Separation Effect Comparison Of Circulating Tumor Cells In Colorectal Cancer By Functional Immunomagnetic Microspheres And Preliminary Clinical Application

To investigate the separation,identification and evaluation of colorectal cancer circulation tumor cells（CTCs）,the polylactic acid-glycolic acid（PLGA）magnetic beads to identify epithelial cell adhesion molecule（EpCAM）and epidermal growth factor receptor（EGFR）were prepared.The carboxylic polylactic acid-glycolic acid（PLGA-COOH）and chitosan hexadecyltrimethylammonium（HQCMC）were used to encapsulate Fe₃O₄ magnetic nanoparticle,and EpCAM antibody and EGFR antibody were connected to them to form immunomagnetic beads（IMMS）and compose the EpCAM immune magnetic kit（EPM）and EGFR immune magnetic kit（GPM）.The test on colorectal cancer CTCs were carried out by these kits to capture the EpCAM and EGFR on tumor cell surface.The results of these capture and identification tests showed EPM kit and GPM kit has higher efficiency in capture of colorectal cancer cell line HT-29 and can be used in tests of colorectal cancer CTCs.Imaging by dye of prussian blue showed the captured cells surface were attached with a layer of magnetic beads.The capture efficiency among the different colorectal cancer cells number was 86%for EPM kit and80%for GPM kit.There was no significance difference between these two methods（p>0.05）.MTT test showed the captured cells remain the cell viability.Based on the results of colorectal cancer cell line test,the study of colorectal cancer patients CTCs was performed.The fresh blood of 35 cases of colorectal cancer patients was collected and detected in 3.75 ml of blood sample.The CTCs captured by EpCAM immunomagnetic beads（EPM）and EGFR immunomagnetic beads（GPM）were counted by IMMS separation technology and compared with the patient’s clinical pathological data.Once the CTC was captured in the blood sample,the patient was CTC positive.The results showed that both EPM and GPM can capture the CTCs efficiently in colorectal cancer blood sample,the positive rate of captured CTCs by EPM and GPM was 85.7%（30/35）and71%（25/35）,respectively.Among the stage ofI^II,III and IV,the positive rate of captured CTCs by EPM was 64.29%（9/14）,100%（6/6）and 100%（14/14）,recpectively.There were significant difference between different stagings（p=0.015）;The positive rates of captured CTCs in metastatic colorectal cancer patient（M1）was 100%（14/14）,higher than that in non metastatic patients（M0）,which was 75%（15/20）,（P=0.043）.Staging by metastasis of lymph nodes N0,N1,N2,N3,the positive rates of captured CTCs were61.54%（8/13）,100%（12/12）,100%（8/8）and 100%（1/1）,respectively.There were significant difference between different staging（p=0.024）.No significant differences were found in GPM captured CTCs among the different patients stages.Analysis of correlationship between captured CTCs and tumor markers,only CEA was significant correlated with number of captured CTCs both by EPM and GPM.（r=0.958,P=0.00 and r=0.397,P=0.018）.Other tumor markers like CA125,CA199 had no correlation with CTCs number.