| Tumor microenvironment(TME),as the internal environment in which tumor cells produce and live,has the characteristics of complex cell components and high heterogeneity.Increasing evidence has demonstrated that TME plays key role in the tumorigenesis,progression and metastasis of cancer.Of which,tumor-associated macrophages(TAMs),as the most important component of tumor immune microenvironment,have been proven to interact with tumor cells to form “crosstalk” of “tumor-immune cells”,thereby participating in various biological processes of malignant tumors.Epithelial-mesenchymal transition(EMT)is a key step in the distant metastasis of cancer.Through its occurrence,epithelial-derived tumor cells acquire a mesenchymal phenotype,and thus enhance their invasive and migrative abilities.Accumulating studies have found that the crosslinking between tumor cells and TAMs is a complex process involving multiple factors,in which EMT plays an important role.In recent,our group has revealed that tumor cells and macrophages can mediate the tumor cells’ EMT and macrophage polarization through the secretion of chemokines and cytokines: on one hand,TAMs could induce EMT of CRC cells by secreting interleukin-6(IL-6);on the other hand,EMT-programmed CRC cells could activate the expression of different cytokines in cancer cells such as chemokines(CC motif)ligand 2(CCL2)and interleukin-4(IL-4)in cancer cells to enhance the macrophages recruitment and promote their M2-like polarization Nowadays,in addition to soluble cytokines,exosomes(Exos)are also considered as the another important mediator for orchestrating intercellular communication.However,the role and potential molecular mechanisms of Exos secreted by EMT-CRC cells in M2-type polarization of macrophages remain largely unknown.Based on the above research background and our previous findings,we hypothesized that EMT-CRC cells can promote the M2-subtype polarization of macrophages by secreting Exos,which in turn affects the malignant biological behaviours of CRC cells,thereby establishing the “crosstalk” between EMT-tumor cells and M2 macrophages,which in consequence promotes the progression and metastasis of CRC.In order to validate the above hypothesis,the present study used a series of cell biology and molecular biology techniques to investigate the molecular mechanisms of EMT-CRC cells promoted the M2-subtype polarization of macrophages in both in vitro and in vivo levels.Furthermore,we performed a preliminary exploration of their clinical significance through detection and analyses of human clinical samples.This topic mainly includes three parts,as the following:Part Ⅰ The molecular mechanism of tumor cells-derived exosomes promoting the invasion and migration of colorectal cancer by activating “crosstalk” between EMT tumor cells and M2-subtype macrophagesObjective To explore the molecular mechanism of tumor cells-derived Exos promote the invasion and migration of CRC by activating “crosstalk” between EMT tumor cells and M2-subtype macrophages.Methods Exos from normal(Nor-Exos)and EMT-CRC cells(EMT-Exos)supernatant were isolated by ultracentrifugation,and the shape,size and density of Exos were identified by using Transmission electron microscopy(TEM),Nanoparticle tracking analysis(NTA)and Western blotting.Subsequently,Flow cytometry,RT-q PCR and ELISA were used to investigate the effect of Nor-Exos and EMT-Exos on macrophages polarization.RNA sequencing was then preformed to identify the differentially expressed mi RNAs profiles between EMT-Exos and Nor-Exos.Furthermore,western blotting,luciferase reporter assay and pathway inhibitor were used to uncover the underlying mechanism of EMT-Exos mediating M2 polarization of macrophages by transmitting mi RNAs.In addition,an co-culture assay was used to explore the effects of EMT-Exos-modulated macrophages on CRC cells’ EMT,migration and invasion in vitro.Results EMT-CRC cells could promote the M2-like polarization of macrophages by secreting Exos in vitro.RNA Sequencing results confirmed that mi R-106-5p was most significantly up-regulated in EMT-Exos.Mechanically,EMT-Exos could mediate the PI3Kγ/AKT/m TOR signalling cascade by delivering mi R-106-5p to posttranscriptionally suppress the expression of programmed cell death 4(PDCD4)inmacrophages,thereby contributing to the M2 polarization of macrophages.Co-culture assay in vitro showed that activated M2 macrophages could promote the EMT of CRC cells in a positive feedback manner,thus enhancing their invasion and migration capabilities.Conclusions EMT-CRC cell-derived exosomal mi R-106-5p promotes the M2 polarization of macrophages by regulating the PDCD4/PI3Kγ/AKT/m TOR axis in macrophages,thereby establishing the “crosstalk” between EMT-tumor cells and M2 macrophages,to promote the invasion and migration of CRC cells.Part Ⅱ In vivo validation of EMT tumor cells-derived exosomes induce M2-subtype polarization of macrophages by delivering mi R-106b-5p to promote colorectal cancer metastasisObjective To verify EMT tumor cells-derived exosomes induce M2-subtype polarization of macrophages by delivering mi R-106b-5p to promote colorectal cancer metastasis in vivo by using the nude mice model with subcutaneous tumorigenesis and caudal vein injection.Methods o examine the effects of EMT-Exos-derived mi R-106b-5p on macrophages polarization in vivo,clodronate liposomes were intravenously injected into each mouse for macrophage ablation.Then,HCT116 cells were subcutaneously injected into the right flank region of each mouse.After 10 days,PBS,Nor-Exos,EMT-Exos,mi RAgomir-NC or mi R-106b-5p Agomir was directly injected into the implanted tumor.Meanwhile,THP1-induced macrophages were injected into the mice via tail vein interval 3 days.Mice were euthanized after 30 days,immunohistochemistry and RTq PCR were used to evaluate the polarization status of macrophages in TME.To examine the effects of the conditioned macrophages on EMT of CRC cells in vivo,clodronate liposomes were intravenously injected into each mouse for macrophage ablation.Then,HCT116 cells were subcutaneously injected into the right flank region of each mouse.After 10 days,HCT116 cells mixed with the conditioned macrophages treated with PBS,Nor-Exos and EMT-Exos for 48 h or transfected by mi R-106b-NC and mimics were injected into the mice via tail vein.Mice were euthanized after 30 days,immunohistochemistry and RT-q PCR were used to evaluate the EMT status of CRC cells,and blood samples of mice were collected through the way of orbital blood to identify the number of circulating tumor cells(CTCs).To examine the effects of the conditioned macrophages on the metastasis of CRC cells in vivo,HCT116 cells mixed with the conditioned macrophages treated with PBS,Nor-Exos and EMT-Exos for 48 h or transfected by mi R-106b-NC and mimics were injected into the mice via tail vein.After 6 weeks,all mice were euthanized.Then,the liver and lung tissues of all mice were performed with H&E staining to examine the suspicious metastases sites.Results Immunohistochemical results showed that compared with other groups,downregulated expression of PDCD4 and E-cadherin,and up-regulated expression of CD163 and Vimentin were found in the tumor surrounding stroma of mice treated with EMTExos and mi R-106b-5p Agomir.Except for no significant changes in PDCD4 m RNA,other results from RT-q PCR were consistent with immunohistochemical results.CTCs detection and quantitative analysis showed that the number of CTCs were significantly increased in the group treated with EMT-Exos or mi R-106b-5p Agomir compared with other three groups.Furthermore,H&E staining showed that more visible liver and lung and metastatic nodules and significantly decreased weight of mice were observed in the group treated with EMT-Exos or transfected by mi R-106b-5p minics compared with other three groups.Conclusion EMT-Exos-derived mi R-106b-5p induces M2-subtype polarization of macrophages to promote the EMT of tumor cells,thereby facilitating the liver and lung metastasis of CRC in vivo.Part Ⅲ Preliminary exploration of the expression and clinical significance of mi R-106b-5p in tissues and plasma exosomes of patients with colorectal cancerObjective Under the foundation of previous in vitro and in vivo results,preliminarily exploring the expression level and clinical significance of mi R-106b-5p in CRC tissues and plasma Exos of CRC patients by analysing patients’ the tissues and blood samples.Methods RT-q PCR was first used to detect the expression level of mi R-106b-5p in the tissues and plasma exosomes of CRC patients.A total of 91 tumor tissue specimens were serially sectioned,and the expression levels of mi R-106b-5p,PDCD4,M2-subtype TAMs markers(CD163)and EMT markers(E-cadherin and Vimentin)was detected by in situ hybridization and immunohistochemistry.Meanwhile,the number of CTCs in peripheral blood of patients was detected,and relationship between mi R-106b-5p expression level in plasma Exos and the number of CTCs was analysed.Finally,the correlation between the expression level of mi R-106b-5p in tumor tissues and plasma exosomes and the clinicopathological characteristics and prognosis of patients was statistically analysed.Results RT-qPCR results showed that the expression of mi R-106b-5p level was upregulated in CRC tissues compared with adjacent normal tissues(n=91,P<0.05).ISH and IHC results further confirmed that mi R-106b-5p was closely related to M2 macrophages polarization and the EMT of tumor cells in the TME.Clinicopathological features analyses showed that upregulated mi R-106b-5p levels in CRC tissues were positively association with poor tumor grade,deep tumor invasion,lymph node metastasis,advanced tumor stage,and positive preoperative CTCs status(P<0.05,respectively).Moreover,prognosis analyses indicated that high mi R-106b-5p levels in CRC tissues were correlated with reduced recurrence-free survival(RFS)and overall survival(OS)of CRC patients(P<0.05),and high mi R-106b-5p expression level was an independent prognostic factor for affecting the RFS and OS of CRC patients(P<0.05).Similar to the results in tumor tissues,the level of mi R-106b-5p expression in plasma exosomes was significantly up-regulated in CRC patients compared with healthy controls(P<0.05),and increased exosomal mi R-106b-5p expression was positively correlated with the number of CTCs in the peripheral blood of CRC patients(P<0.05).After tumor resection,plasma exosomal mi R-106b-5p levels were decreased in most CRC patients.Further analysis revealed that plasma sxosomal mi R-106b-5p upregulation was correlated with multiple unfavourable clinicopathological parameters(P<0.05),such as poor tumor grade,lymphovascular invasion,deep tumor invasion,lymph node metastasis,advanced tumor stage,and positive preoperative CTCs status in CRC patients.Conclusion The level of mi R-106b-5p expression in tissue and plasma Exos of CRC patients is significantly up-regulated,and its increased expression was closely related to multiple unfavourable clinicopathological features and patients’ poor prognosis,indicating mi R-106b-5p might be served as a potential biomarker for treatment and prognosis evaluation in patients with CRC. |
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