Sorting And Analysis Of Rare Tumor Cells In Body Fluid Based On Microfluidic Technology

As one of the main causes of death,malignant tumor is a serious threat to human life and health,and its incidence is increasing.Tumor cells shed into body fluids during the growth,invasion and metastasis of malignant tumor,which are organ-specific and have rich biomarkers.So the exfoliated tumor cells have potential research value for tumor screening,disease diagnosis and prognosis evaluation.In clinical trials,pathologists mainly rely on cytology to identify exfoliated tumor cells in body fluid.But this method is susceptible to human factors and the tumor cells identified are inactivated,making it difficult to conduct further research.Therefore,it is urgent to develop a sensitive,convenient and time-saving method for the separation and application of rare tumor cells in body fluids.Microfluidic technology enables the integration of single cell sampling,isolation and detection onto a micrometer-scale chip.Due to the complex types of cells in body fluids and the small number of tumor cells,simple operations such as centrifugation and filtration are easy to lose tumor cells.Moreover,tumor cells in body fluids are different from other cells in physical characteristics(such as size,deformation and dielectric constant)and biological characteristics(such as biomarkers),which lays a foundation for the application of microfluidic technology in the separation of tumor cells in body fluids.At present,there are many studies on the separation of rare tumor cells from body fluids based on microfluidic technology and its clinical application.Based on this,we proposed an integrated microfluidic device.The device includes sorting chip and single-cell trapper chip.First,the sorting chip was based on the inertial focusing of the spiral channel and the deterministic lateral displacement(DLD)of the micro-pillar channel,which can separate large-sized tumor cells from blood or ascites.Then,the sorted cells were transferred to the single-cell trapper chip,where in situ immunofluorescence and cytological staining of single cell was performed.This method has the advantages of less invasiveness,multiple sampling and real-time monitoring of the patients’condition.The research content of the thesis is as follows:The first chapter is the introduction,which mainly introduces the current research of separation of tumor cells in blood,malignant pleural effusion,malignant ascites and other fluid based on microfluidic technology.And tumor cells in the body fluids in the clinical application that include tumor early diagnosis of,real-time monitoring of therapeutic efficacy and prognosis evaluation,exploration of the therapeutic targets and resistance mechanism.The second chapter is combined molecular and morphological imaging of CTCs for HER2-targeted chemotherapy guidance.Since target protein changes dynamically with tumor evolution and drug treatment,it is necessary to real-time evaluate target protein expression for subsequent effective molecule-targeted chemotherapy guidance.However,routine invasive biopsy is unrealistic to perform frequently because the pathological tissue is not easy to obtain repeatedly.Here,we present a non-invasive method for target protein HER2 detection based on circulating tumor cells(CTCs)analysis using an integrated microfluidic device.The CTCs were isolated and in situ confirmed on the microfluidic device by a new proposed complementary imaging method combining immunofluorescence for specific biomarker expression evaluation and hematoxylin-eosin(HE)staining for morphology analysis.And the CTCs were accurately identified as CK19~+CD45~-HE~+.Finally,the HER2 immunofluorescence signal of a single CTC was detected,this method can provide supplementary information for pathological tissue analysis and guide the use of targeted drugs.The third chapter is separation and single-cell analysis for free gastric cancer cells in ascites and peritoneal lavages based on microfluidic chips.Detecting free cancer cells from ascites and peritoneal lavages is crucial for early-stage diagnosing gastric cancer.But separating cancer cells from the large amount of background cells with high throughput and high purity still presents a difficult problem.Therefore,it is urgent to establish a new method to isolate cancer cells from ascites in order to improve the early diagnosis of peritoneal metastasis.Here,we develop a label-free,rapid,high-throughput technique using an integrated microfluidic device to successfully isolate cancer cells from ascites samples from 12 patients with gastric cancer.Next,we performed in situ immunofluorescence detection and cytological examination for separated cancer cells at single-cell trapper chip.The cancer cells were Ep CAM~+CD45~-or Ep CAM~+YAP1~+CD45~-.Finally,we also performed a serial YAP1 and HER2 expression analysis in nontumorous adjacent tissues(NATs),primary cancer lesions,metastatic cancer tissues,and ascites cancer cells,and revealed that YAP1 and HER2 can have discordant expression during the metastatic processes.This study provides a new method for the early diagnosis of peritoneal metastasis in patients with gastric cancer and to explore a new therapeutic targets and strategies for peritoneal metastasis.