Prostate Tumor-Derived Exosomes Down-Regulate NKG2D Expression on Natural Killer Cells and CD8+ T Cells: Mechanism of Immune Evasion

Tumor-derived exosomes, which are nanometer-sized extracellular vesicles of endosomal origin, have emerged as promoters of tumor immune evasion but their role in prostate cancer (PC) progression is poorly understood. In this study, we investigated the ability of prostate tumor-derived exosomes to downregulate NKG2D expression on natural killer (NK) and CD8⁺ T cells. NKG2D is an activating cytotoxicity receptor whose aberrant loss in cancer plays an important role in immune suppression. Using flow cytometry, we found that exosomes produced by human PC cells express ligands for NKG2D on their surface. The NKG2D ligand-expressing prostate tumor-derived exosomes selectively induced downregulation of NKG2D on NK and CD8⁺ T cells in a dose-dependent manner, leading to impaired cytotoxic function in vitro. Consistent with these findings, patients with castration-resistant PC (CRPC) showed a significant decrease in surface NKG2D expression on circulating NK and CD8⁺ T cells compared to healthy individuals. Tumor-derived exosomes are likely involved in this NKG2D downregulation, since incubation of healthy lymphocytes with exosomes isolated from serum or plasma of CRPC patients triggered downregulation of NKG2D expression in effector lymphocytes. These data suggest prostate tumor-derived exosomes as down-regulators of the NKG2D-mediated cytotoxic response in PC patients, thus promoting immune suppression and tumor escape.     

Cancer exosomes express CD39 and CD73, which suppress T cells through adenosine production

Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune responses. Adenosine production from extracellular ATP has attracted attention as a mechanism of regulatory T cell-mediated immune regulation. In this study, we examined whether small vesicles secreted by cancer cells, called exosomes, contribute to extracellular adenosine production and hence modulate immune effector cells indirectly. We found exosomes from diverse cancer cell types exhibit potent ATP- and 5'AMP-phosphohydrolytic activity, partly attributed to exosomally expressed CD39 and CD73, respectively. Comparable levels of activity were seen with exosomes from pleural effusions of mesothelioma patients. In such fluids, exosomes accounted for 20% of the total ATP-hydrolytic activity. Exosomes can perform both hydrolytic steps sequentially to form adenosine from ATP. This exosome-generated adenosine can trigger a cAMP response in adenosine A(2A) receptor-positive but not A(2A) receptor-negative cells. Similarly, significantly elevated cAMP was also triggered in Jurkat cells by adding exosomes with ATP but not by adding exosomes or ATP alone. A proportion of healthy donor T cells constitutively express CD39 and/or CD73. Activation of T cells by CD3/CD28 cross-linking could be inhibited by exogenously added 5'AMP in a CD73-dependent manner. However, 5'AMP converted to adenosine by exosomes inhibits T cell activation independently of T cell CD73 expression. This T cell inhibition was mediated through the adenosine A(2A) receptor. In summary, the data highlight exosome enzymic activity in the production of extracellular adenosine, and this may play a contributory role in negative modulation of T cells in the tumor environment.     

Activated T cell exosomes promote tumor invasion via Fas signaling pathway

Activated T cells release bioactive Fas ligand (FasL) in exosomes, which subsequently induce self-apoptosis of T cells. However, their potential effects on cell apoptosis in tumors are still unknown. In this study, we purified exosomes expressing FasL from activated CD8(+) T cell from OT-I mice and found that activated T cell exosomes had little effect on apoptosis and proliferation of tumor cells but promoted the invasion of B16 and 3LL cancer cells in vitro via the Fas/FasL pathway. Activated T cell exosomes increased the amount of cellular FLICE inhibitory proteins and subsequently activated the ERK and NF-κB pathways, which subsequently increased MMP9 expression in the B16 murine melanoma cells. In a tumor-invasive model in vivo, we observed that the activated T cell exosomes promoted the migration of B16 tumor cells to lung. Interestingly, pretreatment with FasL mAb significantly reduced the migration of B16 tumor cells to lung. Furthermore, CD8 and FasL double-positive exosomes from tumor mice, but not normal mice, also increased the expression of MMP9 and promoted the invasive ability of B16 murine melanoma and 3LL lung cancer cells. In conclusion, our results indicate that activated T cell exosomes promote melanoma and lung cancer cell metastasis by increasing the expression of MMP9 via Fas signaling, revealing a new mechanism of tumor immune escape.     

Natural extracellular nanovesicles and photodynamic molecules: is there a future for drug delivery?

Photodynamic molecules represent an alternative approach for cancer therapy for their property (i) to be photo-reactive; (ii) to be not-toxic for target cells in absence of light; (iii) to accumulate specifically into tumour tissues; (iv) to be activable by a light beam only at the tumour site and (v) to exert cytotoxic activity against tumour cells. However, to date their clinical use is limited by the side effects elicited by systemic administration. Extracellular vesicles are endogenous nanosized-carriers that have been recently introduced as a natural delivery system for therapeutic molecules. We have recently shown the ability of human exosomes to deliver photodynamic molecules. Therefore, this review focussed on extracellular vesicles as a novel strategy for the delivery of photodynamic molecules at cancer sites. This completely new approach may enhance the delivery and decrease the toxicity of photodynamic molecules, therefore, represent the future for photodynamic therapy for cancer treatment.     

Exosomes Derived from Hypoxic Leukemia Cells Enhance Tube Formation in Endothelial Cells

Hypoxia plays an important role during the evolution of cancer cells and their microenvironment. Emerging evidence suggests communication between cancer cells and their microenvironment occurs via exosomes. This study aimed to clarify whether hypoxia affects angiogenic function through exosomes secreted from leukemia cells. We used the human leukemia cell line K562 for exosome-generating cells and human umbilical vein endothelial cells (HUVECs) for exosome target cells. Exosomes derived from K562 cells cultured under normoxic (20%) or hypoxic (1%) conditions for 24 h were isolated and quantitated by nanoparticle tracking analysis. These exosomes were then cocultured with HUVECs to evaluate angiogenic activity. The exosomes secreted from K562 cells in hypoxic conditions significantly enhanced tube formation by HUVECs compared with exosomes produced in normoxic conditions. Using a TaqMan low-density miRNA array, we found a subset of miRNAs, including miR-210, were significantly increased in exosomes secreted from hypoxic K562 cells. We demonstrated that cancer cells and their exosomes have altered miRNA profiles under hypoxic conditions. Although exosomes contain various molecular constituents such as proteins and mRNAs, altered exosomal compartments under hypoxic conditions, including miR-210, affected the behavior of endothelial cells. Our results suggest that exosomal miRNA derived from cancer cells under hypoxic conditions may partly affect angiogenic activity in endothelial cells.     

Let-7 microRNA family is selectively secreted into the extracellular environment via exosomes in a metastatic gastric cancer cell line

Background

Exosomes play a major role in cell-to-cell communication, targeting cells to transfer exosomal molecules including proteins, mRNAs, and microRNAs (miRNAs) by an endocytosis-like pathway. miRNAs are small noncoding RNA molecules on average 22 nucleotides in length that regulate numerous biological processes including cancer pathogenesis and mediate gene down-regulation by targeting mRNAs to induce RNA degradation and/or interfering with translation. Recent reports imply that miRNAs can be stably detected in circulating plasma and serum since miRNAs are packaged by exosomes to be protected from RNA degradation. Thus, profiling exosomal miRNAs are in need to clarify intercellular signaling and discover a novel disease marker as well.

Methodology/Principal Findings

Exosomes were isolated from cultured cancer cell lines and their quality was validated by analyses of transmission electron microscopy and western blotting. One of the cell lines tested, a metastatic gastric cancer cell line, AZ-P7a, showed the highest RNA yield in the released exosomes and distinctive shape in morphology. In addition, RNAs were isolated from cells and culture media, and profiles of these three miRNA fractions were obtained using microarray analysis. By comparing signal intensities of microarray data and the following validation using RT-PCR analysis, we found that let-7 miRNA family was abundant in both the intracellular and extracellular fractions from AZ-P7a cells, while low metastatic AZ-521, the parental cell line of AZ-P7a, as well as other cancer cell lines showed no such propensity.

Conclusions/Significance

The enrichment of let-7 miRNA family in the extracellular fractions, particularly, in the exosomes from AZ-P7a cells may reflect their oncogenic characteristics including tumorigenesis and metastasis. Since let-7 miRNAs generally play a tumor-suppressive role as targeting oncogenes such as RAS and HMGA2, our results suggest that AZ-P7a cells release let-7 miRNAs via exosomes into the extracellular environment to maintain their oncogenesis.     

Mesenchymal stroma/stem‐like cells of GARP knockdown inhibits cell proliferation and invasion of mouse colon cancer cells (MC38) through exosomes

Mesenchymal stroma/stem‐like cells (MSCs) have antitumour activity, and MSC‐derived exosomes play a role in the growth, metastasis and invasion of tumour cells. Additionally, glycoprotein A repetition predominant (GARP) promotes oncogenesis in breast cancer. Therefore, GARP is speculated to be a target gene for cancer therapy. We aimed to explore the therapy role of MSC‐derived exosomes targeting GARP in mouse colon cancer cell MC38. We successfully established a GARP knockdown system using three kinds of siRNA‐GARP in MSC cells. Exosomes were isolated from MSC and siGARP‐MSC cells, and verified by the exosome surface protein markers CD9, CD63 and CD81. GARP expression was significantly decreased in siGARP‐MSC exosomes compared with that of MSC exosomes. We found that siGARP‐MSC exosomes inhibited cell proliferation, migration and invasion of MC38 cells, using CCK‐8, colony formation, wound‐healing and Transwell invasion assays. Furthermore, siGARP‐MSC exosomes impeded IL‐6 secretion and partly inactivated JAK1/STAT3 pathway, measured using ELISA and RT‐qPCR. In conclusion, MSC‐derived exosomes targeting GARP are a potential strategy for cancer therapy.     

Exosomes Derived from Mesenchymal Stem Cells Suppress Angiogenesis by Down-Regulating VEGF Expression in Breast Cancer Cells

Exosomes are small membrane vesicles released by a variety of cell types. Exosomes contain genetic materials, such as mRNAs and microRNAs (miRNAs), implying that they may play a pivotal role in cell-to-cell communication. Mesenchymal stem cells (MSCs), which potentially differentiate into multiple cell types, can migrate to the tumor sites and have been reported to exert complex effects on tumor progression. To elucidate the role of MSCs within the tumor microenvironment, previous studies have suggested various mechanisms such as immune modulation and secreted factors of MSCs. However, the paracrine effects of MSC-derived exosomes on the tumor microenvironment remain to be explored. The hypothesis of this study was that MSC-derived exosomes might reprogram tumor behavior by transferring their molecular contents. To test this hypothesis, exosomes from MSCs were isolated and characterized. MSC-derived exosomes exhibited different protein and RNA profiles compared with their donor cells and these vesicles could be internalized by breast cancer cells. The results demonstrated that MSC-derived exosomes significantly down-regulated the expression of vascular endothelial growth factor (VEGF) in tumor cells, which lead to inhibition of angiogenesis in vitro and in vivo. Additionally, miR-16, a miRNA known to target VEGF, was enriched in MSC-derived exosomes and it was partially responsible for the anti-angiogenic effect of MSC-derived exosomes. The collective results suggest that MSC-derived exosomes may serve as a significant mediator of cell-to-cell communication within the tumor microenvironment and suppress angiogenesis by transferring anti-angiogenic molecules.     

Induction of ATM/ATR pathway combined with Vγ2Vδ2 T cells enhances cytotoxicity of ovarian cancer cells

Many ovarian cancer cells express stress-related molecule MICA/B on their surface that is recognized by Vγ2Vδ2 T cells through their NKG2D receptor, which is transmitted to downstream stress-signaling pathway. However, it is yet to be established how Vγ2Vδ2 T cell-mediated recognition of MICA/B signal is transmitted to downstream stress-related molecules. Identifying targeted molecules would be critical to develop a better therapy for ovarian cancer cells. It is well established that ATM/ATR signal transduction pathways, which is modulated by DNA damage, replication stress, and oxidative stress play central role in stress signaling pathway regulating cell cycle checkpoint and apoptosis. We investigated whether ATM/ATR and its down stream molecules affect Vγ2Vδ2 T cell-mediated cytotoxicity. Herein, we show that ATM/ATR pathway is modulated in ovarian cancer cells in the presence of Vγ2Vδ2 T cells. Furthermore, downregulation of ATM pathway resulted downregulation of MICA, and reduced Vγ2Vδ2 T cell-mediated cytotoxicity. Alternately, stimulating ATM pathway enhanced expression of MICA, and sensitized ovarian cancer cells for cytotoxic lysis by Vγ2Vδ2 T cells. We further show that combining currently approved chemotherapeutic drugs, which induced ATM signal transduction, along with Vγ2Vδ2 T cells enhanced cytotoxicity of resistant ovarian cancer cells. These findings indicate that ATM/ATR pathway plays an important role in tumor recognition, and drugs promoting ATM signaling pathway might be considered as a combination therapy together with Vγ2Vδ2 T cells for effectively treating resistant ovarian cancer cells.     

Characterization of Human Thymic Exosomes

Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.     

Exosome-mediated transfer of long noncoding RNA H19 induces doxorubicin resistance in breast cancer

Development of the acquired resistance is one major obstacle during chemotherapy for cancer patients. Exosomes mediate intercellular communication and cause environmental changes in tumor progression by transmitting active molecules. In this study, the role of long noncoding RNA H19 within exosomes is elucidated in terms of regulating doxorubicin (DOX) resistance of breast cancer. As a result, increased H19 expression was observed in DOX-resistant breast cancer cells in comparison with the corresponding parental cells. Suppression of H19 significantly lowered DOX resistance by decreasing cell viability, lowering colony-forming ability, and inducing apoptosis. Moreover, extracellular H19 could be moved to sensitive cells via being incorporated into exosomes. Treating sensitive cells with exosomes from resistant cells increased the chemoresistance of DOX, while downregulation of H19 in sensitive cells abated this effect. Taken together, H19 could be delivered by exosomes to sensitive cells, leading to the dissemination of DOX resistance. Our finding highlights the potential of exosomal H19 as a molecular target to reduce DOX resistance.     

Exosomes transmit T790M mutation‐induced resistance in EGFR‐mutant NSCLC by activating PI3K/AKT signalling pathway

Emerging evidence has shown that exosomes derived from drug‐resistant tumour cells are able to horizontally transmit drug‐resistant phenotype to sensitive cells. However, whether exosomes shed by EGFR T790M‐mutant–resistant NSCLC cells could transfer drug resistance to sensitive cells has not been investigated. We isolated exosomes from the conditioned medium (CM) of T790M‐mutant NSCLC cell line H1975 and sensitive cell line PC9. The role and mechanism of exosomes in regulating gefitinib resistance was investigated both in vitro and in vivo. Exosome‐derived miRNA expression profiles from PC9 and H1975 were analysed by small RNA sequencing and confirmed by qRT‐PCR. We found that exosomes shed by H1975 could transfer gefitinib resistance to PC9 both in vitro and in vivo through activating PI3K/AKT signalling pathway. Small RNA sequencing and RT‐PCR confirmed that miR‐3648 and miR‐522‐3p were the two most differentially expressed miRNAs and functional study showed that up‐regulation of miR‐522‐3p could induce gefitinib resistance in PC9 cell. The findings of our study reveal an important mechanism of acquired resistance to EGFR‐TKIs in NSCLC.     

Identification of novel subdominant epitopes on the carcinoembryonic antigen recognized by CD4+ T cells of lung cancer patients

The carcinoembryonic Ag (CEA) is an attractive target for immunotherapy because of its expression profile and role in tumor progression. To verify the existence of spontaneous anti-CEA CD4+ T cells in lung cancer patients, we first identified CEA sequences forming naturally processed epitopes, and then used the identified epitopes to test their recognition by CD4+ T cells from the patients. We had previously identified CEA(177-189/355-367) as an immunodominant epitope recognized by CD4+ T cells in association with several HLA-DR alleles. In this study, we identified four additional subdominant CEA sequences (CEA(99-111), CEA(425-437), CEA(568-582), and CEA(666-678)), recognized in association with one or more HLA-DR alleles. Peptide-specific CD4+ T cells produced proinflammatory cytokines when challenged with the native protein and CEA-expressing tumor cells, thus demonstrating that the identified CEA sequences contain naturally processed epitopes. However, CEA is expressed in the thymus and belongs to the CD66 family that comprises highly homologous molecules expressed on hemopoietic cells, raising concerns about tolerance interfering with the in vivo development of anti-CEA immunity. We thus tested the spontaneous reactivity to the identified epitopes of peripheral blood CD4+ T lymphocytes from eight early-stage lung cancer patients bearing CEA-positive tumors. We found GM-CSF- and IFN-gamma-producing CD4+ T cells in two patients. Our data indicate that CD4+ immune responses against CEA develop in neoplastic patients, suggesting that tolerance toward CEA or cross-reactive CD66 homologous molecules might be either not absolute or be overcome in the neoplastic disease.     

Exosome circ_0044516 promotes prostate cancer cell proliferation and metastasis as a potential biomarker

Circular RNAs (circRNAs) are important regulators in cancer growth and progression. Exosomes carry various molecules including RNA, protein, and lipid from one cell to another cell. But the role of circRNAs from the exosomes from prostate cancer patients are not elucidated. In this study, circ_0044516 was found upregulated in prostate cancer and the roles and molecular mechanism of Hsa_circ_0044516 (circ_0044516) was investigated. Firstly, the exosomes of prostate cancer patients were collected for human circRNAs microarray to screen the circRNA expression profile. There were 35 significantly expressed circRNAs with more than fivefolds from microarray analysis. Circ_0044516 was verified to be significantly upregulated in the exosomes from prostate cancer patients and the cell lines. Further investigation demonstrated that circ_0044516 downregulation inhibited the proliferation and metastasis of prostate cancer cells. By bioinformatics and luciferase reporter assays, circ_0044516 was verified to downregulate miR-29a-3p expression and negatively related to miR-29a-3p expression levels in prostate cancer. In a summary, the study indicated that circ_0044516 played an important role in prostate cancer cell survival and metastasis, which suggested that an oncogenic role of circ_0044516 in prostate cancer.     

Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes

Exosomes proteins and microRNAs have gained much attention as diagnostic tools and biomarker potential in various malignancies including prostate cancer (PCa). However, the role of exosomes and membrane-associated receptors, particularly epidermal growth factor receptor (EGFR) as mediators of cell proliferation and invasion in PCa progression remains unexplored. EGFR is frequently overexpressed and has been associated with aggressive forms of PCa. While PCa cells and tissues express EGFR, it is unknown whether exosomes derived from PCa cells or PCa patient serum contains EGFR. The aim of this study was to detect and characterize EGFR in exosomes derived from PCa cells, LNCaP xenograft and PCa patient serum. Exosomes were isolated from conditioned media of different PCa cell lines; LNCaP xenograft serum as well as patient plasma/serum by differential centrifugation and ultracentrifugation on a sucrose density gradient. Exosomes were confirmed by electron microscopy, expression of exosomal markers and NanoSight^™ analysis. EGFR expression was determined by western blot analysis and ELISA. This study demonstrates that exosomes may easily be derived from PCa cell lines, serum obtained from PCa xenograft bearing mice and clinical samples derived from PCa patients. Presence of exosomal EGFR in PCa patient exosomes may present a novel approach for measuring of the disease state. Our work will allow to build on this finding for future understanding of PCa exosomes and their potential role in PCa progression and as minimal invasive biomarkers for PCa.     

Different phenotypes of colon carcinoma cells interacting with endothelial cells: role of E-selectin and ultrastructural data

Adhesion molecules are intimately involved in the process of tumour progression. Among them, E-selectin is an inducible endothelial cell adhesion molecule that plays a role in the interactions of neoplastic cells with the endothelium. These interactions are required for the trans-endothelial migration of tumour cells that leads to the growth at the new sites. Since the detailed events in the early phase of metastasis still remain poorly defined, our study has undertaken an electron-microscopic analysis of the interactions of human colon carcinoma cells with endothelial cells as well as an analysis of the effect of recombinant purified E-selectin in the cell signalling involved in colon cancer cell malignant phenotype. Results revealed that SW480 and T84 colon cancer cell lines show different features, different adhesion kinetics, a different cytoskeletal organization, and a different tyrosine phosphorylation pattern when seeded on an endothelial cell monolayer or recombinant E-selectin. In particular T84 cancer cells adhere more efficiently to the E-selectin and this interaction is associated with pronounced morphological changes, actin redistribution and filopodial processes, and an increase in tyrosine phosphorylation of different proteins. These data support the hypothesis that E-selectin ligand is not only a cell-cell adhesion molecule but also initiates a signalling transduction pathway inside the cells.     

Exosomal annexin A6 induces gemcitabine resistance by inhibiting ubiquitination and degradation of EGFR in triple-negative breast cancer

Exosomes are carriers of intercellular information that regulate the tumor microenvironment, and they have an essential role in drug resistance through various mechanisms such as transporting RNA molecules and proteins. Nevertheless, their effects on gemcitabine resistance in triple-negative breast cancer (TNBC) are unclear. In the present study, we examined the effects of exosomes on TNBC cell viability, colony formation, apoptosis, and annexin A6 (ANXA6)/EGFR expression. We addressed their roles in gemcitabine resistance and the underlying mechanism. Our results revealed that exosomes derived from resistant cancer cells improved cell viability and colony formation and inhibited apoptosis in sensitive cancer cells. The underlying mechanism included the transfer of exosomal ANXA6 from resistant cancer cells to sensitive cancer cells. Isobaric peptide labeling–liquid chromatography–tandem mass spectrometry and western blotting revealed that ANXA6 was upregulated in resistant cancer cells and their derived exosomes. Sensitive cancer cells exhibited resistance with increased viability and colony formation and decreased apoptosis when ANXA6 was stably overexpressed. On the contrary, knockdown ANXA6 restored the sensitivity of cells to gemcitabine. Co-immunoprecipitation expression and GST pulldown assay demonstrated that exosomal ANXA6 and EGFR could interact with each other and exosomal ANXA6 was associated with the suppression of EGFR ubiquitination and downregulation. While adding lapatinib reversed gemcitabine resistance induced by exosomal ANXA6. Moreover, ANXA6 and EGFR protein expression was correlated in TNBC tissues, and exosomal ANXA6 levels at baseline were lower in patients with highly sensitive TNBC than those with resistant TNBC when treated with first-line gemcitabine-based chemotherapy. In conclusion, resistant cancer cell-derived exosomes induced gemcitabine resistance via exosomal ANXA6, which was associated with the inhibition of EGFR ubiquitination and degradation. Exosomal ANXA6 levels in the serum of patients with TNBC might be predictive of the response to gemcitabine-based chemotherapy.Subject terms: Breast cancer, Predictive markers     

Exosomal miR-375 promotes the activity of osteoblasts in prostate cancer

Studies have shown that exosomes influence tumour metastasis, diagnosis, and treatment. It has been found that exosomal miRNAs are closely linked to the metastatic microenvironment. However, the regulatory role of exosomes from prostate cancer (PCa) cells in bone metastasis remains poorly understood. Here, exosomes were isolated and purified by ultracentrifugation, total RNA from cells and total miRNA from exosomes were isolated, and the level of miR-375 was analyzed by RT-PCR. Exosome libraries from LNCaP cells and RWPE-1 cells were sequenced and filtered with an Illumina HiSeq^TM 2500 system. The activity of alkaline phosphatase, the extent of extracellular matrix mineralization, and the expression of osteoblast activity-related marker genes were measured to evaluate osteoblast activity. Morphological observation, particle size analysis, and molecular phenotyping confirmed that the isolated extracts contained exosomes. Differential expression analysis confirmed the high expression of miR-375 in LNCaP cell-derived exosomes. This study suggest that exosomes could enter osteoblasts and increase their miR-375 level. In addition, exosomal miR-375 could significantly promote the activity of osteoblasts.This study lays the foundation for further investigations on the function of exosomal miR-375 in the activation and differentiation of osteoblasts and the mechanism of bone metastasis in PCa.