitro functional analysis of HBB revealed that its expression in the MDA subpopulations was associated with a reduced production of hydrogen peroxide. Expression of HBB was detected in primary BrCa tissue but not in normal breast epithelial cells. Metastatic lymph nodes were frequently more positive for HBB compared to the corresponding primary tumors, whereas BO metastases had a lower expression than BV metastases, suggesting a positive correlation between HBB and ability of bone metastasis to rapidly spread to other organs. We propose that HBB, along with other genes involved in oxygen metabolism, confers a more aggressive metastatic phenotype in BrCa cells disseminated to bone.

title: Bone Metastases: An Overview
abstract: Bone is a frequent site of metastases and typically indicates a short-term prognosis in cancer patients. Once cancer has spread to the bones it can rarely be cured, but often it can still be treated to slow its growth. The majority of skeletal metastases are due to breast and prostate cancer. Bone metastasis is actually much more common than primary bone cancers, especially in adults. The diagnosis is based on signs, symptoms and imaging. New classes of drugs and new interventions are given a better quality of life to these patients and improved the expectancy of life. It is necessary a multidisciplinary approach to treat patients with bone metastasis. In this paper we review the types, clinical approach and treatment of bone metastases.

title: Mechanisms of multiple myeloma bone disease.
abstract: Multiple myeloma is the second most common hematological malignancy and the most frequent cancer to involve the skeleton. Multiple myeloma bone disease (MMBD) is characterized by abnormal bone remodeling with dysfunction of both bone resorption and bone formation, and thus can be used as a paradigm for other inflammatory bone diseases, and the regulation of osteoclasts and osteoblasts in malignancy. Studies of MMBD have identified novel regulators that increase osteoclastogenesis and osteoclast function, repress osteoblast differentiation, increase angiogenesis, or permanently alter stromal cells. This review will discuss the current understanding of mechanisms of osteoclast and osteoblast regulation in MMBD, and therapeutic approaches currently in use and under development that target mediators of bone destruction and blockade of bone formation for myeloma patients, including new anabolic therapies.

title: Abnormal bone remodelling in patients with myelomatosis and normal biochemical indices of bone resorption
abstract: We studied bone biopsies from 26 patients with myelomatosis with apparently normal skeletal metabolism. Quantitative histomorphometric measurements suggested that skeletal disease was progressive despite normocalcaemia and normal urinary excretion rates of calcium and hydroxyproline. When biopsies were divided according to the involvement of marrow by plasma cells, bone resorption — as judged by the eroded surface — increased significantly the greater plasma cell burden. Osteoclasts were frequent with moderate tumour burdens, but there was no further increase in the number of osteoclasts when plasma cell infiltration increased by more than 50% of bone marrow. Contrary to expectation, the numbers of osteoblasts and bone formation rates were increased with bone biopsies with moderate tumour burden, but were markedly lower when plasma cell infiltration occupied more than 50% of bone marrow, due to a decreased functional capacity of osteoblasts. We conclude that skeletal bone disease in myeloma is commonly progressive despite apparently stable bone disease as judged by biochemical measurements. The major mechanism of bone loss in myelomatosis is increased osteoclastic resorption but decreased bone formation contributes to bone loss with heavy plasma cell burdens. Urinary excretion of calcium and hydroxyproline provide insensitive indices of bone resorption in myelomatosis.

title: Pathologic fractures correlate with reduced survival in patients with malignant bone disease
abstract: Data from randomized, controlled trials of zoledronic acid were retrospectively analyzed to assess the effect of pathologic fractures on survival in patients with malignant bone disease.

title: Classic Hodgkin lymphoma with osseous involvement mimicking Langerhans cell histiocytosis in a child.
abstract: Hodgkin lymphoma (HL) commonly presents superficial lymphadenopathy. In addition, HL cells can arise in various organs including the liver and spleen as an extranodal lymphoma. HL in bone is unusual at the initial diagnosis, although some cases show late-stage localization of lymphoma cells to bone. We report the rare case of a young patient with cranial bone classic HL, presumably originating from the skull without any involvement of lymph nodes. As the main clinical manifestation was only tumor mass in the skull without osteoscopic pain, the tentative diagnosis of Langerhans cell histiocytosis was histologically confirmed by an excisional biopsy. Before the final pathological diagnosis as classic HL, we noticed several small lesions in extranodal regions through systemic surveys, suggesting that the cranial lesion appeared antecedent to those lesions. This is a rare and instructive case of cranial bone HL for which a histological diagnosis has been meticulously made.

title: Calcitriol Production in Hypercalcemic and Normocalcemic Patients with Non-Hodgkin Lymphoma
abstract: As many as 15% of patients with non-Hodgkin lymphoma develop hypercalcemia at some time during the course of their disease [1-3]. However, the pathogenesis of this complication is not understood. Anecdotal reports have implicated calcitriol as a possible mediator in some patients [4-10], but no large studies have been done. Under physiologic conditions, the kidney, under the control of parathyroid hormone, is the sole source of circulating calcitriol [11]. Hypercalcemia inhibits parathyroid hormone production and thereby diminishes renal calcitriol synthesis [11, 12]. An elevated serum calcium level itself also inhibits renal calcitriol production [13], as does hyperphosphatemia [11]. Unless an autonomous source of parathyroid hormone is present, as in primary hyperparathyroidism [14, 15], serum calcitriol levels are suppressed during hypercalcemia; a failure to observe such suppression is evidence of disordered regulation of calcitriol synthesis and usually indicates extrarenal production. Such extrarenal calcitriol production causes hypercalcemia in sarcoidosis, in which activated macrophages within the granuloma synthesize calcitriol [16, 17]. The available reference ranges for serum calcitriol levels are derived from normocalcemic persons, and the appropriate serum level in the setting of hypercalcemia is unknown. Thus, recognizing abnormal calcitriol regulation in the absence of a frank elevation of the serum level is problematic. To better determine the incidence of abnormal calcitriol production during hypercalcemia that complicates non-Hodgkin lymphoma, we studied hypercalcemic patients with myeloma as a control group. Healthy persons rendered severely hypercalcemic by calcium infusion also would have been appropriate, but such study is not ethically possible. However, neither patients with primary hyperparathyroidism nor those with hypercalcemia that complicates solid tumors are suitable as controls. In both of these disorders, extraneous factors substantially influence calcitriol metabolism. In primary hyperparathyroidism, the elevated serum level of parathyroid hormone enhances calcitriol production [14, 15]. In patients with hypercalcemia that complicates solid tumors, the serum level of the novel hormone parathyroid hormonerelated protein is often elevated [18-21]. Parathyroid hormonerelated protein, like parathyroid hormone, can increase renal calcitriol production [22, 23]. Despite this capacity, serum calcitriol levels are rarely elevated in these patients [24], possibly because of a tumor-derived humoral factor that inhibits renal calcitriol production . In patients with myeloma, however, it is known that calcitriol levels are suppressed during episodes of hypercalcemia and return to normal when hypercalcemia resolves after bisphosphonate treatment [26]. Our study had two aims: 1) to establish a reference range for serum calcitriol during hypercalcemia in the control group and, using this reference range, to determine the frequency of elevated serum levels of calcitriol in hypercalcemic patients with non-Hodgkin lymphoma and 2) to investigate the frequency of abnormal calcium metabolism in normocalcemic patients with non-Hodgkin lymphoma.  Patients From July 1992 to May 1993, we determined the serum calcium levels in patients with lymphoma and myeloma to identify those who had non-Hodgkin lymphoma or myeloma and hypercalcemia. During this time, approximately 345 new patients were seen in the lymphoma clinic and 90 were seen in the myeloma clinic, although both newly diagnosed patients and patients with relapse were eligible for this study. Hypercalcemia was defined as a corrected serum calcium level of 11.0 mg/dL (2.75 mmol/L) or greater, in which corrected serum calcium (mg/dL) = total serum calcium (mg/dL) + 0.8(4.0 serum albumin [g/dL]) . Corrected serum calcium in mg/dL can be converted to mmol/L by multiplying by 0.25. We identified 28 patients with non-Hodgkin lymphoma and hypercalcemia and excluded 6 from the study for the following reasons: One patient had a B-cell, diffuse large-cell lymphoma and a corrected serum calcium level of 2.94 mmol/L (11.8 mg/dL) and could not be studied; 3 patients had elevated serum levels of intact parathyroid hormone (144, 63, and 221 pg/mL, respectively), suggesting primary hyperparathyroidism; 1 patient had idiopathic absorptive hypercalciuria; and 1 patient had hypercalcemia caused by human T-cell lymphotrophic virus type 1related T-cell leukemia-lymphoma, in which parathyroid hormonerelated protein is known to mediate hypercalcemia [28]. The remaining 22 patients were studied within 48 hours of the documentation of hypercalcemia and before corticosteroids or cytotoxic drugs were administered. Patients usually received intravenous hydration during this interval, which may explain why 5 patients had corrected serum calcium values of less than 2.74 mmol/L (11.0 mg/dL) at the time of full evaluation. One patient had received bisphosphonate treatment one day before the study but was included because serum levels of parathyroid hormone, parathyroid hormonerelated protein, and calcitriol are known not to be influenced by bisphosphonate therapy during this period [29-31]. We excluded this patient from the analysis of urinary values because these may change rapidly after therapy with bisphosphonates [32]. We applied identical inclusion criteria for the control patients with myeloma. We identified 26 hypercalcemic patients and evaluated 17. One patient was excluded because of an elevated serum intact parathyroid hormone level (56 pg/mL); 16 patients thus remained for further study. All patients provided informed consent for investigational studies in accordance with institutional guidelines, no patients had preexisting chronic renal impairment (with a serum creatinine level of 132.6 mmol/L [1.5 mg/dL]), and none reported receiving vitamin D supplements, lithium, or thiazide diuretics before the study.  We determined serum electrolyte, albumin, and creatinine levels using an automated analyzer (Kodak, Rochester, New York). Urine was collected as a random fasting specimen, and urinary calcium excretion was calculated as the product of urinary calcium and serum creatinine levels divided by the urinary creatinine level, where all values are in mg/dL with a normal range of less than 0.14 mg/dL (<0.035 mmol/L) of glomerular filtrate . We calculated the renal tubular phosphate threshold (TmPo 4 3-), an index of phosphate resorption, using the method of Walton and Bijvoet [34]. The reference range for TmPo 4 3- is 2.5 to 4.2 mg/dL (0.80 to 1.35 mmol/L) of glomerular filtrate; a lower value indicates a greater degree of phosphaturia [34]. We measured serum levels of intact parathyroid hormone using a two-site chemiluminometric immunoassay (CIBA-Corning Diagnostics, Medfield, Massachusetts), with a normal range of 11 to 54 pg/mL and a lower limit of detection of 3 pg/mL. We extracted calcitriol from serum, purified it as described [35], and measured it using the chicken intestine cytosolic receptor competitive binding assay with tritium-labelled calcitriol standards of known concentration (SmithKline Beecham, Houston, Texas). The reference range for serum calcitriol during normocalcemia is 20 to 76 pg/mL; the assay used has a lower limit of detection of 5 pg/mL and a coefficient of variation of 15% at concentrations of 26 to 90 pg/mL. We measured serum 25-hydroxyvitamin D levels using a competitive protein-binding assay (SmithKline Beecham); the reference range is 10 to 55 ng/mL (25 to 138 nmol/L). Parathyroid hormonerelated protein in serum was measured using an immunoradiometric assay (Nichols Institute, San Juan, California). The assay uses a solid-phase antiparathyroid hormonerelated protein37-74 antibody and Iodine-125-labeled antiparathyroid hormonerelated protein1-36 as tracer and has no cross-reactivity with parathyroid hormone; it has a reference range of less than 1.5 pmol/L and a lower limit of detection of 0.2 pmol/L [21]. Statistical Analysis We compared categorical data using the chi-square test or the Fisher Exact test and ordinal data using the Wilcoxon two-sample test or the Kruskal-Wallis test as appropriate. Sequential values were compared using the paired student t-test. We evaluated correlations using the Spearman rank correlation coefficient and compared them on the basis of the slope determined from the linear regression analysis using the least-squares method. All P values are two-sided.  Control Group The median age of the hypercalcemic patients with myeloma was 60 years (range, 37 to 79 years); nine (56%) were male, and all had lytic bone lesions. Their mean serum calcitriol level was 11.6 10.0 pg/mL, and the highest level observed was 32 pg/mL . We defined the upper limit of expected serum calcitriol in hypercalcemia as the mean plus 3 standard deviations of the serum calcitriol levels measured in the control patients (<42 pg/mL). No patient in the control group had an elevated serum level of parathyroid hormonerelated protein (median, 0.2 pmol/L; range, <0.2 to 1.5 pmol/L). Other biochemical features are given in Figure 1 and Table 1. Table 1. Biochemical Features of Control Patients and Patients with Non-Hodgkin Lymphoma according to Serum Calcitriol Level* Figure 1. Distribution of serum levels of parathyroid hormonerelated protein (PTHrP), intact parathyroid hormone (PTH), and calcitriol and corrected serum calcium of control patients with multiple myeloma (right column) and of patients with non-Hodgkin lymphoma and either elevated (left column) or normal (center column) serum calcitriol levels. Hypercalcemic Patients with Non-Hodgkin Lymphoma We next applied the derived reference range for serum calcitriol levels during hypercalcemia to the 22 hypercalcemic patients with non-Hodgkin lymphoma and used the upper limit to divide the patients into two groups: those with elevated serum c

title: Amphiregulin contained in NSCLC-exosomes induces osteoclast differentiation through the activation of EGFR pathway
abstract: Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths worldwide. The majority of patients are diagnosed in advanced disease stage. Bone metastasis is the most frequent complication in NSCLC resulting in osteolytic lesions. The perfect balance between bone-resorbing osteoclasts and bone-forming osteoblasts activity is lost in bone metastasis, inducing osteoclastogenesis. In NSCLC, the epidermal growth factor receptor (EGFR) pathway is constitutively activated. EGFR binds Amphiregulin (AREG) that is overexpressed in several cancers such as colon, breast and lung. Its levels in plasma of NSCLC patients correlate with poor prognosis and AREG was recently found as a signaling molecule in exosomes derived from cancer cell lines. Exosomes have a key role in the cell-cell communication and they were recently indicated as important actors in metastatic niche preparation. In the present work, we hypothesize a role of AREG carried by exosomes derived from NSCLC in bone metastasis induction. We observed that NSCLC-exosomes, containing AREG, induce EGFR pathway activation in pre-osteoclasts that in turn causes an increased expression of RANKL. RANKL is able to induce the expression of proteolytic enzymes, well-known markers of osteoclastogenesis, triggering a vicious cycle in osteolytic bone metastasis.

title: Lung adenocarcinoma cell-derived exosomal miR-21 facilitates osteoclastogenesis.
abstract: Osteoclastogenesis is the rate-limiting step in tumor osteolytic metastasis. MicroRNAs play crucial roles in tumor progression and osteoclastogenesis regulation. Recent studies have demonstrated that exosomes are able to function as messengers that deliver microRNAs between cells. However, the effects of lung adenocarcinoma cell-derived exosomal miRNAs in osteoclastogenesis remain poorly understood. In this study, we found that exosomes derived from A549 cells facilitate osteoclastogenesis. As miR-21 was involved in tumorigenesis and osteoclastogenesis, we further proved the existence of miR-21 in A549 cell-derived exosomes and investigated its function. MiR-21 overexpression in A549 cells led to increased levels of miR-21 in exosomes and facilitated osteoclastogenesis. Conversely, miR-21 depletion in A549 cells down-regulated miR-21 in exosomes and alleviated osteoclastogenesis. Mechanical experiments demonstrated that exosomal miR-21 exerted its promoting effects on osteoclastogenesis via targeting Pdcd4, which is a known target of miR-21 and a regulator of osteoclastogenesis. Finally, clinical data showed that higher miR-21 levels were associated with a worse overall survival in lung adenocarcinoma patients. In conclusion, we found that lung adenocarcinoma derived exosomal miR-21 may facilitate osteoclastogenesis, which suggests that it is a potential therapeutic target of bone metastasis.

title: miRNA cargo within exosome‐like vesicle transfer influences metastatic bone colonization
abstract: Bone metastasis represents one of the most deleterious clinical consequences arising in the context of many solid tumors. Severe osteolysis results from tumor cell colonization of the bone compartment, a process which entails reciprocal exchange of soluble signals between tumor cells and their osseous microenvironment. Recent evidence indicates that tumor‐intrinsic miRNAs are pleiotropic regulators of gene expression. But they are also frequently released in exosome‐like vesicles (ELV). Yet the functional relevance of the transference of tumor‐derived ELV and their miRNA cargo to the extracellular milieu during osseous colonization is unknown.

title: Tumor Cell-Derived Exosomes from the Prostate Cancer Cell Line TRAMP-C1 Impair Osteoclast Formation and Differentiation
abstract: Skeletal metastatic disease is a deleterious consequence of dissemination of tumor cells from numerous primary sites, such as prostate, lung and breast. Skeletal metastases are still incurable, resulting in development of clinical complications and decreased survival for cancer patients with metastatic disease. During the last decade, tumor cell-derived microvesicles have been identified and suggested to be involved in cancer disease progression. Whether cancer exosomes are involved in tumor and bone cell interactions in the metastatic site is still, however, a rather unexplored field. Here we show that exosomes isolated from the murine prostate cancer cell line TRAMP-C1 dramatically decrease fusion and differentiation of monocytic osteoclast precursors to mature, multinucleated osteoclasts. The presence of tumor cell-derived exosomes also clearly decreased the expression of established markers for osteoclast fusion and differentiation, including DC-STAMP, TRAP, cathepsin K, and MMP-9. In contrast, exosomes derived from murine fibroblastic cells did not affect osteoclast formation. Our findings suggest that exosomes released from tumor cells in the tumor-bone interface are involved in pathological regulation of bone cell formation in the metastatic site. This further strengthens the role of tumor cell-derived microvesicles in cancer progression and disease aggressiveness.

title: Cavin-1/PTRF alters prostate cancer cell-derived extracellular vesicle content and internalization to attenuate extracellular vesicle-mediated osteoclastogenesis and osteoblast proliferation
abstract: Tumour-derived extracellular vesicles (EVs) play a role in tumour progression; however, the spectrum of molecular mechanisms regulating EV secretion and cargo selection remain to be fully elucidated. We have reported that cavin-1 expression in prostate cancer PC3 cells reduced the abundance of a subset of EV proteins, concomitant with reduced xenograft tumour growth and metastasis.  We examined the functional outcomes and mechanisms of cavin-1 expression on PC3-derived EVs (PC3-EVs).  PC3-EVs were internalized by osteoclast precursor RAW264.7 cells and primary human osteoblasts (hOBs) in vitro, stimulating osteoclastogenesis 37-fold and hOB proliferation 1.5-fold, respectively. Strikingly, EVs derived from cavin-1-expressing PC3 cells (cavin-1-PC3-EVs) failed to induce multinucleate osteoblasts or hOB proliferation. Cavin-1 was not detected in EVs, indicating an indirect mechanism of action. EV morphology, size and quantity were also not affected by cavin-1 expression, suggesting that cavin-1 modulated EV cargo recruitment rather than release. While cavin-1-EVs had no osteoclastogenic function, they were internalized by RAW264.7 cells but at a reduced efficiency compared to control EVs. EV surface proteins are required for internalization of PC3-EVs by RAW264.7 cells, as proteinase K treatment abolished uptake of both control and cavin-1-PC3-EVs. Removal of sialic acid modifications by neuraminidase treatment increased the amount of control PC3-EVs internalized by RAW264.7 cells, without affecting cavin-1-PC3-EVs. This suggests that cavin-1 expression altered the glycosylation modifications on PC3-EV surface. Finally, cavin-1 expression did not affect EV in vivo tissue targeting as both control and cavin-1-PC3-EVs were predominantly retained in the lung and bone 24 hours after injection into mice.  Taken together, our results reveal a novel pathway for EV cargo sorting, and highlight the potential of utilizing cavin-1-mediated pathways to attenuate metastatic prostate cancer.

title: Cancer-secreted hsa-miR-940 induces an osteoblastic phenotype in the bone metastatic microenvironment via targeting ARHGAP1 and FAM134A
abstract: Prostate cancer is one of most common cancers in men worldwide, and osteoblastic bone metastasis is frequently observed in prostate cancer patients. However, the mechanisms responsible for the predominantly osteoblastic phenotype have not been fully elucidated. Cancer-secreted microRNAs (miRNAs) were recently shown to be significant in the modification of the tumor microenvironment. Here, hsa-miR-940, which was highly secreted by prostate cancer cells, promoted osteogenic differentiation of human mesenchymal stem cells in vitro, and induced extensive osteoblastic lesions in the bone metastatic microenvironment in vivo. Our study provides a demonstration that osteoblastic bone metastasis can be induced by miRNAs secreted by cancer cells in the bone microenvironment. Bone metastatic lesions are classified as osteoblastic or osteolytic lesions. Prostate and breast cancer patients frequently exhibit osteoblastic-type and osteolytic-type bone metastasis, respectively. In metastatic lesions, tumor cells interact with many different cell types, including osteoblasts, osteoclasts, and mesenchymal stem cells, resulting in an osteoblastic or osteolytic phenotype. However, the mechanisms responsible for the modification of bone remodeling have not been fully elucidated. MicroRNAs (miRNAs) are transferred between cells via exosomes and serve as intercellular communication tools, and numerous studies have demonstrated that cancer-secreted miRNAs are capable of modifying the tumor microenvironment. Thus, cancer-secreted miRNAs can induce an osteoblastic or osteolytic phenotype in the bone metastatic microenvironment. In this study, we performed a comprehensive expression analysis of exosomal miRNAs secreted by several human cancer cell lines and identified eight types of human miRNAs that were highly expressed in exosomes from osteoblastic phenotype-inducing prostate cancer cell lines. One of these miRNAs, hsa-miR-940, significantly promoted the osteogenic differentiation of human mesenchymal stem cells in vitro by targeting ARHGAP1 and FAM134A. Interestingly, although MDA-MB-231 breast cancer cells are commonly known as an osteolytic phenotype-inducing cancer cell line, the implantation of miR-940–overexpressing MDA-MB-231 cells induced extensive osteoblastic lesions in the resulting tumors by facilitating the osteogenic differentiation of host mesenchymal cells. Our results suggest that the phenotypes of bone metastases can be induced by miRNAs secreted by cancer cells in the bone microenvironment.

title: Exosomal miR-141-3p regulates osteoblast activity to promote the osteoblastic metastasis of prostate cancer
abstract: Exosomes from cancer cells, which contain microRNA and reach metastasis loci prior to cancer cells, stimulate the formation of a metastatic microenvironment. Previous studies have shown that exosomal miR-141-3p is associated with metastatic prostate cancer (PCa). However, the role and regulatory mechanism of miR-141-3p in the microenvironment of bone metastases require further study. In this study, we performed a series of experiments in vivo and in vitro to determine whether exosomal miR-141-3p from MDA PCa 2b cells regulates osteoblast activity to promote osteoblastic metastasis. We demonstrate that extracts obtained from cell culture supernatants contained exosomes and that miR-141-3p levels were significantly higher in MDA PCa 2b cell exosomes. Via confocal imaging, numerous MDA PCa 2b exosomes were observed to enter osteoblasts, and miR-141-3p was transferred to osteoblasts through MDA PCa 2b exosomes in vitro. Exosomal miR-141-3p from MDA PCa 2b promoted osteoblast activity and increased osteoprotegerin OPG expression. miR-141-3p suppressed the protein levels of the target gene DLC1, indicating its functional significance in activating the p38MAPK pathway. In animal experiments, exosomal miR-141-3p had bone-target specificity and promoted osteoblast activity. Mice injected with miR-141-3p-mimics exosomes developed apparent osteoblastic bone metastasis. Exosomal miR-141-3p from MDA PCa 2b cells promoted osteoblast activity and regulated the microenvironment of bone metastases, which plays an important role in the formation of bone metastases and osteogenesis damage in PCa. Clarifying the specific mechanism of bone metastasis will help generate new possibilities for the treatment of PCa.

title: Communication of prostate cancer cells with bone cells via extracellular vesicle RNA; a potential mechanism of metastasis
abstract: The role of extracellular vesicles (EVs) as vehicles for cell-to-cell communication between a tumour and its environment is a relatively new concept. The hypothesis that EVs may be critical in co-opting tissues by tumours to generate distant metastatic niches is particularly pertinent to prostate cancer (PCa), where metastatic-tropism to bone predominates over other tissue types. The potential role of EVs as a means of communication between PCa cells and cells of the bone stroma such as osteoblasts, is yet to be fully explored. In this study, we demonstrate that PCa cell EVs both enhance osteoblast viability and produce a significantly more supportive growth environment for PCa cells when grown in co-culture with EV-treated osteoblasts (p < 0.005). Characterisation of the RNA cargo of EVs produced by the bone-metastatic PCa cell line PC3, highlights the EV-RNA cargo is significantly enriched in genes relating to cell surface signalling, cell–cell interaction, and protein translation (p < 0.01). Using novel techniques to track RNA, we demonstrate the delivery of a set of PCa-RNAs to osteoblast via PCa-EVs and show the effect on osteoblast endogenous transcript abundance. Taken together, by using proof-of-concept studies we demonstrate for the first time the contribution of the RNA element of the PCa EV cargo, providing evidence to support PCa EV communication via RNA molecules as a potential novel route to mediate bone metastasis. We propose targeting PCa EVs could offer a potentially important preventative therapy for men at risk of metastatic PCa.

title: Cancer Dormancy: A Model of Early Dissemination and Late Cancer Recurrence
abstract: Cancer dormancy is a stage in tumor progression in which residual disease remains occult and asymptomatic for a prolonged period of time. Dormant tumor cells can be present as one of the earliest stages in tumor development, as well as a stage in micrometastases, and/or minimal residual disease left after an apparently successful treatment of the primary tumor. The general mechanisms that regulate the transition of disseminated tumor cells that have lain dormant into a proliferative state remain largely unknown. However, regulation of the growth from dormant tumor cells may be explained in part through the interaction of the tumor cell with its microenvironment, limitations in the blood supply, or an active immune system. An understanding of the regulatory machinery of these processes is essential for identifying early cancer biomarkers and could provide a rationale for the development of novel agents to target dormant tumor cells. This review focuses on the different signaling models responsible for early cancer dissemination and tumor recurrence that are involved in dormancy pathways.

title: Notch2 pathway mediates breast cancer cellular dormancy and mobilisation in bone and contributes to haematopoietic stem cell mimicry
abstract: Recurrence after >5-year disease-free survival affects one-fifth of breast cancer patients and is the clinical manifestation of cancer cell reactivation after persistent dormancy. We investigated cellular dormancy in vitro and in vivo using breast cancer cell lines and cell and molecular biology techniques. We demonstrated cellular dormancy in breast cancer bone metastasis, associated with haematopoietic stem cell (HSC) mimicry, in vivo competition for HSC engraftment and non-random distribution of dormant cells at the endosteal niche. Notch2 signal implication was demonstrated by immunophenotyping the endosteal niche-associated cancer cells and upon co-culture with sorted endosteal niche cells, which inhibited breast cancer cell proliferation in a Notch2-dependent manner. Blocking this signal by in vivo acute administration of the γ-secretase inhibitor, dibenzazepine, induced dormant cell mobilisation from the endosteal niche and colonisation of visceral organs. Sorted Notch2HIGH breast cancer cells exhibited a unique stem phenotype similar to HSCs and in vitro tumour-initiating ability in mammosphere assay. Human samples confirmed the existence of a small Notch2HIGH cell population in primary and bone metastatic breast cancers, with a survival advantage for Notch2HIGH vs Notch2LOW patients. Notch2 represents a key determinant of breast cancer cellular dormancy and mobilisation in the bone microenvironment.

title: Exosomes from bone marrow mesenchymal stem cells contain a microRNA that promotes dormancy in metastatic breast cancer cells
abstract: The bone marrow delivers dormancy signals in exosomes to metastatic cancer cells.  Exosomes Metastatic disease often develops long after treatment of the primary tumor because of cells that spread into metastatic niches, particularly the bone marrow. Because these “dormant” cells have a slow rate of cell cycling, they are not killed by traditional chemotherapies that target rapidly dividing cells. Ono et al. examined how cells present in the bone marrow niche trigger dormancy of metastatic breast cancer cells. They found that exosomes (vesicles shed by cells) from bone marrow–derived mesenchymal stem cells were taken up by cancer cells, in which they inhibited proliferation and promoted other characteristics associated with dormancy, in part through the delivery of a microRNA that suppressed the expression of a gene encoding a protein that promotes cell proliferation and motility. Thus, the bone marrow niche releases exosomes that deliver signals promoting metastatic cancer cell dormancy. Breast cancer patients often develop metastatic disease years after resection of the primary tumor. The patients are asymptomatic because the disseminated cells appear to become dormant and are undetectable. Because the proliferation of these cells is slowed, dormant cells are often unresponsive to traditional chemotherapies that exploit the rapid cell cycling of most cancer cells. We generated a bone marrow–metastatic human breast cancer cell line (BM2) by tracking and isolating fluorescent-labeled MDA-MB-231 cells that disseminated to the bone marrow in mice. Coculturing BM2 cells with bone marrow mesenchymal stem cells (BM-MSCs) isolated from human donors revealed that BM-MSCs suppressed the proliferation of BM2 cells, decreased the abundance of stem cell–like surface markers, inhibited their invasion through Matrigel Transwells, and decreased their sensitivity to docetaxel, a common chemotherapy agent. Acquisition of these dormant phenotypes in BM2 cells was also observed by culturing the cells in BM-MSC–conditioned medium or with exosomes isolated from BM-MSC cultures, which were taken up by BM2 cells. Among various microRNAs (miRNAs) increased in BM-MSC–derived exosomes compared with those from adult fibroblasts, overexpression of miR-23b in BM2 cells induced dormant phenotypes through the suppression of a target gene, MARCKS, which encodes a protein that promotes cell cycling and motility. Metastatic breast cancer cells in patient bone marrow had increased miR-23b and decreased MARCKS expression. Together, these findings suggest that exosomal transfer of miRNAs from the bone marrow may promote breast cancer cell dormancy in a metastatic niche.

title: Mesenchymal Stem Cell-Derived Exosomes Stimulate Cycling Quiescence and Early Breast Cancer Dormancy in Bone Marrow.
abstract: Dormant breast cancers resurge as metastatic disease after a long dormancy period in the bone marrow, where cancer cells interact with mesenchymal stem cells (MSC). However, the nature of early interactions between breast cancer cells and MSCs in the bone marrow microenvironment that facilitate adaptation to a quiescent state remains poorly understood. Here, we report that breast cancer cells prime MSC to release exosomes containing distinct miRNA contents, such as miR-222/223, which in turn promotes quiescence in a subset of cancer cells and confers drug resistance. Building on these results, we developed a novel, nontoxic therapeutic strategy to target dormant breast cancer cells based on systemic administration of MSC loaded with antagomiR-222/223. In an immunodeficient mouse model of dormant breast cancer, this therapy sensitized breast cancer cells to carboplatin-based therapy and increased host survival. Overall, our findings illuminate the nature of the regulatory interactions between breast cancer cells and MSCs in the evolution of tumor dormancy and resurgence in the micrometastatic microenvironment of the bone marrow. ; 5832-44.

title: The Biodistribution and Immune Suppressive Effects of Breast Cancer-Derived Exosomes.
abstract: Small membranous secretions from tumor cells, termed exosomes, contribute significantly to intercellular communication and subsequent reprogramming of the tumor microenvironment. Here, we use optical imaging to determine that exogenously administered fluorescently labeled exosomes derived from highly metastatic murine breast cancer cells distributed predominantly to the lung of syngeneic mice, a frequent site of breast cancer metastasis. At the sites of accumulation, exosomes were taken up by CD45+ bone marrow-derived cells. Subsequent long-term conditioning of naïve mice with exosomes from highly metastatic breast cancer cells revealed the accumulation of myeloid-derived suppressor cells in the lung and liver. This favorable immune suppressive microenvironment was capable of promoting metastatic colonization in the lung and liver, an effect not observed from exosomes derived from nonmetastatic cells and liposome control vesicles. Furthermore, we determined that breast cancer exosomes directly suppressed T-cell proliferation and inhibited NK cell cytotoxicity, and hence likely suppressed the anticancer immune response in premetastatic organs. Together, our findings provide novel insight into the tissue-specific outcomes of breast cancer-derived exosome accumulation and their contribution to immune suppression and promotion of metastases. Cancer Res; 76(23); 6816-27.

title: Pre-metastatic cancer exosomes induce immune surveillance by patrolling monocytes at the metastatic niche
abstract: Metastatic cancers produce exosomes that condition pre-metastatic niches in remote microenvironments to favor metastasis. In contrast, here we show that exosomes from poorly metastatic melanoma cells can potently inhibit metastasis to the lung. These “non-metastatic” exosomes stimulate an innate immune response through the expansion of Ly6Clow patrolling monocytes (PMo) in the bone marrow, which then cause cancer cell clearance at the pre-metastatic niche, via the recruitment of NK cells and TRAIL-dependent killing of melanoma cells by macrophages. These events require the induction of the Nr4a1 transcription factor and are dependent on pigment epithelium-derived factor (PEDF) on the outer surface of exosomes. Importantly, exosomes isolated from patients with non-metastatic primary melanomas have a similar ability to suppress lung metastasis. This study thus demonstrates that pre-metastatic tumors produce exosomes, which elicit a broad range of PMo-reliant innate immune responses via trigger(s) of immune surveillance, causing cancer cell clearance at the pre-metastatic niche.Exosomes are extracellular vesicles that can favor tumor development and metastasis. Here, the authors show that cancer exosomes may also exert a suppressive function; in fact, exosomes from non-metastatic melanoma cells can lead to the recruitment of patrolling monocytes, which clear cancer cells at the pre-metastatic niche.

title: Exosome release by primary B cells.
abstract: Exosomes are subcellular nanoparticles derived from the endosomal pathway. It is now becoming clear that a potential major in vivo source of exosomes is the B cell. Although it has been widely assumed that exosome release is a constitutive activity of most cell types, recent work has emphasized the role of cellular activation in the release of exosomes from primary cells. Like other lymphocytes, B cells undergo extensive cellular physiologic changes during the process of differentiation into effector cells. One newly identified feature of this process is exosome synthesis, which is initiated following the receipt of activation signals, particularly T-cell "help" via CD40 and IL-4 signaling. B-cell-derived exosomes contain immunoglobulin, which traffics antigen bound by the surface B-cell receptor (BCR) into the endosomal/exosomal pathway and finally into the extracellular space. Exosomes have been implicated in viral transmission, cell signaling, and antigen presentation, as well as in the disposal of effete or defective cellular components. However, the possible targets of B-cell-derived exosomes remain unknown. This review focuses on the synthesis and release of exosomes derived from activated and malignant B cells and explores the possible functions of B-cell-derived exosomes in immune function.

title: Role of T cell-derived exosomes in immunoregulation
abstract: Exosomes are small membrane vesicles of endocytic origin that are secreted by most cells. They are composed of a lipid bilayer containing transmembrane proteins and enclosing cytosolic proteins and RNA, mediating intercellular communication between different cell types in the body, and thus influencing various physiological and pathological functions of both recipient and parent cells. For their nanolevel structures with a stable nature and various biological functions, studies of exosomes have been the subject of increasing interest in the past few years. It is widely known that different T cell subsets play important roles in cellular and humoral immunity, and their exosomes were also reported to exert similar biological functions. While several groups reported the secretion of exosomes by various T cells, the systematic summary involved in these exosomes are deficient. In this review, we will summarize the structure and functions of exosomes derived from T cells in recent reports, discuss emerging therapeutic opportunities, and consider the associated challenges.

title: Bone marrow stromal cell-derived exosomes as communicators in drug resistance in multiple myeloma cells.
abstract: The interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells performs a crucial role in MM pathogenesis by secreting growth factors, cytokines, and extracellular vesicles. Exosomes are membranous vesicles 40 to 100 nm in diameter constitutively released by almost all cell types, and they mediate local cell-to-cell communication by transferring mRNAs, miRNAs, and proteins. Although BMSC-induced growth and drug resistance of MM cells has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here we investigate the effect of BMSC-derived exosomes on the viability, proliferation, survival, migration, and drug resistance of MM cells, using the murine 5T33MM model and human MM samples. BMSCs and MM cells could mutually exchange exosomes carrying certain cytokines. Both naive and 5T33 BMSC-derived exosomes increased MM cell growth and induced drug resistance to bortezomib. BMSC-derived exosomes also influenced the activation of several survival relevant pathways, including c-Jun N-terminal kinase, p38, p53, and Akt. Exosomes obtained from normal donor and MM patient BMSCs also induced survival and drug resistance of human MM cells. Taken together, our results demonstrate the involvement of exosome-mediated communication in BMSC-induced proliferation, migration, survival, and drug resistance of MM cells.

title: Acute myeloid leukemia transforms the bone marrow niche into a leukemia-permissive microenvironment through exosome secretion
abstract: Little is known about how leukemia cells alter the bone marrow (BM) niche to facilitate their own growth and evade chemotherapy. Here, we provide evidence that acute myeloid leukemia (AML) blasts remodel the BM niche into a leukemia growth-permissive and normal hematopoiesis-suppressive microenvironment through exosome secretion. Either engrafted AML cells or AML-derived exosomes increased mesenchymal stromal progenitors and blocked osteolineage development and bone formation in vivo. Preconditioning with AML-derived exosomes ‘primed’ the animals for accelerated AML growth. Conversely, disruption of exosome secretion in AML cells through targeting Rab27a, an important regulator involved in exosome release, significantly delayed leukemia development. In BM stromal cells, AML-derived exosomes induced the expression of DKK1, a suppressor of normal hematopoiesis and osteogenesis, thereby contributing to osteoblast loss. Conversely, treatment with a DKK1 inhibitor delayed AML progression and prolonged survival in AML-engrafted mice. In addition, AML-derived exosomes induced a broad downregulation of hematopoietic stem cell-supporting factors (for example, CXCL12, KITL and IGF1) in BM stromal cells and reduced their ability to support normal hematopoiesis. Altogether, this study uncovers novel features of AML pathogenesis and unveils how AML cells create a self-strengthening leukemic niche that promotes leukemic cell proliferation and survival, while suppressing normal hematopoiesis through exosome secretion.

title: Exosome-mediated transfer of lncRUNX2-AS1 from multiple myeloma cells to MSCs contributes to osteogenesis
abstract: Multiple myeloma (MM) is characterized by the decreased osteogenic potential of mesenchymal stem cells (MSCs). Communication between cancer cells and cancer stromal cells is a driving factor in tumor progression. Understanding the myeloma–stroma interactions is critical to the development of effective strategies that can reverse bone diseases. Here we identified that bioactive lncRNA RUNX2-AS1 in myeloma cells could be packed into exosomes and transmitted to MSCs, thus repressing the osteogenesis of MSCs. RUNX2-AS1, which arises from the antisense strand of RUNX2, was enriched in MSCs derived from MM patients (MM-MSCs). RUNX2-AS1 was capable of forming an RNA duplex with RUNX2 pre-mRNA at overlapping regions and this duplex transcriptionally repressed RUNX2 expression by reducing the splicing efficiency, resulting in decreased osteogenic potential of MSCs. In vivo mouse models, administered an inhibitor of exosome secretion, GW4869, was found to be effective in preventing bone loss, sustained by both bone formation and anticatabolic activities. Therefore, exosomal lncRNA RUNX2-AS1 may serve as a potential therapeutic target for bone lesions in MM. In summary, our results indicated a key role of exosomal lncRUNX2-AS1 in transferring from MM cells to MSCs in osteogenic differentiation, through a unique exosomal lncRUNX2-AS1/RUNX2 pathway.

title: Multiple myeloma-derived exosomes are enriched of amphiregulin (AREG) and activate the epidermal growth factor pathway in the bone microenvironment leading to osteoclastogenesis
abstract: BackgroundMultiple myeloma (MM) is a clonal plasma cell malignancy associated with osteolytic bone disease. Recently, the role of MM-derived exosomes in the osteoclastogenesis has been demonstrated although the underlying mechanism is still unknown. Since exosomes-derived epidermal growth factor receptor ligands (EGFR) are involved in tumor-associated osteolysis, we hypothesize that the EGFR ligand amphiregulin (AREG) can be delivered by MM-derived exosomes and participate in MM-induced osteoclastogenesis.MethodsExosomes were isolated from the conditioned medium of MM1.S cell line and from bone marrow (BM) plasma samples of MM patients. The murine cell line RAW264.7 and primary human CD14+ cells were used as osteoclast (OC) sources.ResultsWe found that AREG was specifically enriched in exosomes from MM samples and that exosomes-derived AREG led to the activation of EGFR in pre-OC, as showed by the increase of mRNA expression of its downstream SNAIL in both RAW264.7 and CD14+ cells. The presence of neutralizing anti-AREG monoclonal antibody (mAb) reverted this effect. Consequently, we showed that the effect of MM-derived exosomes on osteoclast differentiation was inhibited by the pre-treatment of exosomes with anti-AREG mAb. In addition, we demonstrated the ability of MM-derived AREG-enriched exosomes to be internalized into human mesenchymal stromal cells (MSCs) blocking osteoblast (OB) differentiation, increasing MM cell adhesion and the release of the pro-osteoclastogenic cytokine interleukin-8 (IL8). Accordingly, anti-AREG mAb inhibited the release of IL8 by MSCs suggesting that both direct and indirect effects are responsible for AREG-enriched exosomes involvement on MM-induced osteoclastogenesis.ConclusionsIn conclusion, our data indicate that AREG is packed into MM-derived exosomes and implicated in OC differentiation through an indirect mechanism mediated by OBs.

title: Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.
abstract: In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.

title: Ceramide Triggers Budding of Exosome Vesicles into Multivesicular Endosomes
abstract: Intraluminal vesicles of multivesicular endosomes are either sorted for cargo degradation into lysosomes or secreted as exosomes into the extracellular milieu. The mechanisms underlying the sorting of membrane into the different populations of intraluminal vesicles are unknown. Here, we find that cargo is segregated into distinct subdomains on the endosomal membrane and that the transfer of exosome-associated domains into the lumen of the endosome did not depend on the function of the ESCRT (endosomal sorting complex required for transport) machinery, but required the sphingolipid ceramide. Purified exosomes were enriched in ceramide, and the release of exosomes was reduced after the inhibition of neutral sphingomyelinases. These results establish a pathway in intraendosomal membrane transport and exosome formation.

title: Blockade of exosome generation with GW4869 dampens the sepsis-induced inflammation and cardiac dysfunction.
abstract: Sepsis is an infection-induced severe inflammatory disorder that leads to multiple organ failure. Amongst organs affected, myocardial depression is believed to be a major contributor to septic death. While it has been identified that large amounts of circulating pro-inflammatory cytokines are culprit for triggering cardiac dysfunction in sepsis, the underlying mechanisms remain obscure. Additionally, recent studies have shown that exosomes released from bacteria-infected macrophages are pro-inflammatory. Hence, we examined in this study whether blocking the generation of exosomes would be protective against sepsis-induced inflammatory response and cardiac dysfunction. To this end, we pre-treated RAW264.7 macrophages with GW4869, an inhibitor of exosome biogenesis/release, followed by endotoxin (LPS) challenge. In vivo, we injected wild-type (WT) mice with GW4869 for 1h prior to endotoxin treatment or cecal ligation/puncture (CLP) surgery. We observed that pre-treatment with GW4869 significantly impaired release of both exosomes and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in RAW264.7 macrophages. At 12h after LPS treatment or CLP surgery, WT mice pre-treated with GW4869 displayed lower amounts of exosomes and pro-inflammatory cytokines in the serum than control PBS-injected mice. Accordingly, GW4869 treatment diminished the sepsis-induced cardiac inflammation, attenuated myocardial depression and prolonged survival. Together, our findings indicate that blockade of exosome generation in sepsis dampens the sepsis-triggered inflammatory response and thereby, improves cardiac function and survival.

title: Depletion of microglia and inhibition of exosome synthesis halt tau propagation
abstract: Accumulation of pathological tau protein is a major hallmark of Alzheimer's disease. Tau protein spreads from the entorhinal cortex to the hippocampal region early in the disease. Microglia, the primary phagocytes in the brain, are positively correlated with tau pathology, but their involvement in tau propagation is unknown. We developed an adeno-associated virus–based model exhibiting rapid tau propagation from the entorhinal cortex to the dentate gyrus in 4 weeks. We found that depleting microglia dramatically suppressed the propagation of tau and reduced excitability in the dentate gyrus in this mouse model. Moreover, we demonstrate that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that microglia and exosomes contribute to the progression of tauopathy and that the exosome secretion pathway may be a therapeutic target.

title: Exosome-mediated transfer of miR-10b promotes cell invasion in breast cancer
abstract: IntroductionExosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions including tumor cell invasion, cell-cell communication and antigen presentation through transfer of proteins, mRNAs and microRNAs. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme encoded by the smpd3 gene that triggers exosome secretion. However, whether exosome-mediated microRNA transfer plays any role in cell invasion remains poorly understood. Thus, the aim of this study was to identify the exosomal microRNAs involved in breast cancer invasion.MethodsThe expression level of endogenous and exosomal miRNAs were examined by real time PCR and the expression level of target proteins were detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study its uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-10b was estimated by invasion assay.ResultsIn this study, we demonstrate that microRNA carrying exosomes can be transferred among different cell lines through direct uptake. miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cancer cells or non-malignant breast cells; it is actively secreted into medium via exosomes. In particular, nSMase2 or ceramide promotes the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppresses this secretion. Moreover, upon uptake, miR-10b can suppress the protein level of its target genes such as HOXD10 and KLF4, indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could induce the invasion ability of non-malignant HMLE cells.ConclusionTogether, our results suggest that a set of specific microRNAs may play an important role in modulating tumor microenvironment through exosomes. Thus, a better understanding of this process may aid in the development of novel therapeutic agents.

title: Rab27a and Rab27b control different steps of the exosome secretion pathway
abstract: Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo.

title: New insights into the function of Rab GTPases in the context of exosomal secretion
abstract: In the last two decades, extracellular vesicle-mediated communication between cells has become a major field in cell biology. However, the function of extracellular vesicles is far from clear, especially due to the disparity of released vesicles by cells. Basically, one must consider vesicles budding from the cell plasma membrane (ectosomes) and vesicles released upon fusion of an endosomal multivesicular compartment (exosomes). Moreover, even for exosomes, we report and discuss here the possibility that different routes regulated by specific Rab GTPases might produce exosomes having various biologic functions.

title: Rab11 Promotes Docking and Fusion of Multivesicular Bodies in a Calcium‐Dependent Manner
abstract: Multivesicular bodies (MVBs) are membranous structures within 60–100 nm diameter vesicles accumulate. MVBs are generated after invagination and pinching off of the endosomal membrane in the lumen of the vacuole. In certain cell types, fusion of MVBs with the plasma membrane results in the release of the internal vesicles called exosomes. In this report we have examined how an increase in cytosolic calcium affects the development of MVBs and exosome release in K562 cells overexpressing GFP‐Rab11 wt or its mutants. In cells overexpressing the Rab11Q70 L mutant or Rab11 wt, an increase in the cytosolic calcium concentration induced by monensin caused a marked enlargement of the MVBs. This effect was abrogated by the membrane permeant calcium chelator BAPTA‐AM. We also examined the behavior of MVBs in living cells by time lapse confocal microscopy. Many MVBs, decorated by wt or Q70L mutant GFP‐Rab11, were docked and ready to fuse in the presence of a calcium chelator. This observation suggests that Rab11 is acting in the tethering/docking of MVBs to promote homotypic fusion, but that the final fusion reaction requires the presence of calcium. Additionally, a rise in intracellular calcium concentration enhanced exosome secretion in Rab11 wt overexpressing cells and reversed the inhibition of the mutants. The results suggest that both Rab11 and calcium are involved in the homotypic fusion of MVBs.

title: Rab27a supports exosome-dependent and -independent mechanisms that modify the tumor microenvironment and can promote tumor progression.
abstract: During progression from single cancer cells to a tumor mass and metastases, tumor cells send signals that can subvert their tissue microenvironment. These signals involve soluble molecules and various extracellular vesicles, including a particular type termed exosomes. The specific roles of exosomes secreted in the tumor microenvironment, however, is unclear. The small GTPases RAB27A and RAB27B regulate exocytosis of multivesicular endosomes, which lead to exosome secretion, in human HeLa cells. Here, we used mouse models to show that Rab27a blockade in mammary carcinoma cells decreased secretion of exosomes characterized by endocytic markers, but also of matrix metalloproteinase 9, which is not associated with exosomes. Rab27a blockade resulted in decreased primary tumor growth and lung dissemination of a metastatic carcinoma (4T1), but not of a nonmetastatic carcinoma (TS/A). Local growth of 4T1 tumors required mobilization of a population of neutrophil immune cells induced by Rab27a-dependent secretion of exosomes together with a specific combination of cytokines and/or metalloproteinases. Our findings offer in vivo validation of the concept that exosome secretion can exert key pathophysiologic roles during tumor formation and progression, but they also highlight the idiosyncratic character of the tumor context.

title: Dynasore, a cell-permeable inhibitor of dynamin.
abstract: Dynamin is essential for clathrin-dependent coated vesicle formation. It is required for membrane budding at a late stage during the transition from a fully formed pit to a pinched-off vesicle. Dynamin may also fulfill other roles during earlier stages of vesicle formation. We have screened about 16,000 small molecules and have identified 1, named here dynasore, that interferes in vitro with the GTPase activity of dynamin1, dynamin2, and Drp1, the mitochondrial dynamin, but not of other small GTPases. Dynasore acts as a potent inhibitor of endocytic pathways known to depend on dynamin by rapidly blocking coated vesicle formation within seconds of dynasore addition. Two types of coated pit intermediates accumulate during dynasore treatment, U-shaped, half formed pits and O-shaped, fully formed pits, captured while pinching off. Thus, dynamin acts at two steps during clathrin coat formation; GTP hydrolysis is probably needed at both steps.

title: Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity
abstract: Exosome-mediated intercellular transfer of proteins and nucleic acids has attracted considerable attention as exosomes may promote the development of cancer and other pathological conditions; however, the mechanism of exosome uptake by target cells and how this may be inhibited remain as important questions. We provide evidence that heparan sulfate proteoglycans (HSPGs) function as receptors of cancer cell-derived exosomes. Importantly, our data indicate that the HSPG-dependent uptake route is highly relevant for the biological activity of exosomes, and thus a potential target for inhibition of exosome-mediated tumor development. Given that several viruses have previously been shown to enter cells through HSPGs, our data implicate HSPG as a convergence point during cellular uptake of endogenous vesicles and virus particles. Extracellular vesicle (EV)-mediated intercellular transfer of signaling proteins and nucleic acids has recently been implicated in the development of cancer and other pathological conditions; however, the mechanism of EV uptake and how this may be targeted remain as important questions. Here, we provide evidence that heparan sulfate (HS) proteoglycans (PGs; HSPGs) function as internalizing receptors of cancer cell-derived EVs with exosome-like characteristics. Internalized exosomes colocalized with cell-surface HSPGs of the syndecan and glypican type, and exosome uptake was specifically inhibited by free HS chains, whereas closely related chondroitin sulfate had no effect. By using several cell mutants, we provide genetic evidence of a receptor function of HSPG in exosome uptake, which was dependent on intact HS, specifically on the 2-O and N-sulfation groups. Further, enzymatic depletion of cell-surface HSPG or pharmacological inhibition of endogenous PG biosynthesis by xyloside significantly attenuated exosome uptake. We provide biochemical evidence that HSPGs are sorted to and associate with exosomes; however, exosome-associated HSPGs appear to have no direct role in exosome internalization. On a functional level, exosome-induced ERK1/2 signaling activation was attenuated in PG-deficient mutant cells as well as in WT cells treated with xyloside. Importantly, exosome-mediated stimulation of cancer cell migration was significantly reduced in PG-deficient mutant cells, or by treatment of WT cells with heparin or xyloside. We conclude that cancer cell-derived exosomes use HSPGs for their internalization and functional activity, which significantly extends the emerging role of HSPGs as key receptors of macromolecular cargo.

title: Routes and mechanisms of extracellular vesicle uptake
abstract: Extracellular vesicles (EVs) are small vesicles released by donor cells that can be taken up by recipient cells. Despite their discovery decades ago, it has only recently become apparent that EVs play an important role in cell-to-cell communication. EVs can carry a range of nucleic acids and proteins which can have a significant impact on the phenotype of the recipient. For this phenotypic effect to occur, EVs need to fuse with target cell membranes, either directly with the plasma membrane or with the endosomal membrane after endocytic uptake. EVs are of therapeutic interest because they are deregulated in diseases such as cancer and they could be harnessed to deliver drugs to target cells. It is therefore important to understand the molecular mechanisms by which EVs are taken up into cells. This comprehensive review summarizes current knowledge of EV uptake mechanisms. Cells appear to take up EVs by a variety of endocytic pathways, including clathrin-dependent endocytosis, and clathrin-independent pathways such as caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft–mediated internalization. Indeed, it seems likely that a heterogeneous population of EVs may gain entry into a cell via more than one route. The uptake mechanism used by a given EV may depend on proteins and glycoproteins found on the surface of both the vesicle and the target cell. Further research is needed to understand the precise rules that underpin EV entry into cells.

title: Tumor-Derived Microvesicles Induce Proangiogenic Phenotype in Endothelial Cells via Endocytosis
abstract: Increasing evidence indicates that tumor endothelial cells (TEC) differ from normal endothelial cells (NEC). Our previous reports also showed that TEC were different from NEC. For example, TEC have chromosomal abnormality and proangiogenic properties such as high motility and proliferative activity. However, the mechanism by which TEC acquire a specific character remains unclear. To investigate this mechanism, we focused on tumor-derived microvesicles (TMV). Recent studies have shown that TMV contain numerous types of bioactive molecules and affect normal stromal cells in the tumor microenvironment. However, most of the functional mechanisms of TMV remain unclear.  Here we showed that TMV isolated from tumor cells were taken up by NEC through endocytosis. In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC. Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore. Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis.  We for the first time showed that endocytosis of TMV contributes to tumor angiogenesis. These findings offer new insights into cancer therapies and the crosstalk between tumor and endothelial cells mediated by TMV in the tumor microenvironment.

title: Cell-specific uptake of mantle cell lymphoma-derived exosomes by malignant and non-malignant B-lymphocytes.
abstract: Mantle cell lymphoma (MCL) is an aggressive and incurable mature B cell neoplasm. The current treatments are based on chemotherapeutics and new class of drugs (e.g. Ibrutinib(®)), which in most cases ends with tumor resistance and relapse. Therefore, further development of novel therapeutic modalities is needed. Exosomes are natural extracellular vesicles, which play an important role in intercellular communication. The specificity of exosome uptake by different target cells remains unknown. In this study, we observed that MCL exosomes are taken up rapidly and preferentially by MCL cells. Only a minor fraction of exosomes was internalized into T-cell leukemia and bone marrow stroma cell lines, when these cells were co-cultured with MCL cells. Moreover, MCL patients' exosomes were taken up by both healthy and patients' B-lymphocytes with no apparent internalization to T lymphocytes and NK cells. Exosome internalization was not inhibited by specific siRNA against caveolin1 and clathrin but was found to be mediated by a cholesterol-dependent pathway. These findings demonstrate natural specificity of exosomes to B-lymphocytes and ultimately might be used for therapeutic intervention in B cells malignancies.

title: Dendritic cell-derived exosomes for cancer therapy.
abstract: DC-derived exosomes (Dex) are nanometer-sized membrane vesicles that are secreted by the sentinel antigen-presenting cells of the immune system: DCs. Like DCs, the molecular composition of Dex includes surface expression of functional MHC-peptide complexes, costimulatory molecules, and other components that interact with immune cells. Dex have the potential to facilitate immune cell-dependent tumor rejection and have distinct advantages over cell-based immunotherapies involving DCs. Accordingly, Dex-based phase I and II clinical trials have been conducted in advanced malignancies, showing the feasibility and safety of the approach, as well as the propensity of these nanovesicles to mediate T and NK cell-based immune responses in patients. This Review will evaluate the interactions of Dex with immune cells, their clinical progress, and the future of Dex immunotherapy for cancer.

title: Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes
abstract: To realize the therapeutic potential of RNA drugs, efficient, tissue-specific and nonimmunogenic delivery technologies must be developed. Here we show that exosomes—endogenous nano-vesicles that transport RNAs and proteins—can deliver short interfering (si)RNA to the brain in mice. To reduce immunogenicity, we used self-derived dendritic cells for exosome production. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide. Purified exosomes were loaded with exogenous siRNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease, in wild-type mice.

title: Genetically Engineered Microvesicles Carrying Suicide mRNA/Protein Inhibit Schwannoma Tumor Growth
abstract: Microvesicles (MVs) play an important role in intercellular communication by carrying mRNAs, microRNAs (miRNAs), non-coding RNAs, proteins, and DNA from cell to cell. To our knowledge, this is the first report of delivery of a therapeutic mRNA/protein via MVs for treatment of cancer. We first generated genetically engineered MVs by expressing high levels of the suicide gene mRNA and protein–cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT) in MV donor cells. MVs were isolated from these cells and used to treat pre-established nerve sheath tumors (schwannomas) in an orthotopic mouse model. We demonstrated that MV-mediated delivery of CD-UPRT mRNA/protein by direct injection into schwannomas led to regression of these tumors upon systemic treatment with the prodrug (5-fluorocytosine (5-FC)), which is converted within tumor cells to 5-fluorouracil (5-FU)–an anticancer agent. Taken together, these studies suggest that MVs can serve as novel cell-derived “liposomes” to effectively deliver therapeutic mRNA/proteins to treatment of diseases.

title: SiRNA delivery with exosome nanoparticles
abstract: A natural system for ferrying RNA between cells is used to transport siRNA to the mouse brain.

title: A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy.
abstract: Targeted drug delivery vehicles with low immunogenicity and toxicity are needed for cancer therapy. Here we show that exosomes, endogenous nano-sized membrane vesicles secreted by most cell types, can deliver chemotherapeutics such as doxorubicin (Dox) to tumor tissue in BALB/c nude mice. To reduce immunogenicity and toxicity, mouse immature dendritic cells (imDCs) were used for exosome production. Tumor targeting was facilitated by engineering the imDCs to express a well-characterized exosomal membrane protein (Lamp2b) fused to αv integrin-specific iRGD peptide (CRGDKGPDC). Purified exosomes from imDCs were loaded with Dox via electroporation, with an encapsulation efficiency of up to 20%. iRGD exosomes showed highly efficient targeting and Dox delivery to αv integrin-positive breast cancer cells in vitro as demonstrated by confocal imaging and flow cytometry. Intravenously injected targeted exosomes delivered Dox specifically to tumor tissues, leading to inhibition of tumor growth without overt toxicity. Our results suggest that exosomes modified by targeting ligands can be used therapeutically for the delivery of Dox to tumors, thus having great potential value for clinical applications.

title: Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugs from the nasal region to the brain.
abstract: In this study, exosomes used to encapsulate curcumin (Exo-cur) or a signal transducer and activator of transcription 3 (Stat3) inhibitor, i.e., JSI124 (Exo-JSI124) were delivered noninvasively to microglia cells via an intranasal route. The results generated from three inflammation-mediated disease models, i.e., a lipopolysaccharide (LPS)-induced brain inflammation model, experimental autoimmune encephalitis and a GL26 brain tumor model, showed that mice treated intranasally with Exo-cur or Exo-JSI124 are protected from LPS-induced brain inflammation, the progression of myelin oligodendrocyte glycoprotein (MOG) peptide induced experimental autoimmune encephalomyelitis (EAE), and had significantly delayed brain tumor growth in the GL26 tumor model. Intranasal administration of Exo-cur or Exo-JSI124 led to rapid delivery of exosome encapsulated drug to the brain that was selectively taken up by microglial cells, and subsequently induced apoptosis of microglial cells. Our results demonstrate that this strategy may provide a noninvasive and novel therapeutic approach for treating brain inflammatory-related diseases.

title: Malignant effusions and immunogenic tumour-derived exosomes
abstract: Exosomes derived from tumours are small vesicles released in vitro by tumour cell lines in culture supernatants. To assess the role of these exosomes in vivo, we examined malignant effusions for their presence. We also investigated whether these exosomes could induce production of tumour-specific T cells when pulsed with dendritic cells.



We isolated exosomes by ultracentrifugation on sucrose and D(2)O gradients of 11 malignant effusions. We characterised exosomes with Western blot analyses, immunoelectron microscopy, and in-vitro stimulations of autologous T lymphocytes.



Malignant effusions accumulate high numbers of membrane vesicles that have a mean diameter of 80 nm (SD 30). These vesicles have antigen-presenting molecules (MHC class-I heat-shock proteins), tetraspanins (CD81), and tumour antigens (Her2/Neu, Mart1, TRP, gp100). These criteria, including their morphological characteristics, indicate the similarities between these vesicles and exosomes. Exosomes from patients with melanoma deliver Mart1 tumour antigens to dendritic cells derived from monocytes (MD-DCs) for cross presentation to clones of cytotoxic T lymphocytes specific to Mart1. In seven of nine patients with cancer, lymphocytes specific to the tumour could be efficiently expanded from peripheral blood cells by pulsing autologous MD-DCs with autologous ascitis exosomes. In one patient tested, we successfully expanded a restricted T-cell repertoire, which could not be recovered carcinomatosis nodules.



Exosomes derived from tumours accumulate in ascites from patients with cancer. Ascitis exosomes are a natural and new source of tumour-rejection antigens, opening up new avenues for immunisation against cancers.