| ObjectiveIn order to establish a simple but efficient method of enrichment and detection of CTC(Circulating Tumor Cell, CTC) in cancer patients and apply this method to clinical use to count CTC number in the peripheral blood of cancer patients efficiently, this research uses Opti Prep density gradient centrifugation and immunomagnetic negative screening to enrich combined with multicolor immunofluorescence and fluorescence in situ hybridization to detect circulating tumor cells. Meanwhile, this research tries to build up integrated technology of fluorescent marker and targeted identification to obtain single-cell to establish single-cell genomics research for specific CTC or cell populations. We identify CTC by microscopy then perform micro-dissection and micro-absorption to obtain single cell to do molecular identification, so this research will further improve CTC molecular identification and will provide the base of exploring new treatment target, which will eventually lead to more efficient cancer treatment protocol.Method1. To verify the accumulation layer of monocytes, we use different sedimentation coefficient in cells with different density and different concentrations of Opti Prep to form density gradient to centrifuge and separate peripheral blood cells. 7701 and other different tumor cell lines were selected and mixed into peripheral blood to mimic circulating tumor cells in vivo. We also compare the rate of enrichment, recovery, activity between Opti Prep solution and Ficoll solution.We combine density gradient centrifugation and immunomagnetic to establish optimal circulating tumor cell enrichment system.2. Select the five kinds of tumor cell lines: hepatoma cells(QGY-7701, Huh-7, SMMC-7721),ovarian cancer HO-8910, A549 cells, immunofluorescence staining them with CD45 and CK18,respectively, and use CIK cells as controls. We use FISH to label the number of chromosome of CIK. The tumor cells were mixed into normal human peripheral blood to mimic circulating tumor cells in peripheral blood, then enrich them and use immunofluorescence and FISH detection to accurate identify the tumor cells.3. We combine enrichment and detection methods to accurately detect circulating tumor cells in peripheral blood of cancer patients in order to conduct validation and evaluation methodology.Based on the CTC number detection and clinical prognosis analysis, we have established the CTC detection method for evaluation of cancer patient prognosis.4. In trying to do microscopic microdissection and gain single target cell and pure cell populations, we apply Carl Zeiss PALM Combi System and Cell Ector Plus, these two systems to capture individual cells, and examine tumor cells gene expression by RT-PCR.Result1. Opti Prep solution can effectively isolate monocytes, and capture 7701 tumor cells in white film. Hepatoma cells(QGY-7701, Huh-7, SMMC-7721), ovarian cancer HO-8910, A549 lung cancer cells enrichment is more efficient in Opti Prep solution than in Ficoll solution, the differences between the two groups were statistically significance(P <0.05). Single cell manipulation taking about 50 tumor cells and mixing into the blood to mimic circulating tumor cells, and the recovery rate turn out to the same between Opti Prep solution and Ficoll solution.2. Immunofluorescence result shows only CIK cells were observed in CD45 red fluorescence,and CIK cells were the only cells without green fluorescence. Fluorescence in situ hybridization result shows this method can mark chromosomes clearly, therefore can identify chromosomal abnormalities cells.3. By using the method mentioned above to examine the peripheral blood of 33 enrolled cancer patients, were found the presence of circulating tumor cells in all cases, indicating that this method can effectively detect CTC in blood of cancer patient. Our results show that the higher number of CTC corresponding to the shorter lifetime and poorer prognosis.4. In this study, according to the experimental needs, we involve two identifiable obtain fluorescently labeled single-cell technology, and the results show these two methods both can quickly and accurately obtain single target cell, solve cell heterogeneity problem, and gain pure target cell, therefore lay foundation to the next step of a small amount CTC molecular characterization, and show a good prospect for clinical application.ConclusionThis research successfully establish a method using Opti Prep density gradient centrifugation combined with immunomagnetic negative screening to enrich CTC as well as multicolor immunofluorescence and fluorescence in situ hybridization to detect CTC. This method can be used in testing CTC in peripheral blood of cancer patients. Also, this research tries the single cell capture technology to obtain the scarce CTC, and gives a direction for single cell target gene identification and gene sequencing, therefore provides reference for cancer prognosis, individual treatment and rational use of drugs. |
