Establishment Of The Method To Efficiently Detect The Circulating Tumor Cells And Its Clinical Significance For Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the cancers with higher morbidity andmortality. Although the inspection and treatment are increasing, its mortality is still high,the main reasons are the recurrence and metastasis. One of the mechanisms of tumorrecurrence and metastasis at present is circulating tumor cells (CTC). Circulating tumorcells shed from the primary tumor or metastatic tumor into the peripheral blood, andcirculate to everywhere of the body, then settle down in a proper place and form themetastasis.The common enrichment methods of CTC include density gradient centrifugation,filtration, immunomagnetic beads, and so on. The density gradient centrifugation has lowspecificity and lower CTC purity. Immunomagnetic beads method includes positiveenrichment and negative enrichment method. The immunomagnetic method has higherspecificity, but it’s more expensive.The ditection methods of CTC areimmunocytochemistry, Reverse Transcription Polymerase Chain Reaction （RT-PCR）,flow cytometry, and so on. Now, The FDA approved method is CellSearch, using thepositive immunomagnetic enrichment to capture CTCs with the immunomagnetic beads ofanti-EpCAM antibody, then identifies the CTCs using the CK’s expression. However,studies report that CTCs undergo epithelial-mesenchymal transition (EMT), which makesthat the CTCs that do not express some of the epithelial markers, and the detectionmethods that rely on CTC’s epithelial phenotype have a high false negative rate, and thepositive immunomagnetic enrichment has more efficiency than the negative one. Becauseof EMT，the existing methods to study CTCs can not effectively enrich and identify theCTCs, and remain to be improved; CTCs are also tumor cells exiting abnormalchromosome. So, perhaps we can identify the CTCs using the combination method withthe characteristic of chromosomal abnormalities，and this is one of the basises of ourresearch. So, method using density gradient centrifugation and the negativeimmunomagnetic beads to enrich the tumor cells, and chromosome analysis andimmunofluorescence staining technique to detect CTCs, may has more efficiency.On theother hand, although the research on CTC is more and more,most of them are about breastcancer、colorectal cancer and prostate cancer, fewer is about HCC, and this is the otherbasis of our research.Based on the above two points, we started this study from the following three aspects:the first part, to explore the enrichment efficiency of density gradient centrifugation combining with immunomagnetic negative enrichment to CTCs in peripheral blood; thesecond part, to test the detection efficiency of the chromosome fluorescence in situhybridization combining with immunofluorescence staining technique to different tumorcell stains cultured in vitro; the third part, to detect the CTCs in peripheral blood ofpatients with HCC using the former enrichment and detection methods.Part one: Establishment of the CTC enrichment method combining density gradientcentrifugation and immunomagnetic negative enrichmentWe used Green fluorescent mitochondrial probe to label colon cancer cell line SW480,and counted, then a certain amount of tumor cells were mixed with7.5ml peripheral bloodof healthy donors. Then we enriched the tumor cells using density gradient centrifugationcombining with immunomagnetic negative enrichment, and finally calculated the recoveryrate. The results showed that, by using density gradient centrifugation andimmunomagnetic negative enrichment method, we could recover tumor cells in theperipheral blood efficiently, and the recovery efficiency was high, so we believed that thismethod had high enrichment efficiency and could be used to enrich CTCs in the peripheralblood.Part two: Establishment of tumor cell identification method by chromosomefluorescence in situ hybridization combined with immunofluorescence reaction basedon cell surface markersWe selected8cell lines including the human breast cancer cell line MDA-MB-231,human bladder cancer cell line scaber, human ovarian cancer cell line SK-OV-3, renalcarcinoma cell line TK10, hepatocellular carcinoma cell line HepG2, lung cancer cell lineA549, human colon cancer cell line SW480, human gastric carcinoma cell line SNU-16.Firstly，we smeared the top four tumor cells cultured in vitro on the glass slide, thendetected the cells using chromosome fluorescence in situ hybridization(FISH) andimmunofluorescence staining technique. Secondly, we mixed the rest four tumor cellscultured in vitro into the peripheral blood. Then we enriched the tumor cells by densitygradient centrifugation and immunomagnetic negative enrichment method, and detectedthe tumors cells using the former method. The results showed that, chromosomefluorescence in situ hybridization combined with immunofluorescence staining techniquecould effectively detect various cancer cells of different tumor cell lines, and can be usedto detect the CTCs in the peripheral blood of patients with various solid tumors.Part three: Detection and significance of the circulating tumor cells in peripheral blood of patients with hepatocellular carcinoma using the enrichment method ofdensity gradient centrifugation combining with negative immunomagnetic and theidentify method of chromosome in situ hybridization combining withimmunofluorescence stainingWe enrolled35cases of HCC patients, and enriched the circulating tumor cells bydensity gradient centrifugation and immunomagnetic negative enrichment method, thenused the chromosome fluorescence in situ hybridization and immunofluorescence stainingtechnique to detect and identify the CTCs. And finally, we observed and counted the cellsunder fluorescent microscope. The results showed that, the number of CTCs in theperipheral blood had no obvious relationship with the prognosis of HCC patients andCancer embolus, but it was related to lymph node and/or distant metastasis, For thepatients with more than5CTCs,they may be more likely to have lymph node and/ordistant metastasis; for patients in stage III and IV, the number of CTCs had no obviouscorrelation with their satges.In summary, our results suggested the following conclusions:1. We successfully established the method to efficiently detect the circulating tumorcells of malignant tumors including hepatocellular carcinoma (HCC).2. The number of CTCs in the peripheral blood of HCC patients was related to lymphnode and/or distant metastasis. For the HCC in patients with more than5CTCs, the tumormay be more likely to have lymph node and/or distant metastasis.