Novel Microfluidic Chips For Separation And Release Of Circulate Tumor Cells And Their Clinical Application Research

As one of the main research directions of "liquid biopsy",the analysis of circulating tumor cells(CTCs)provides a non-invasive method to obtain biological information of solid tumors.It has not only contributed to the understanding of tumor metastasis,recurrence,and chemotherapy resistance mechanisms,but also facilitated the development of precision medicine.However,CTCs are an extremely rare cell population,which presents a challenge to isolate CTCs from patient’s blood.In addition to the separation of CTCs,high efficiency release of CTCs is one of the current challenges.In recent years,the membrane-based filtration methods based on the differences of size and deformability have attracted extensive attention due to their advantages,mainly including label-free,simple,rapid and high separation efficiency.Microfluidic technology has been applied to CTC separation due to its unique advantages such as integrated miniaturization and automation,low reagent consumption,high throughput,and low sample volume requirement.In this dissertation,the membrane-based filtration method was combined with microfluidic technology to construct a CTC separation system.Ultrathin silicon nitride filtering membranes were used for the high-performance separation of CTCs,and the filtering membranes were modified with polyethylene glycol(PEG)for high efficiency release of CTCs.Finally,the CTCs were applied to gene mutation detection and treatment response monitoring of cancer patients.The contents of this dissertation are summarized as follows:1.The characteristics of tumor cell diameter and stiffness were investigated.The differences in diameter and stiffness between tumor cells and white blood cells(WBCs)were clarified,which further enrich the theoretical basis of CTC separation method based on cell size and deformability.Next,optimization of the selection of filtering membrane thickness based on fluid simulation,the optimal filtering membrane thickness(200 nm)was obtained with less damage to the cells.This is a reference for designing next-generation microfilters with higher throughput and less cell damage.2.By optimizing the design parameters of filtering membranes,silicon nitride was selected as the raw material of the filtering membranes and standard microfabrication techniques were used to fabricate ultrathin filtering membranes.Ultrathin silicon nitride filtering membranes were used for high-performance separation of CTCs.By using the filtering membrane with pore width of 6 μm,a capture efficiency of～96%was obtained.In addition,the filtering membrane with high porosity(34%)was used for CTCs separation with～99.99%WBC depletion rate.Finally,the ultrathin(thickness:200 nm)filtering membrane facilitated the viability of CTCs,and～90%of CTCs were viable cells.In clinical applications,CTCs were successfully isolated from whole blood samples of cancer patients using silicon nitride filtering membranes,while CTCs were not detected in whole blood samples from healthy donors.3.It is briefly described that high efficiency(～85%)release of CTCs was achieved by modifying the filtering membrane with hydrophilic polymer PEG,which facilitated the reduction of non-specific cell adhesion on the filtering membrane surface.In subsequent analysis of the released cells,the released A549 cells was subjected to re-culture study,which showed that A549 cells exhibited normal adhesion and division in culture flasks.Next,epidermal growth factor receptor(EGFR)gene mutations were detected in CTCs released from the blood of lung cancer patients using Sanger sequencing.EGFR gene mutations(exon 21,G>A)were finally detected in CTCs from lung cancer patients.This result well demonstrated that the novel microfluidic chip has the ability to perform molecular analysis of CTCs,and also shows the good potential in studying the molecular mechanisms of tumorigenesis and tumor progression.4.The relationship between baseline CTC count and overall survival(OS)in patients with advanced non-small cell lung cancer(NSCLC)and as a predictor of response to chemotherapy was explored.A significant correlation between baseline CTC count and OS in NSCLC patients was found in the study.Baseline CTC count of more than 6 was a relevant factor for reduced OS.Next,a high baseline CTC count was associated with disease progression after chemotherapy treatment,and patients with baseline CTC count over 6 had poor prognosis or even death.In summary,in this dissertation,we proposed to use ultrathin silicon nitride filtering membranes for high-performance(high separation efficiency,high WBC depletion,and high cell viability)separation of CTCs.In addition,high efficiency release of CTCs was achieved by PEG-modified filtering membranes.Finally,we applied the CTCs for gene mutation detection and prognostic assessment of chemotherapy in lung cancer patients.This dissertation explored and expanded the application of novel microfluidic chips in CTC separation and release,and facilitated the clinical application of CTC-based liquid biopsy technology.