A Novel Approach For Detection Of Circulating Tumor Cells By Replication-selective Oncolytic Adenovirus In Digestive Cancer

Background and ObjectivesAccording to many research, the detection of circulating tumor cells (CTCs) may contribute to discovering the micrometastasis, monitoring the recurrence or metastasis, providing potential prognostic value, guiding the treatments, judging the therapeutic efficacy and monitoring the recurrence or metastasis.CTC are rare and have not specific markers, so the development of CTC detection is hard. There are some methods for the detection of CTCs, typically including two parts:enrichment technique and analysis technique. Each of them has its own advantages and disadvantages, and there is not a unified protocol and standard.With the development of studying on cancer and virus molecular biology, we reformed wild type virus or discovered congenital oncolytic virus to get the replication-selective oncolytic adenovirus (RSOA). RSOA can replication-selective in tumor cells and not replication in normal cells or low. In the RSOA, Adenoviral vectors has many strengths:wide infect cell types; high infect efficiency; stabilization of physical and chemical character and easy to prepare high drops degree virus; not intergrate into the cell genome and low genetic toxicity; infect both division and non-division stage cells. Our experimental research was going to optimize a novel approach which was using the replication-selective characteristics of RSOA, and rebuilt the virus’ structure,in order to help us identifying the circulating tumor cells easier in millions of hemocytes.Green fluorescen protein(GFP) is a kind of bioluminescent protein which is exist in coelenterate. It is a kind of acid, the ball, soluble natural fluorescent protein, nature extremely stable, easy to tolerate high temperature treatment, both formaldehyde fixed and paraffin do not affect its fluorescence properties. Our experimental research had inserted this protein gene into the RSOA genome successfully, and used them to infect tumor cells. With the virus replicating in the tumor cells, the GFP expression was more and more, in right time,we could identify the rare circulating tumor cells easier under a fluorescence microscopy. This is a simple and efficient approach for detection of circulating tumor cells in digestive cancer.Materials and methodsHuman esophageal cancer cell line EC-1, gastric cancer cell line AGS, colorectal cancer cell line SW620 and pancreatic cancer cell line suit2 were experimental subjects. The tumor cells were counted and mixed with normal blood, and then incubated with different virus gradient at different time gradient. After stained by DAPI, GFP-positive tumor cells were counted under a fluorescence microscopy. The blank control was normal blood without tumor cells, the positive control was naked tumor cells. The statistics of results were treated by statistical software SPSS 17.0Results1. The rebuilt RSOA which we used in our experimental research could not infect and replicate in human leucocytes.2. In our experimental research, the rebuilt RSOA could specifically infect tumor cells and replicate massively in tumor cells. According to the positive rate and fluorescence intensity, we found that:the effect by the virus dose was significant; incubation time was also an important factor, with the time gone,the living cell number tail away, but the positive rate and fluorescence intensity were strengthen.3. In the experiments of Human esophageal cancer cell line EC-1, gastric cancer cell line AGS, colorectal cancer cell line SW620 and pancreatic cancer cell line suit2, the positive rate was respectively 76.6%、73.3%、76.6% and 80.0%, and x2 test was used to contrast the positive rate and the results show no significant differences in each other. (x2=0.373,p=0.946)ConclusionsOur rebuilt RSOA Ad11 shows a satisfactory marker to label malignant cells in preclinical studies, with high infection efficiency, high sensitivity, and specificity, which is a simple and effective new method of detecting CTCs in the digestive malignancy.