Esophageal Cancer-Derived Extracellular Vesicle miR-21-5p Contributes to EMT of ESCC Cells by Disorganizing Macrophage Polarization.
ABSTRACT: The disorganized polarization of tumor-associated macrophages (TAMs) exerts a critical effect on tumor progression. MicroRNAs (miRNAs) in extracellular vesicles (EVs) secreted from cancer cells may contribute to this process. However, the relationship between TAMs and EVs-miRNAs-mediated regulation in esophageal squamous cell carcinoma (ESCC) remains unclear. In the present study, immunoaffinity magnetic beads combined with antiepithelial cell adhesion molecules (EpCAM) were used to isolate and identify EVs-miR-21-5p from the plasma of ESCC patients. An in vitro coculture system was designed to evaluate the effect of esophageal cancer cells with miR-21-5p overexpression on macrophage polarization. We found that phorbol myristate acetate-induced THP-1 macrophages took up EVs-miR-21-5p from EC109 or EC9706 cells and were transformed into M2 macrophages. This, in turn, contributed to the excessive migration and invasion of esophageal cancer cells. The mechanism underlying these changes may involve activation of M2 macrophages by upregulated ESCC-derived EVs-miR-21-5p through the PTEN/AKT/STAT6 pathway. This may result in esophageal cancer cell epithelial-mesenchymal transition (EMT) via TGF-β/Smad2 signaling. Our results indicate positive feedback between M2 macrophage polarization and EMT of esophageal cancer cells in the tumor microenvironment via shuttling of miR-21-5p in tumor-derived EVs.
Project description:BACKGROUND:The poor prognosis of esophageal squamous cell carcinoma (ESCC) highlights the need for novel strategies against this disease. Our previous study suggested the involvement of CCL2 and tumor associated macrophages (TAMs) in esophageal carcinogenesis. Despite the recognition of TAMs as a promising target for cancer treatment, mechanisms underlying its infiltration, activation and tumor-promotive function in ESCC remain unknown. METHODS:Human esophageal tissue array and TCGA database were used to evaluate the clinical relevance of CCL2 and TAMs in ESCC. F344 rats and C57BL/6 mice were treated with N-nitrosomethylbenzylamine (NMBA) to establish orthotopic models of esophageal carcinogenesis. CCL2/CCR2 gene knockout mice and macrophage-specific PPARG gene knockout mice were respectively used to investigate the role of infiltration and polarization of TAMs in ESCC. CCL2-mediated monocyte chemotaxis was estimated in malignantly transformed Het-1A cells. THP-1 cells were used to simulate TAMs polarization in vitro. RNA-sequencing was performed to uncover the mechanism. RESULTS:Increasing expression of CCL2 correlated with TAMs accumulation in esophageal carcinogenesis, and they both predicts poor prognosis in ESCC cohort. Animal studies show blockade of CCL2-CCR2 axis strongly reduces tumor incidence by hindering TAMs recruitment and thereby potentiates the antitumor efficacy of CD8+ T cells in the tumor microenvironment. More importantly, M2 polarization increases PD-L2 expression in TAMs, resulting in immune evasion and tumor promotion through PD-1 signaling pathway. CONCLUSION:This study highlights the role of CCL2-CCR2 axis in esophageal carcinogenesis. Our findings provide new insight into the mechanism of immune evasion mediated by TAMs in ESCC, suggesting the potential of TAMs-targeted strategies for ESCC prevention and immunotherapy.
Project description:<h4>Background</h4>Human papillomavirus (HPV)-positive oral squamous cell carcinoma (OSCC) is increasing worldwide with typically higher grade and stage, while better prognosis. microRNAs (miRNAs) has been shown to play a critical role in cancer, however, their role in HPV-positive OSCC progression remains unclear.<h4>Methods</h4>miRNA microarray was performed to identify differentially expressed miRNAs. qRT-PCR and FISH were performed to determine the relative expression of miR-550a-3-5p. CCK-8, Flow cytometry, Wound healing, Cell invasion assays and xenograft experiments were conducted to analyze the biological roles of miR-550a-3-5p. Tumor-associated macrophages (TAMs) generation, co-culturing of cancer cells with TAMs, Western blot, Dual-luciferase reporter gene assay, Immunohistochemistry and animal studies were performed to explore the mechanisms underlying the functions of miR-550a-3-5p.<h4>Results</h4>We identified 19 miRNAs differentially expressed in HPV-positive OSCC specimens and miR-550a-3-5p was down-regulated. The low expression of miR-550a-3-5p correlated with higher tumor size and nodal metastasis of HPV-positive OSCC patients. Then, we found that miR-550a-3-5p suppressed the migration, invasion and EMT of HPV-positive OSCC cells dependent on decreasing M2 macrophages polarization. Moreover, miR-550a-3-5p, down-regulated by E6 oncoprotein, inhibited M2 macrophages polarization by YAP/CCL2 signaling, which in turn abrogating EMT program in HPV-positive OSCC cells. In addition, in both xenografts and clinical HPV-positive OSCC samples, miR-550a-3-5p levels were inversely associated with YAP, CCL2 expressions and the number of M2 macrophages.<h4>Conclusions</h4>E6/miR-550a-3-5p/YAP/CCL2 signaling induces M2 macrophages polarization to enhance EMT and progression, revealing a novel crosstalk between cancer cells and immune cells in HPV-positive OSCC microenvironment.
Project description:Pathological angiogenesis is necessary for tumor development and metastasis. Tumor-derived extracellular vesicles (EVs) play an important role in mediating the crosstalk between cancer cells and vascular endothelial cells. To date, whether and how microRNAs (miRNAs) encapsulated in tumor-derived EVs affect angiogenesis in esophageal squamous cell carcinoma (ESCC) remains unclear. Here, we showed that miR-181b-5p, an angiogenesis-promoting miRNA of ESCC, can be transferred from ESCC cells to vascular endothelial cells via EVs. In addition, ESCC-derived EVs-miR-181b-5p dramatically induced angiogenesis by targeting PTEN and PHLPP2, and thereby facilitated tumor growth and metastasis. Moreover, miR-181b-5p was highly expressed in ESCC tissues and serum EVs. High miR-181b-5p expression level in ESCC patients was well predicted for poor overall survival. Our work suggests that intercellular crosstalk between tumor cells and vascular endothelial cells is mediated by tumor-derived EVs. miR-181b-5p-enriched EVs secreted from ESCC cells are involved in angiogenesis that control metastasis of ESCC, providing a potential diagnostic biomarker or drug target for ESCC patients.
Project description:Hepatocellular carcinoma (HCC) is a common malignancy. CD8<sup>+</sup> T cell-mediated immune response is critical for the inhibition of HCC progression. M2 macrophages participate in HCC progression. This study set out to investigate the effect of M2 macrophage-derived extracellular vesicles (EVs) on CD8<sup>+</sup> T cell exhaustion in HCC. M2 macrophage-derived EVs were isolated and identified. The murine model of primary HCC was established through DEN/CCl<sub>4</sub> induction, and model mice were injected with EVs. Peripheral blood mononuclear cells (PBMCs) were isolated from the mouse liver and CD8<sup>+</sup> T cells were sorted. The expressions of immune checkpoint inhibitory receptors and effector cytokines on CD8<sup>+</sup> T cells were detected, followed by the evaluation of CD8<sup>+</sup> T cell proliferation and killing function. miR-21-5p expression in M2 macrophage-derived EVs was detected. The binding relationship between miR-21-5p and YOD1 was verified. The activation of the YAP/β-catenin pathway was detected. Consequently, M2 macrophage-derived EVs promoted CD8<sup>+</sup> T cell exhaustion in HCC mice. miR-21-5p expression was upregulated in M2 macrophage-derived EVs, and EVs carried miR-21-5p into HCC tissues. miR-21-5p targeted YOD1. Inhibition of miR-21-5p or overexpression of YOD1 annulled the promoting effect of EVs on CD8<sup>+</sup> T cell exhaustion. YOD1 inactivated the YAP/β-catenin pathway. In conclusion, M2 macrophage-derived EVs facilitated CD8<sup>+</sup> T cell exhaustion via the miR-21-5p/YOD1/YAP/β-catenin axis. This study may confer novel insights into the immunotherapy of HCC.
Project description:BACKGROUND:Tumor microenvironment (TME) is a complex environment containing tumor cells, tumor-associated macrophages (TAMs), interstitial cells, and non-cellular components. Epithelial-mesenchymal transition (EMT), as a major actor in cancer tumorigenicity and metastasis, was involved in the interaction between TAMs and tumor cells. However, the potential mechanisms of EMT and how EMT-programmed tumor cells affect M2-like TAMs still need further exploration. METHODS:An integrated analysis of nine CRC miRNA expression datasets was performed. Functional assays, including the EdU, clone formation, wound healing, and transwell assays, were used to determine the anticancer role of miR-195-5p in human CRC progression. Furthermore, RNA immunoprecipitation, RNA decay, and dual-luciferase reporter assays were used to determine the mechanism of miR-195-p CRC progression. Then co-culture, migration, and ELISA assays were applied to determine the role of miR-195-5p in macrophage recruitment and alternative polarization. Xenograft mouse models were used to determine the role of miR-195-5p in CRC tumorigenicity and TAM polarization in vivo. RESULTS:An integrated analysis confirmed that miR-195-5p was significantly downregulated in CRC tissues, and patients with a low level of miR-195-5p had significantly shortened overall survival as revealed by the TCGA-COAD dataset. Altered miR-195-5p in colon cancer cells led to distinct changes of proliferation, migration, invasion, and EMT. Mechanistically, miR-195-5p regulated NOTCH2 expression in a post-transcriptional manner by directly binding to 3'-UTR of the Notch2 mRNA. Subsequently, miR-195-5p/NOTCH2 suppressed GATA3-mediated IL-4 secretion in CRC cells and ultimately inhibited M2-like TAM polarization. CONCLUSIONS:miR-195-5p may play a vital role in regulating NOTCH2-mediated tumor cell EMT, thereby affecting IL-4-related M2-like TAM polarization in CRC.
Project description:Osteoarthritis (OA) is a degenerative illness that greatly impacts the life quality of patients. Currently, the therapeutic approaches for OA are very limited in clinical. The extracellular vesicles (EVs) derived from different mesenchymal stem cells displayed a prominent therapeutic effect on OA. But most EVs have limited resources and the risks of host rejection, immunological response, and etc. Human umbilical cord mesenchymal stem cells (hUCMSCs) hold the advantages of easy availability, minimal immune rejection, and excellent immunomodulatory effects, although hUCMSCs-EVs have seldom been applied in OA. Herein, we investigated the potential immunomodulatory and anti-inflammatory effects of hUCMSCs-EVs on the treatment of OA. In our results, the treatment of hUCMSCs-EVs promoted the polarization of M2-type macrophages and the expression of anti-inflammation-related cytokines (IL-10). Notably, the supernate of M2 macrophages induced by hUCMSCs-EVs inhibited the level of inflammation-associated factors in OA chondrocytes caused by IL-1β. Further, injection of hUCMSCs-EVs in the articular lumen ameliorated progression of OA and exerted chondroprotective potential based on the OA joint model created by the surgical transection of the anterior cruciate ligament (ACLT). In addition, we found five highly enriched miRNAs in hUCMSCs-EVs, including has-miR-122-5p, has-miR-148a-3p, has-miR-486-5p, has-miR-let-7a-5p, and has-miR-100-5p by High-throughput sequencing of miRNAs, with targeted genes mainly enriched in the PI3K-Akt signaling pathway. Furthermore, we also detected the protein abundance of hUCMSCs-EVs using liquidation chromatography with tandem quadrupole mass spectrometry (LC-MS/MS) analysis. Thus, our study indicates that hUCMSCs-EVs can alleviate cartilage degradation during the OA progression, mechanically may through delivering key proteins and modulating the PI3K-Akt signaling pathway mediated by miRNAs to promote polarization of M2 macrophage, exhibiting potent immunomodulatory potential. The current findings suggest that hUCMSCs-EVs might serve as a new reagent for the therapy of OA.
Project description:OBJECTIVE:To investigate the lung cancer-promoting mechanism of mesenchymal stem cell-secreted extracellular vesicles (MSC-EV). METHODS:EV were isolated from culture media of human bone marrow-derived MSCs that were pre-challenged with or without hypoxia (referred to as H-EV and N-EV, respectively). After treatment with N-EV or H-EV, A549 and H23 cell proliferation, apoptosis, trans-well invasion and epithelial-to-mesenchymal transition (EMT) were examined. Polarization of human primary monocytes-derived macrophages with or without N-EV or H-EV induction were analyzed by flow cytometry and ELISA. PTEN, PDCD4 or RECK gene was overexpressed in A549 cells, while miR-21-5p was knocked down in MSCs, A549 or H23 lung cancer cells or primary monocytes by miR-21-5p inhibitor transfection. Protein level of PTEN, PDCD4, RECK, AKT or STAT3 as well as phosphorylation level of AKT or STAT3 protein were assayed by western blot. Tumorigenicity of A549 and H23 cells with or without MSC-EV co-injection was assayed on immunocompromised mice. The xenograft tumor were examined for cell proliferation, angiogenesis, apoptosis and intra-tumoral M1/M2 macrophage polarization. RESULTS:Comparing to N-EV, H-EV treatment significantly increased A549 and H23 cell proliferation, survival, invasiveness and EMT as well as macrophage M2 polarization. MiR-21-5p knocked down significantly abrogated the cancer-promoting and macrophage M2 polarizing effects of H-EV treatment. H-EV treatment downregulated PTEN, PDCD4 and RECK gene expression largely through miR-21-5p. Overexpressing PTEN, PDCD4 and RECK in A549 cells significantly reduced the miR-21-5p-mediated anti-apoptotic and pro-metastatic effect of H-EV, while overexpressing PTEN in monocytes significantly reduced macrophage M2 polarization after induction with the presence of H-EV. H-EV co-injection significantly increased tumor growth, cancer cell proliferation, intra-tumoral angiogenesis and M2 polarization of macrophages in vivo partially through miR-21-5p. CONCLUSIONS:Increased miR-21-5p delivery by MSC-EV after hypoxia pre-challenge can promote lung cancer development by reducing apoptosis and promoting macrophage M2 polarization.
Project description:Esophageal squamous cell carcinoma (ESCC) has a poor prognosis when diagnosed at an advanced stage, and early detection and treatment are essential to improve survival. However, intraobserver and interobserver variation make the diagnosis of superficial ESCC difficult, and suitable biomarkers are urgently needed. Here, we compared the microRNA (miRNA) expression profiles of superficial ESCC tissues and adjacent normal tissues obtained immediately before esophageal endoscopic submucosal dissection. We found that ESCC and normal tissues differed in their miRNA expression profiles. In particular, miR-21-5p and miR-146b-5p were significantly upregulated and miR-210-3p was significantly downregulated in tumor tissues compared with normal tissues. We also detected significant associations between miRNA expression and ESCC invasion depth and lymphovascular invasion. The same differential expression of miR-21-5p, miR-146b-5p, and miR-210-3p was detected in ESCC cell lines compared with normal esophageal epithelial cells in vitro. However, transfection of ESCC cells with miR-210-3p and miR-21-5p mimics or inhibitors had partial effects on cell proliferation and invasion in vitro. These results indicate that miRNA expression is significantly deregulated in superficial ESCC, and suggest that the potential contribution of differentially expressed miRNAs to the malignant phenotype should be further investigated.
Project description:Epithelial-mesenchymal transition (EMT) is a major event during cancer progression and metastasis; however, the definitive role of EMT in remodeling tumor microenvironments (TMEs) is unclear. Tumor-associated macrophages (TAMs) are a major type of host immune cells in TMEs, and they perform a wide range of functions to regulate tumor colonization and progression by regulating tumor invasiveness, local tumor immunity, and angiogenesis. TAMs are considered to have an M2-like, i.e., alternatively activated, phenotype; however, how these EMT-undergoing cancer cells promote M2 polarization of TAMs as a crucial tumor-host interplay during cancer progression is unclear. In this study, we investigated the mechanism of EMT-mediated TAM activation. Here, we demonstrate that the EMT transcriptional factor Snail directly activates the transcription of MIR21 to produce miR-21-abundant tumor-derived exosomes (TEXs). The miR-21-containing exosomes were engulfed by CD14+ human monocytes, suppressing the expression of M1 markers and increasing that of M2 markers. Knockdown of miR-21 in Snail-expressing human head and neck cancer cells attenuated the Snail-induced M2 polarization, angiogenesis, and tumor growth. In head and neck cancer samples, a high expression of miR-21 was correlated with a higher level of SNAI1 and the M2 marker MRC1. This study elucidates the mechanism of EMT-mediated M2 polarization through delivery of the miR-21-abundant exosomes, which may serve as a candidate biomarker of tumor progression and provide a potential target for intercepting EMT-mediated TME remodeling.
Project description:BACKGROUND:Extracellular vesicles (EVs) are endogenous membrane vesicles with a diameter of 30-200?nm. It has been reported that hypoxic cancer cells can release numerous EVs to mediate multiple regional and systemic effects in the tumor microenvironment. METHODS:In this study, we used ultracentrifugation to extract EVs secreted by TE-13, an esophageal squamous carcinoma (ESCC) cell line during normoxia and hypoxia and performed high-throughput sequencing to detect exosomal miRNAs. Gene ontology (GO) and KEGG pathway analyses were used to reveal pathways potentially regulated by the miRNAs. RESULTS:A total of 10 810 miRNAs were detected; 50 were significantly upregulated and 34 were significantly downregulated under hypoxic environment. GO analysis identified enrichment of protein binding, regulation of transcription (DNA-templated), and membrane as molecular function, biological process, and cellular component, respectively. KEGG pathway analysis revealed cancer-associated pathways, phospholipase D signaling pathway, autophagy, focal adhesion and AGE-RAGE signaling as the key pathways. Further verification experiment from qRT-PCR indicated that miR-128-3p, miR-140-3p, miR-340-5p, miR-452-5p, miR-769-5p and miR-1304-p5 were significantly upregulated in EVs from hypoxia TE-13 cells while miR-340-5p was significantly upregulated in two other ESCC cells, ECA109 and TE-1. CONCLUSION:This study, for the first time reveals changes in the expression of exosomal miRNAs in hypoxic ESCC cells and these findings will act as a resource to study the hypoxic tumor microenvironment and ESCC EVs.