High Efficient Capture Of Breast Cancer Cell MDA-MB-231 By Using Microfluidic Chip And Re-culture

In recent years, breast cancer is one of the most common malignant tumors in women, and the incidence of this disease is quite high. Most of the desease were closely associated with the mutation of suppressor gene, leading to normal cells to be cancerous.The transfer of malignant tumor is the main reason that giving rise to the death or relapse in cancer patients. Serum tumor markers and imaging diagnosis of cancer early detection and so on are traditional methods that used for the determination of tumor,however, the relevance ratio is very low. Detecting tumor desease by micrometastasis of early clinical medicine has always been an enormous challenge.Circulating tumor cells (circulating tumor cells, CTCs) have been confirmded existing in many cancer patients bodies. CTCs are caused from the primary pathogenic stove, and then enters the human body circulation system. CTCs are metastasized to distant tissues or organs and even all over the body through peripheral blood.Circulating tumor cells have the biology characteristics of epithelial-mesenchymal transition(EMT) and some of them even express the features of stem cells.All of these circulating tumor cells can traverse blood vessels to reach the circulation system and rapid proliferation form the metastatic tumor. Many studies have shown that the migration- and adhesion of circulating tumor cell have close relationship with cancer metastasis and recurrence afte prognosis. Therefore, circulating tumor cells has very important clinical significance, such as for early diagnosis, prognostic assessment and curative effect detection. What’s more, circulating tumor cells can also provide theoretical basis for the development of targeted drugs.Circulating tumor cells is very difficult to separating, detecting and purifying for the bare number compared with the blood cells in whole blood. At present, the separation methods of circulating tumor cells mainly based on the cell size, cell morphology, cell density and the unique biological characteristics of tumor cells. The methods to seperate circulating tumor cells including filtration, density gradient centrifugation, charge on the surface of the cells and immune methods such as magnetic beads. Cell count and nucleic acid analysis are main the techniques of detecting circulating tumor cells. Researchers have been devoted to the study of circulating tumor cells enrichment, detection and purification, hoping to find efficient capture method and appling to clinical detection.Microfluidic chip is developed rapidly in recent years, a rapid detection and control technology of CTCs. It has controlled unit mode and micro-scale structure, which can realize the sample processing and detecting integration, Microfluidic chip can reduce the consumption of reagent and sample, and can reach rapid, efficient and high throughput analysis. In the end,it cuold get more information about CTCs.MUC1 antibody is a kind of macromolecule glycoprotein, it has lower expression in normal cells. It has two existing status in the cancer tissue incuding deformity glycosylation and uncompletely glycosylation. The two status makes the core protein of MUC1 revealed new protein epitope antigen and distributed across the surface of cancer cells, can be identified by specific antibodies. MUC1 plays an important role in adenocarcinoma progression, especially for breast cancer. It has high expression in breast cancer cells, also has relevance to metastasis and recurrence of breast cancer.In this paper, to achieve an effective capture efficiency of breast cancer cell (MDA-MB-231) by the microfluidic chip which was designed in our lab. Then to analyse the changes of the target genes FN1, ITGA6, LAMB3, ERBB2, RUNX2 and MMP3 expressing level in the tumor cells. Methods:The substrate of microfluidic chip was coupled with MUC1 antibodies, which could capture the tumor cells by the antigen on the cell surface. The conditions of cell capture were optimized to gain high cell capture efficiency. The captured cells were released by trypsin digestion and then the released cells were collected and re-cultured. The normal and re-cultivated cells were incubated with lum doxorubicin for 24h respectively. Then the RNA in the cells was extracted and reversed to synthesize DNA and the target genes FN1, ITGA6, LAMB3, ERBB2, RUNX2 and MMP3 were amplified by reverse transcriptase polymerase chain reaction(RT-PCR). Results:The tumor cells can be effectively captured by the microfluidic bio-chip after MUC1 antibodies were modified on the surface of chip and the capture rate can reach 80%. The cells release efficiency was as high as 98% and the released cells still had high viability which could be re-cultured. Doxorubicin could inhibit the expression quantity of FN1, ITGA6, LAMB3, ERBB2, RUNX2 and MMP3 in normal and re-cultivated MDA-MB-231 cells. Conclusion:The tumor cells can be captured effectively by the microfluidic chip and re-cultured. The expressions of genes of the cells were not interfered remarkably for pre or post-captured. All of these would lay the foundation for the following related research such as biochemistry and molecular biology research, the efficacy evaluation analysis of antitumor drugs and so on.