The Prognostic Value Of CTC In Colorectal Cancer And A Preliminary Study Of Its Mechanism

Relapse and metastasis are still the main causes of death in patients with colorectal cancer.With the deepening of basic clinical research,the role of circulating tumor cells(CTC)in metastasis and recurrence of colorectal cancer has drawn increasing attention.Current studies have confirmed that CTC has a high correlation with the recurrence and metastasis of solid tumors,and it is also an independent prognostic factor that affects the survival period of patients.The biological characteristics of epithelial-mesenchymal transition(EMT)is the main direction of CTC research.As an important physiological and pathological process,it plays an important role in the metastasis,relapse and drug resistance of malignant tumors.Therefore,dynamic monitoring of CTC and EMT-CTC changes can provide an important basis for tumor recurrence and efficacy evaluation.Exosomes has received extensive attention as a new type of intercellular communication model.Exosomes are a type of membranous vesicles with a diameter of about 30-100 nm released into the extracellular matrix by active cells.They are widely distributed and sourced.They carry biologically active molecules such as proteins and genetic carriers and are important intercellular compartments.Material and information exchange tools.Tumor-derived exosomes regulate tumor cells in the microenvironment via transfer proteins,microRNAs(miRNAs),and Long Non-coding RNAs(LncRNAs),enabling the delivery of EMT phenotypes.MicroRNAs(miRNAs)are a class of non-coding RNAs that can regulate the expression level of target genes in the post-transcriptional period.More and more evidence shows that EMT plays a key role in tumors,greatly affecting the ability of tumor invasion and metastasis.Therefore,starting from the clinical point of view,this study first explores the prognostic value of CTC number changes in colorectal cancer;next,we explore the CTC phenotype,epithelial phenotype CTC(ECTC)and interstitial phenotype CTC(MCTC)dynamic changes in colorectal cancer.Finally,the role of exosomes in the transmission of EMT phenotypes will be studied mainly from the following two aspects:1.Establish stability the occurrence of EMT colorectal cancer cells,extraction and identification of exosomes,to explore the EMT cell exosomes on the biological behavior of colorectal cancer.2.The miR-148a-3p was screened by sequencing analysis to further explore its influence on the biological behavior of colorectal cancer and the prediction and validation of its target genes.This topic mainly includes the three parts,as the following:Part Ⅰ:Prognostic value of circulating tumor cells number changes in colorectal cancer patients treated Background:Circulating tumor cells(CTCs)has been regarded as a promising biomarker for colorectal cancer(CRC),however,the prognostic value of post-operative(op)CTCs is still unclear.This study aimed to compare the recurrence prediction value of pre-and post-op CTCs in CRC patients treated with curative resection.Methods:Consecutive CRC patients treated with curative resection from January 2014 to March 2015 were identified.CTCs from peripheral blood were enumerated with an ISETdevice-CTCBIOPSY(?) before and after surgery.Based on the status of pre-and post-op CTCs,included patients were grouped into 4 cohorts:pre-and post-op CTCs-,pre-op CTCs-but post-op CTCs+,pre-op CTCs+but post-op CTCs-,and pre-and post-op CTCs+.Three-year recurrence-free survival(RFS)rate of patients was analyzed.Results:A total of 138 patients(79[57.2%]male;median age,62[43-75]years)were enrolled.Patients with pre-op CTCs-had 19.2%higher 3-year RFS rate(86.2%)than the combined cohorts with pre-op CTCs+(67.0%)(P=0.038).Patients with post-op CTCs+had 25.6%lower 3-year RFS rate(57.1%)than the combined cohorts with post-op CTCs-(82.7%)(P=0.001).Moreover,patients with pre-and post-op CTCs+had 25.1%lower 3-year RFS rate(53.8%)than patients with pre-op CTCs+but post-op CTCs-(78.9%)(P = 0.004).Multivariate analyses confirmed that post-op CTCs+(hazard ratio[HR]=2.82,95%CI:1.39-5.75,P = 0.004),but not but pre-op CTCs+(HR=2.17,95%CI:0.75-6.31,P=0.153),was independently associated with shorter 3-year RFS rate.Conclusion:Post-op CTCs+,but not pre-op CTCs+,is an independent indicator of poor prognosis for CRC patients treated with curative resection.Patients with post-op CTCs+have a higher risk of recurrence than that of pre-op CTCs+.Evaluation of post-op,rather than pre-op,CTCs is warranted.Part Ⅱ:Prognostic value of circulating tumor cells phenotype changes in colorectal cancerBackground:Circulating tumor cells(CTCs)are composed of different subpopulations of epithelial phenotype and interstitial phenotype.However,the prognostic value of these different subtypes of CTC before and after treatment of colorectal cancer(CRC)remains unclear.This section aims to investigate the prognostic value of epithelial phenotype and interstitial phenotypic changes in CTC in colorectal cancer.Methods:This study was mainly to enrich CTC by membrane filtration method using tumor cell size,and to identify the epithelial or mesenchymal phenotype of CTC by immunocytochemical staining method,thereby monitoring the epithelial phenotype of 89 patients with colorectal cancer before and after treatment.CTC(ECTC)and interstitial phenotype CTC(MCTC)vary dynamically.Kaplan-Meier(KM)and univariate and multivariate COX regression survival analyses were used to study the association of ECTC and MCTC with patient recurrence-free survival(RFS).All statistical tests were performed using SPSS v19.0 software and were considered significant when P<0.05.Results:The phenotypic changes of CTC in 89 patients with colorectal cancer were monitored before and after treatment:it was found that the detection rate of ECTC was decreased during the treatment and the MCTC detection rate did not change.ECTC+and MCTC+ were associated with RFS in both patients before and after treatment.Further analysis showed that the median time of RFS in the ECTC+ group before and after treatment was 25.6 months,which was significantly higher than that in the MCTC+group before and after treatment whose median time was 18.1 months(P<0.001).Conclusions:Epithelial and mesenchymal CTC exist in peripheral blood of patients with colorectal cancer.Interstitial phenotype CTC detection had higher prognostic value in identifying high-risk relapse patients.Part Ⅲ:Mechanism of exosomes-derived miR-148a-3p mediated EMT in colorectal cancerBackground:In the previous study,it was found that MCTC detection is more clinically significant.CTC is defined as a tumor cell derived from a primary tumor or metastatic tumor that acquires the ability to detach from the basement membrane and invade the tissue through the tissue matrix.Therefore,we further analyzed the EMT phenotype of CTC+-derived tumor tissue samples.The extent of EMT in the invasion front and the center(cancer nest)was different in cancer tissues,while the invasion front EMT was more significant in MCTC+ tissues.At present,many studies have confirmed that the spatial heterogeneity of EMT in tumor tissues is closely related to the microenvironment of tumors,and exosomes is an important part of tumor microenvironment,and its role in EMT is increasingly causing researchers’ Concerned,in this part we will proceed from the tumor microenvironment,exosome as the entry point,and conduct a preliminary exploration of the mechanism of EMT spatial heterogeneity in tumor tissue.Methods:Immunohistochemical staining of E-cadherin and Vimentin was performed in 56 patients with positive ECTC and MCTC before treatment.The degree of EMT in the invasion front and cancer nest was observed,and the relationship between ECTC and MCTC was analyzed.Next,we established the HCT116 EMT model by treating epithelial colorectal cancer cell line HCT116 with IL6.The exosomes(E-exo)were extracted from the normal culture supernatant and the exudates(M-exo)were extracted from the supernatant of the HCT116 that had undergone EMT,and were added to the normal cultured HCT116,respectively,to detect the increase of colorectal cancer cells.Changes in metastasis and invasion reflect the function of M-exo in human colorectal cancer.The miRNA expression profiles in E-exo and M-exo were analyzed using Illumina HiSeq 2500 High-throughput Sequencing(miRNA-seq)to determine the up-regulation of miR-148a-3p in M-exo,followed by experiments.We will use the bioinformatics website to predict the miR-148a-3p target and validate it through a series of experiments.Results:1 The EMT phenotype of the aggressive front of MCTC positive patients is more significant2.After IL-6 induced HCT116,the morphology of HCT116 cells changed.WB results showed that IL6 could induce EMT in HVT116.3.The projection electron microscopy and WB results suggested that exosomes was extracted by ultracentrifugation and the sample was 50-200 nm in diameter.The specific expressed protein on exosomes was further verified by WB.4.Cross-hatch experiments and Transwell showed that M-exo added HCT116,can increase the migration and invasion of HCT116.5.WB results show that M-exo can induce EMT in HCT116.Conclusion:IL6 can induce EMT in cells;E-exo can increase the invasion and migration of HCT116;E-exo can induce EMT in HCT116.6.Transwell assay and scratch test results showed that after up-regulating miRNA-148a-3p expression,the invasion ability of HCT116 cells was significantly enhanced.Down-regulation of miRNA-148a-3p expression,E-exo induced HCT116 cell invasion was weakened.7.Up-regulation of miR-148a-3p induces EMT in cells.WB and qPCR showed a negative correlation between miR-148a-3p and PTEN expression,suggesting that PTEN may be the downstream target gene of miR-148a-3pCONCLUSION:EMT phenotypic analysis of tumor tissue samples revealed that the extent of EMT was different between the invasion front and the center(cancer nest)in cancer tissues,while the invasion front EMT was more significant in MCTC tissues.IL6 can induce EMT in cells;M-exo can enhance the invasion and migration of HCT116;M-exo can induce EMT in HCT116;miR-148a-3p can induce EMT in exosomes.The expression of miR-148a-3p and PTEN in cells is negatively correlated,suggesting that PTEN may be a downstream target gene of miR-148a-3p.