Microfluidic Technologies-based Cell Sorting And Single-cell Analysis For Tumor Drug Sensitivity Studies

The differences of patients’ genetic information and living environment lead to significant differences in the sensitivity of different patients to the same drug.In the process of tumor treatment,the difference in drug sensitivity and drug resistance will make it difficult to achieve accurate drug administration,affecting the therapeutic effect of drugs.Accurate drug sensitivity analysis is the prerequisite for precision medicine.The drug sensitivity analysis based on standard cell lines is a commonly used method to evaluate the sensitivity of cells to drugs by detecting changes in cell proliferation or cell viability after drug treatment.However,this method based on standard cell lines does not reflect the real information of drug sensitivity in patients.To explore the true situation of patients’ response to drugs,it is necessary to perform the drug sensitivity analysis of the patient-derived tumor cells.This method provides the average information of cell viability changes after drug treatment,which ignores the intercellular heterogeneity of drug response,masks the important information of some special cells,and fails to obtain the drug sensitivity information comprehensively.Patient-derived tumor cells are surgically obtained from pathological tissues,and this invasive acquisition method cannot meet the requirements of realtime and repeated drug sensitivity analysis.To solve above problems,we need to first identify a type of cells that can reflect the tumor information of pathological tissues,and establish a noninvasive,real-time method for sorting and obtaining these cells.Second,for the obtained tumor cells,it is necessary to develop efficient and precise cell manipulation techniques,as well as highly sensitive analysis methods that can accurately analyze cell susceptibility to drugs at the single-cell level.In addition,to comprehensively obtain the drug sensitivity information of tumor cells,besides the changes of cell viability,we should pay attention to the changes of cell phenotype,cell adhesion,and cell secretion,etc.Microfluidic technology can accurately control fluids at micro and nano scales.In the microfluidic chip,diverse design of microstructures and small volumes of confined spaces can make the small the reaction and the small the confined can accelerate the reaction rate and reduce reagent consumption.Moreover,by combining detection technologies such as light,electricity,and mass spectrometry,the operation of single cell sampling,reaction,separation and detection can be integrated into a single microfluidic chip.Based on the above advantages,microfluidic technologies have been widely used for cell sorting and single-cell analysis,which provide an opportunity to obtain rare patient-derived tumor cells and drug sensitivity studies at single-cell level.Based on microfluidic technologies,the main results of this dissertation are shown as follows:1.An integrated microfluidic chip was proposed for rapid sorting and enrichment of circulating tumor cells(CTCs)from whole blood.CTCs were label-free isolated from blood with a spiral channel by the principle of inertial focusing,and further purified and concentrated by the principle of deterministic lateral deviation(DLD).The chip can rapidly isolate CTCs from whole blood with high purity(92±3%)and high throughput(6 m L of whole blood/hour).By analyzing the number of CTCs in xenograft-bearing mice and colorectal cancer patients with different tumor stages,the correlation between the number of CTCs and drug response was explored.2.Based on the first work,CTCs sorting chip was combined with a detection chip to achieve continuous operation of CTCs sorting,enrichment,online labeling and single cell phenotype analysis.By establishing the single cell quantitative curves of epithelial-mesenchymal transition(EMT)markers Ep CAM and N-cadherin,the quantitative differentiation of EMT phenotype could be achieved at single cell level.The phenotype of CTCs in patients with colorectal cancer was analyzed,and the correlation between EMT phenotype and drug sensitivity was explored.3.Based on a microfluidic chip for label-free isolation of tumor stem cells(CSCs),we utilized the differences in cell adhesion to isolate low adhesion cell subpopulation from population cells and verified their stem cell properties.Low adhesion cells(LACs)were utilized as rare CSCs candidate to evaluate CSCs-specific drugs.Through sphere-forming inhibition assay,the results showed that only CSCs-specific drugs had better inhibition of sphere formation.The LACs isolated from patient-derived breast cancer tissue only respond to CSCs-specific drugs.4.Based on a microfluidic chip containing a microwell array,the simultaneous analysis of tumor cells and extracellular vesicles(EVs)was realized.The differences in the secretion of EVs between drug-resistant and sensitive cells were analyzed,and the relationship between the secretion of EVs and drug resistance was explored.The results confirmed that cells were able to release more EVs to enhance cell survival when stimulated with low concentrations of drugs.We speculate that inhibiting the release of EVs could be a strategy to intervene in tumor cell drug resistance.