Isolation,Characterization Circulating Tumor Cells And The Relevance In Tumor Metastasis Studies

OBJECTIVE:The ability to metastasize is the principal cause of death among cancer patients,and the root cause of cancer metastasis can be traced down to the presence of circulating tumor cells(CTCs)in blood.Therefore,the isolation,characterization and enumeration of CTC has an important intervention in tumor metastasis.In the present study,we aimed at establishing a sensitive and specific isolation,characterization and enumeration method for CTCs in patients with colorectal carcinoma.The correlation between CTC and clinical metastasis of CRC will be researched.The vitro culture conditions of CTCs were preliminarily discussed.And the value of EMT(epithelial mesenchymal transitions)related protein will be explored in predicting the prognosis of CRC(colorectal cancer)patients.METHOD:1.The method that quantitative isolation and characterization of CTCs was established by combining immunomagnetic negative enrichment and fluorescence-activated cell sorting.Blood samples spiked with HCT116 cells(ranging from 1 to 80 cells)were used to determine specificity,recovery and sensitivity.The CD47/CD44 expression of CTC was detected by FACS(Flow cytometer)for exploring the correlation with tumor stage.The sorted CTC was identified by Fluorescence Immunoassay.Co-incubate HUVECs with HCT116 to research the vitro culture condision of CTC.2.The CD324、CD47、CD66 expression between para-carcinoma tissue and cancer tissue of CRC was detected by FACS.The expression of EMT related genes between para-carcinoma tissue and cancer tissue was detected by RT-PCR for exploring the correlation with cancer metastasis.RESULTS:1.By verifing the method that quantitative isolation and characterization of CTCs by combining immunomagnetic negative enrichment and fluorescence-activated cell sorting,the average recovery of HCT116 cells was showed 61%and a correlation coefficient(R2)was 0.992 and the HCT116 can be detected in single cell level.All patients showed CD47 positive with CD44 positive only 4.3-61.2%.The DAPI+CD45-CK+ cell was verified CTC.However,CTCs were not able to proliferate or propagate in the culture plate,although they were found well adhered to the culture plate.2.The expression percentage of CD66 was raised in cancer tissue of CRC,and the RT-PCR result showing that the expression of CDHI was declined while SNAI1,SNAI2,ZEB1,ZEB2 rasied in cancer tissue of CRC.CONCLUSION:1.The method of immune-omagnetic negative enrichment coupled with FACS for detection,isolation,and characterization of colorectal CTCs from patient blood is a sensitive,quantitative,timesaving and effective technique which could also show in parallel other signal biomarkers on CTCs.The number of CTCs and percentage of CD44+ were significantly correlated with TNM(patients’tumor-node-metastasis stage).Co-incubate HUVECs with HCT116 were well adhered.2.The percentage of CD66+ in cancer tissue was significantly correlated with TNM.The expression percentage of SNAI1,SNAI2,ZEB1 and ZEB2 was correlated with EMT.The study on detection,isolation,and characterization of CTC provided the important theoretical foundation for preventing cancer metastasis.