Non–small cell lung cancer-derived exosomes promote proliferation,phagocytosis, and secretion of microglia via exosomal microRNA in the metastatic microenvironment

Non–small cell lung cancer (NSCLC) is the most common tumor that metastasizes to the brain. It is now accepted that the successful colonization and growth of tumor cells are determined by the interaction between tumor cells and the tumor microenvironment (TME). Microglia, brain innate immune cells, have been reported to play a vital role in the establishment of brain metastases. As essential mediators of intercellular communications, tumor-derived exosomes have an important role in the pathogenesis and progression of cancer by transferring their cargos to specific recipient cells. The crosstalk between microglia and tumor-derived exosomes has been extensively described. However, it is still unclear whether metastatic NSCLC cells secret exosomes to microglia and regulate the microglial functions. Here, our results showed that microglia aggregated in the brain metastatic sites. Meanwhile, microglia could take up the exosomes derived from NSCLC cells, leading to alterations of microglial morphology and increased proliferation, phagocytosis, and release of inflammatory cytokines including interleukin-6, interleukin-8, and CXCL1. Further investigation indicated that miR1246 was the most enriched microRNA in NSCLC-derived exosomes and mediated the partial effects of exosomes on microglia. Notably, miR1246 was also upregulated in the plasmatic exosomes of NSCLC patients. These results offer a new insight into the impact of NSCLC-derived exosomes on microglia and provide a new potential biomarker for diagnosing NSCLC.     

Hypoxic stress suppresses lung tumor-secreted exosomal miR101 to activate macrophages and induce inflammation

Hypoxia promotes inflammation in the tumor microenvironment. Although hypoxia-inducible factor 1α (HIF1α) is a master modulator of the response to hypoxia, the exact mechanisms through which HIF1α regulates the induction of inflammation remain largely unclear. Using The Cancer Genome Atlas Lung Squamous Cell Carcinoma (TCGA-LUSC) database, we divided patients with LUSC into two groups based on low or high HIF1α expression. After analyzing the differentially expressed genes in these two groups, we found that HIF1α was positively correlated with interleukin 1A (IL1A) and IL6 expression. Our in vitro study showed that hypoxic stress did not induce IL1A or IL6 expression in tumor cells or macrophages but dramatically enhanced their expression when co-cultured with tumor cells. We then investigated the effect of tumor-derived exosomes on macrophages. Our data suggested that the changes in miR101 in the tumor-derived exosomes played an important role in IL1A and IL6 expression in macrophages, although the hypoxic stress did not change the total amount of exosome secretion. The expression of miR101 in exosomes was suppressed by hypoxic stress, since depletion of HIF1α in tumor cells recovered the miR101 expression in both tumor cells and exosomes. In vitro, miRNA101 overexpression or uptake enriched exosomes by macrophages suppressed their reprogramming into a pro-inflammatory state by targeting CDK8. Injection of miR101 into xenografted tumors resulted in the suppression of tumor growth and macrophage tumor infiltration in vivo. Collectively, this study suggests that the HIF1α-dependent suppression of exosome miR101 from hypoxic tumor cells activates macrophages to induce inflammation in the tumor microenvironment.Subject terms: Small-cell lung cancer, Small-cell lung cancer     

Exosomal miR‐663b targets Ets2‐repressor factor to promote proliferation and the epithelial–mesenchymal transition of bladder cancer cells

Exosomes circulating in biological fluids have the potential to be utilized as cancer biomarkers and are associated with cancer progression and metastasis. MicroRNA (miR)‐663b has been found to be elevated in plasma from patients with bladder cancer (BC). However, the functional role of exosomal miR‐663b in BC processes remains unknown. Here, we isolated exosomes from plasma and found that the miR‐663b level was elevated in exosomes from plasma of patients with BC compared with healthy controls. Exosomal miR‐663b from BC cells promoted cell proliferation and epithelial–mesenchymal transition. Moreover, exosomal miR‐663b targeted Ets2‐repressor factor and acted as a tumor promoter in BC cells. Taken together, our findings suggested that exosomal miR‐663b is a promising potential biomarker and target for clinical detection and therapy in BC.     

Hydrogel microchip as a tool for studying exosomes in human serum

Exosomes are cell-derived vesicles that are secreted by both normal and cancer cells. Over the last decade, a few studies have revealed that exosomes cross talk and/or influence major tumor-related pathways such as angiogenesis and metastasis involving many cell types within the tumor microenvironment. The protein composition of the membrane of an exosome reflects that of the membrane of the cell of origin. Because of this, tumor-derived exosomes differ from exosomes that are derived from normal cells. The detection of tumor exosomes and analysis of their molecular composition hold promise for diagnosis and prognosis of cancer. Here, we present hydrogel microarrays (biochips), which contain a panel of immobilized antibodies that recognize tetraspanins (CD9, CD63, CD81) and prognostic markers for colorectal cancer (A33, CD147). These biochips make it possible to analyze the surface proteins of either isolated exosomes or exosomes that are present in the serum samples without isolation. These biochips were successfully used to analyze the surface proteins of exosomes from serum that was collected from a colorectal cancer patient and healthy donor. Biochip-guided immunofluorescent analysis of the exosomes has made it possible for us to detect the A33 antigen and CD147 in the serum sample of the colorectal cancer patient with normal levels of CEA and CA19-9.     

Oncogenic and Non‐Malignant Pancreatic Exosome Cargo Reveal Distinct Expression of Oncogenic and Prognostic Factors Involved in Tumor Invasion and Metastasis

Exosomes are small extracellular membrane vesicles important in intercellular communication, with their oncogenic cargo attributed to tumor progression and pre‐metastatic niche formation. To gain an insight into key differences in oncogenic composition of exosomes, human non‐malignant epithelial and pancreatic cancer cell models and purified and characterized resultant exosome populations are utilized. Proteomic analysis reveals the selective enrichment of known exosome markers and signaling proteins in comparison to parental cells. Importantly, valuable insights into oncogenic exosomes (362 unique proteins in comparison to non‐malignant exosomes) of key metastatic regulatory factors and signaling molecules fundamental to pancreatic cancer progression (KRAS, CD44, EGFR) are provided. It is reported that oncogenic exosomes contain factors known to regulate the pre‐metastatic niche (S100A4, F3, ITGβ5, ANXA1), clinically‐relevant proteins which correlate with poor prognosis (CLDN1, MUC1) as well as protein networks involved in various cancer hallmarks including proliferation (CLU, CAV1), invasion (PODXL, ITGA3), metastasis (LAMP1, ST14) and immune surveillance escape (B2M). The presence of these factors in oncogenic exosomes offers an understanding of select differences in exosome composition during tumorigenesis, potential components as prognostic and diagnostic biomarkers in pancreatic cancer, and highlights the role of exosomes in mediating crosstalk between tumor and stromal cells.     

Endoplasmic reticulum stress‐induced exosomal miR‐27a‐3p promotes immune escape in breast cancer via regulating PD‐L1 expression in macrophages

Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA‐mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)‐27a‐3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR‐27a‐3p and MAGI2 was predicted using bioinformatic analysis and verified by dual‐luciferase reporter assay. Ectopic expression and inhibition of miR‐27a‐3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co‐cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD‐L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR‐27a‐3p expression. Elevation of miR‐27a‐3p and PD‐L1 levels in macrophages was observed in response to exosomes‐overexpressing miR‐27a‐3p in vivo and in vitro. miR‐27a‐3p could target and negatively regulate MAGI2, while MAGI2 down‐regulated PD‐L1 by up‐regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4⁺, CD8⁺ T cells and IL‐2, and T cells apoptosis were observed in response to co‐culture of macrophages and CD3⁺ T cells. Conjointly, exosomal miR‐27a‐3p promotes immune evasion by up‐regulating PD‐L1 via MAGI2/PTEN/PI3K axis in breast cancer.     

GPC1 exosome and its regulatory miRNAs are specific markers for the detection and target therapy of colorectal cancer

Colorectal cancer (CRC) is the second leading cause of cancer‐related deaths worldwide. However, a biomarker for a sensitive and simple diagnostic test and highly effective target therapy of CRC is still clinically unavailable. This study is to investigate the evidence and significance of plasma GPC1 positive exosomes as a biomarker of CRC. Results showed that GPC1⁺ exosomes were successfully isolated from tissues and plasma. The percentage of GPC1⁺ exosomes and the GPC1 protein expression in exosomes from tumour tissues and plasma of CRC patients before surgical treatment was significantly elevated compared to that in the peritumoural tissues and the plasma of healthy controls. miR‐96‐5p and miR‐149 expression in tumour tissues and plasma of CRC patients as well as in the GPC1⁺ exosomes from CRC patients were significantly decreased compared to that in the peritumoural tissues and the plasma of healthy controls. Two months after surgical treatment, levels of all tested markers significantly normalized. Overexpression of miR‐96‐5p and miR‐149 significantly decreased GPC1 expression in HT‐29 and HCT‐116 cells, xenograft tumours, plasma in mice bearing HT‐29 and HCT‐116 tumours, and the secretion of GPC1⁺ exosomes from the HT‐29 and HCT‐116 cells and xenograft tumours. Overexpression of miR‐96‐5p and miR‐149 significantly decreased cell viability and increased cell apoptosis in HT‐29 and HCT‐116 cells, and inhibited the growth of xenograft HT‐29 and HCT‐116 tumours. In conclusion, the increased plasma GPC1⁺ exosomes and reduced plasma miR‐96‐5p and miR‐149 expression are specific markers for the diagnosis of CRC and targets for the therapy of CRC.     

Isolation and proteomic analysis of exosomes secreted by human cancer cells in vitro

Exosomes are 20-100 nm membrane vesicles of endocytic origin secreted by most cell types in vitro and in vivo. Since exosomes contain both RNA (mRNA and microRNA) and proteins, which can be transferred to another cell, and be functional in that new environment, these vesicles may be involved in the communication between cells. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo suggest their participation in pathological situations. Our purpose here is to describe methods for the production, purification, and proteomic characterization of exosomes derived from human cancer cells in vitro. Based on exosomes' unique lipidic composition, we have developed the new approach to increase production of exosomes by cells in vitro. Secondly, we have developed quality control by laser correlation spectroscopy for exosomal assays based on the amount of MHC class I and CD63 molecules on their surface. At last, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was used after 2D electrophoresis for the proteomic analysis of exosomes derived from cancer cell lines. This study describes the protein composition of brain tumor cell-derived exosomes in more detail.     

Pancreatic cancer cell–derived exosomal microRNA‐27a promotes angiogenesis of human microvascular endothelial cells in pancreatic cancer via BTG2

Pancreatic cancer (PC) remains a primary cause of cancer‐related deaths worldwide. Existing literature has highlighted the oncogenic role of microRNA‐27a (miR‐27a) in multiple cancers. Hence, the current study aimed to clarify the potential therapeutic role of PC cell–derived exosomal miR‐27a in human microvascular endothelial cell (HMVEC) angiogenesis in PC. Initially, differentially expressed genes (DEGs) and miRs related to PC were identified by microarray analysis. Microarray analysis provided data predicting the interaction between miR‐27a and BTG2 in PC, which was further verified by the elevation or depletion of miR‐27a. Next, the expression of miR‐27a and BTG2 in the PC tissues was quantified. HMVECs were exposed to exosomes derived from PC cell line PANC‐1 to investigate the effects associated with PC cell–derived exosomes carrying miR‐27a on HMVEC proliferation, invasion and angiogenesis. Finally, the effect of miR‐27a on tumorigenesis and microvessel density (MVD) was analysed after xenograft tumour inoculation in nude mice. Our results revealed that miR‐27a was highly expressed, while BTG2 was poorly expressed in both PC tissues and cell lines. miR‐27a targeted BTG2. Moreover, miR‐27a silencing inhibited PC cell proliferation and invasion, and promoted apoptosis through the elevation of BTG2. The in vitro assays revealed that PC cell–derived exosomes carrying miR‐27a stimulated HMVEC proliferation, invasion and angiogenesis, while this effect was reversed in the HMVECs cultured with medium containing GW4869‐treated PANC‐1 cells. Furthermore, in vivo experiment revealed that miR‐27a knockdown suppressed tumorigenesis and MVD. Taken together, cell‐derived exosomes carrying miR‐27a promotes HMVEC angiogenesis via BTG2 in PC.     

Curcumin reinforces MSC‐derived exosomes in attenuating osteoarthritis via modulating the miR‐124/NF‐kB and miR‐143/ROCK1/TLR9 signalling pathways

Curcumin treatment was reported to delay the progression of OA, but its underlying mechanism remains unclear. In this study, we aimed to investigate the molecular mechanism underlying the role of curcumin in OA treatment. Accordingly, by conducting MTT and flow cytometry assays, we found that the exosomes derived from curcumin‐treated MSCs helped to maintain the viability while inhibiting the apoptosis of model OA cells. Additionally, quantitative real‐time PCR and Western blot assays showed that the exosomes derived from curcumin‐treated MSCs significantly restored the down‐regulated miR‐143 and miR‐124 expression as well as up‐regulated NF‐kB and ROCK1 expression in OA cells. Mechanistically, curcumin treatment decreased the DNA methylation of miR‐143 and miR‐124 promoters. In addition, the 3’ UTRs of NF‐kB and ROCK1 were proven to contain the binding sites for miR‐143 and miR‐124, respectively. Therefore, the up‐regulation of miR‐143 and miR‐124 in cellular and mouse OA models treated with exosomes remarkably restored the normal expression of NF‐kB and ROCK1. Consequently, the progression of OA was attenuated by the exosomes. Our results clarified the molecular mechanism underlying the therapeutic role of MSC‐derived exosomes in OA treatment.     

Long non‐coding RNA H19 promotes the proliferation and invasion of breast cancer through upregulating DNMT1 expression by sponging miR‐152

Long non‐coding RNA (lncRNA) H19 in tumors played important roles in various biological processes. However, the biological role and molecular mechanism of H19 in breast cancer are unclear. Here, we found that H19 was aberrantly upregulated in human breast tumor tissues and cells. A negative correlation between H19 and miR‐152 and positive correlation between H19 and DNMT1 mRNA were observed. Downregulation of H19 and DNMT1 significantly retarded breast cancer cell proliferation and invasion. H19 act as an endogenous sponge by directly binding to miR‐152. miR‐152 directly targeted DNMT1 and was regulated by H19. Besides, H19 overexpression dramatically relieved the inhibition of miR‐152 on DNMT1 expression. miR‐152 inhibition and DNMT1 overexpression obviously reversed the inhibitory effects of H19 downregulation on cell proliferation and invasion. In conclusion, H19 promoted proliferation and invasion of breast cancer through the miR‐152/DNMT1 axis, providing a novel mechanism about the occurrence and development of breast cancer.     

Exosomal MiR‐500a‐3p promotes cisplatin resistance and stemness via negatively regulating FBXW7 in gastric cancer

Chemoresistance has been a major challenge in advanced gastric cancer (GC) therapy. Exosomal transfer of oncogenic miRNAs implicates important effects in mediating recipient cell chemoresistance by transmitting active molecules. In this study, we found that microRNA‐500a‐3p was highly expressed in cisplatin (DDP) resistant GC cells (MGC803/DDP and MKN45/DDP) and their secreted exosomes than that in the corresponding parental cells. MGC803/DDP‐derived exosomes enhance DDP resistance and stemness properties of MGC803 recipient cells via exosomal delivery of miR‐500a‐3p in vitro and in vivo through targeting FBXW7. However, reintroduction of FBXW7 in MGC803 cells reverses miR‐500a‐3p‐mediated DDP resistance as well as stemness properties. Furthermore, elevated miR‐500a‐3p in the plasma exosomes of GC patients is correlated with DDP resistance and thereby results in poor progression‐free prognosis. Our finding highlights the potential of exosomal miR‐500a‐3p as an potential modality for the prediction and treatment of GC with chemoresistance.     

Heparanase Regulates Secretion,Composition, and Function of Tumor Cell-derived Exosomes

Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.     

Exosomes from mesenchymal stem cells expressing miR‐125b inhibit neointimal hyperplasia via myosin IE

Intercellular communication between mesenchymal stem cells (MSCs) and their target cells in the perivascular environment is modulated by exosomes derived from MSCs. However, the potential role of exosome‐mediated microRNA transfer in neointimal hyperplasia remains to be investigated. To evaluate the effects of MSC‐derived exosomes (MSC‐Exo) on neointimal hyperplasia, their effects upon vascular smooth muscle cell (VSMC) growth in vitro and neointimal hyperplasia in vivo were assessed in a model of balloon‐induced vascular injury. Our results showed that MSC‐Exo were internalised by VSMCs and inhibited proliferation and migration in vitro. Further analysis revealed that miR‐125b was enriched in MSC‐Exo, and repressed the expression of myosin 1E (Myo1e) by targeting its 3? untranslated region. Additionally, MSC‐Exo and exosomally transferred miR‐125b repressed Myo1e expression and suppressed VSMC proliferation and migration and neointimal hyperplasia in vivo. In summary, our findings revealed that MSC‐Exo can transfer miR‐125b to VSMCs and inhibit VSMC proliferation and migration in vitro and neointimal hyperplasia in vivo by repressing Myo1e, indicating that miR‐125b may be a therapeutic target in the treatment of vascular diseases.     

Tumour‐derived exosomal miR‐3473b promotes lung tumour cell intrapulmonary colonization by activating the nuclear factor‐κB of local fibroblasts

Tumour‐derived exosomes have been shown to induce pre‐metastatic niche formation, favoring metastatic colonization of tumour cells, but the underlying molecular mechanism is still not fully understood. In this study, we showed that exosomes derived from the LLC cells could indeed significantly enhance their intrapulmonary colonization. Circulating LLC‐derived exosomes were mainly engulfed by lung fibroblasts and led to the NF‐κB signalling activation. Further studies indicated that the exosomal miR‐3473b was responsible for that by hindering the NFKB inhibitor delta's (NFKBID) function. Blocking miR‐3473b could reverse the exosome‐mediated NF‐κB activation of fibroblasts and decrease intrapulmonary colonization of lung tumour cells. Together, this study demonstrated that the miR‐3473b in exosomes could mediate the interaction of lung tumour cells and local fibroblasts in metastatic sites and, therefore, enhance the metastasis of lung tumour cells.     

Molecular lipidomics of exosomes released by PC-3 prostate cancer cells

The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating an extraordinary discrimination of lipids sorted into these microvesicles. In particular, exosomes are highly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (mol% of total lipids). Furthermore, lipid species, even of classes not enriched in exosomes, were selectively included in exosomes. Finally, it was found that there is an 8.4-fold enrichment of lipids per mg of protein in exosomes. The detailed lipid composition provided in this study may be useful to understand the mechanism of exosome formation, release and function. Several of the lipids enriched in exosomes could potentially be used as cancer biomarkers.     

Exosomes in carcinogenesis: molecular palkis carry signals for the regulation of cancer progression and metastasis

Exosomes, which act as biological cargo vessels, are cell-released, phospholipid-enclosed vesicles. In eukaryotic cells, exosomes carry and exchange biological materials or signals for the benefit or detriment to the cells. Thereby, we consider exosomes to be molecular Palkis (carriers). Although exosomes are currently one of the most popularly researched cellular entities, they have remained largely enigmatic and warrant continued investigation into their structure and functions. These membraned vesicles are between 30 and 150 nm in diameter and are actively secreted by all cell types. While initially considered cellular “trash bags,” recent years have revealed exosomes to be dynamic and multi-functional vesicles that may play a crucial role in cancer development, progression and metastasis. Thereby, they have the potential to be used in development of therapeutic modalities for cancer and other diseases. As more research studies emerge, it’s becoming evident that exosomes are released by cells with a purpose and are representatives of certain cell types and disease conditions. Hence, they may also be used as biomarkers for the detection of cancer initiation, progression and organotropic metastatic growth of cancer cells. This review will focus on the recent developments achieved in identifying the role of exosomes in cancer development and progression as well as therapeutic implications. The review will also discuss the pitfalls of methodologies used for the extraction of exosomes.