Identification Of Circulating Tumor Cells Using A Novel Epithelial-marker-independent Technology

Circulating tumor cells (CTCs) are associated with metastasis and reduced survival, and CTC detection is considered a clinically useful prognostic factor and a surrogate marker for treatment response in multiple cancer types. It is difficult to identify the CTCs as which is an extremely rare phenomenon. oHSV1-hTERT-GFP was constructed by our research group. The virus could proliferate specifically in cells which have telomerase activity and then express GFP.This study aims to utilize the virus to establish a simple, reproducible, and high sensitivity detection approach. The reliability of the test method and the value of clinical application were discussed.We first examined the accuracy of this method by the following experiment:The hTERT expression level of different tumor cell lines, tissues and normal cells were detected by Western blot assay. The detection accuracy was verified by adding different number of tumor cells into 4ml of blood and assured by immunofloresence approche. The results showed that the hTERT was highly expressed in tumor cell lines and tumor tissues. The CD45-/GFP+ cells were defined as CTCs by this approach. The recovery rate of SMMC7721 and Huh7 were >85%. Regression analysis of the number of CD45-/GFP+ cells versus the number of spiked tumor cells showed good relevancy. We identified the ciruculating tumor cells which expressed the GFP protein and CK18 protein simutaniously in peripheral blood of two lung cancer patients by immunofloresence approche.Then the clinical samples, as little as 4ml peripheral blood, were used to detect CTCs by this method. CTCs in 36 hepatoma samples,101 lung cancer samples and 100 healthy samples were evaluated.The true positive rate in hepatocellular carcinoma patients’peripheral blood and lung cancer patients’blood was 91.6%,79.2%. The true negative rate was 97%.The method can specific trace circulating tumor cells, has potential clinical application value.Glioma is the most common human nervous system tumors,accounting for 40% to 60% of the central nervous system tumors, with a high recurrence rate, high mortality and low cure rate. These tumors are considered to be confined to the central nervous system because extracranial metastasis of glioblastomas rarely occurs (0.4 to 0.5% of patients). Several reasons for this low incidence of extracranial metastasis have been discussed, including short survival periods, the blood-brain barrier, suppression ofextracranial growth of glioblastoma cells by the immune system or the inability of GBM cells to invade and loosen connective tissue in the extracranial space.In this study, we took use of the sensitive detection method which has been mentioned above and then we detected the glioma patients’CTCs before surgery and after surgery. We identified that patients’circulating tumor cells could detected before operation(2.4±3.4/4mL blood). Furthermore, after surgical removal of the tumor, the number of circulating tumor cells in patients declined significantly. Then, we analyzed the white blood cell gene expression level between glioma patients and patients with brain metastases. By analyzing microarray data, white blood cell gene expression in brain glioma samples increase in the aspect of immune related gene expression.Above results suggest that blood-brain barrier doesn’t appear to block glioma cells entering into perioheral blood. CTCs countng may be an effective indicator for surgical evaluation.