The Establishment Of New Circulating Tumor Cells Detection Technology And Its Application In Liquid Biopsy Of Hepatocellular Carcinoma

Capture and analysis of rare circulating tumor cells(CTCs)is an important means for monitoring of tumor metastasis and evaluating the efficacy of chemotherapy,peripheral blood CTCs as metastatic seeds are the main cause of tumor spreading from primary to distal organs.The precise separation of CTCs is important for the early diagnosis and prognosis of cancer and downstream analysis.Researchers have developed many different kinds of CTCs technology for separation and analysis,including affinity separation based on CTCs surface markers and unlabeled separation based on CTCs physical and chemical properties,The current trend is towards multitechnology fusion design,because each single method may have its inherent disadvantages,such as selective bias towards cell subsets,the low separation efficiency or capture purity,or the high cost and complexity of operation,the disadvantages limit this important way of understanding and fighting against tumors to really go into the clinicObjective: To find new and better CTCs capture techniques or to combine various single techniques to achieve more accurate and efficient CTCs capture and more beneficial for our subsequent cell analysis and culture.This study aims to establish biocompatible CTCs capture methods that can maximize the viability of the target cells and flexibly integrate the technologies of nano-technology,nucleic acid aptamer technology,fluorescence imaging technology,tumor molecular targeting technology,Optical tweezers technology,micromanipulation technology and microfluidic technology.Methods: the study was divided into three parts,in the first part,We used red blood cells as raw materials to make red blood cell nanovesicles and designed bionic nanovesicles targeting tumor cells,first of all,Folic acid(FA)molecules and luciferin Cy5 are modified on the erythrocyte membrane to target folic acid receptors on the surface of tumor cells,then a liposome extruder was used to physically extrude the erythrocyte membrane into a nanocapsules modified with FA and Cy5(FRVs),FRVs was used to bind CTCs simulated by MCF7 cell lines in blood samples,and then the tumor cells were then isolated and collected by micromanipulation under fluorescence microscopy.In the second part,we use the same FA modified RBC building FA-RBCs,and then CTCs was combined with FA-RBCs to form CC-RBCs in blood,This causes a significant optical constant difference between CTCs and other blood cells(volume size and average refractive index).Then,the modified CTCs were precisely separated by laser irradiation using a photofluidic separation system,and the CTCs were recovered by removing red blood cells;In the third part of the study,we used LY1,a specific nucleic acid aptamer targeting hepatocellular carcinoma cells developed in the previous screening stage,in combination with a specially designed microfluidic chip device,to verify the capture efficiency of this system,CTCs separation experiments were carried out respectively in blood samples of patients with liver cancer and liver cancer cell line HCCLM9,next we use methods of Immunofluorescence and q RT-PCR to analyze the molecular expression of the captured cellsResults: the first part of the study,we successfully established FRVs,a nanomaterial derived from red blood cells,In vitro,FRVs can specifically target tumor cells with high FA receptor expression,and then use micromanipulation for efficient and high-purity CTCs capture,the captured tumor cells were highly viable,In vivo,FRVs can actively target tumor tissues and concentrate on tumor sites,showing a strong Cy5 fluorescence signal and achieving effective tumor imaging.In addition,no toxicity or side effects of FRVs were found in vivo.Therefore,FRVs has a high potential for clinical application;The second part of the study,the experimental results show that CTCs can be effectively combined with modified red blood cells to form a cc-rbcs complex,which is eventually separated from the blood in high purity(over 92%)and high recovery(over 90%),CTCs have been shown to maintain membrane and functional integrity throughout the process.The combination of the RBC capture system and the optofluidic separation system may provide a convenient tool for early diagnosis and therapeutic monitoring of cancer;In the third part of the study,We verified the efficacy of liver cancer targeting nucleic acid aptamer combined with microfluidic chip in the capture of CTCs blood samples,Its capture efficiency is 89.1-92.4% and purity is above 55%,the invasive and tumorigenic abilities and partial molecular phenotypes of the cells captured on the chip were significantly different from those of the outgoing cells.A study of CTCs captured from 61 patients with liver cancer showed that the number of CTCs was significantly correlated with disease progression,and the differences of CTCs expression levels in19 patients with or without metastatic hepatocellular carcinoma were analyzed as well.