Cancer Screening And Primary Exploration Of Immunotherapy Based On Microfluidic Technology

Cancer is a disease that endangers the health of people all over the world.Only in 201 8,18.1 million new cases and 9.6 million deaths were reported.Because of its difficulty in early detection,diagnosis and easy-to-metastasis,the thorough treatment of cancer is still very difficult.Therefore,three methods of non-invasive diagnosis of malignant tumors based on microfluidic technology were constructed and tested in clinical application.At the same time,an immunotherapy approach based on "tumor-on-a-chip" was constructed.Furthermore,the application of exosomes secreted by natural killer cells(NK)to study the effect on tumor cells was preliminarily discussed,which can provide new ideas for clinical cancer screening and treatment.Firstly,an integrated microfluidic filtration device with a size of 5 ?m polycarbonate membrane was developed,which can isolate and enrich exfoliated tumor cells(ETCs)from discarded urine,and then quantified ETCs in urine samples of bladder cancer patients via microchip-ELISA.The detection principle of the device was mainly based on size-exclusion,membrane.At the same time,the isolated cells were analyzed by scanning electron microscopy,fluorescence staining and mutation sequencing to verify that they were exfoliated tumor cells,and the number of ETCs was quantitatively detected on the device by direct ELISA.The results showed that the number of exfoliated tumor cells in urine samples of bladder cancer patients was significantly higher than that of healthy subjects.The ROC analysis showed that the integrated filtration microfluidic device was effective in bladder cancer screening.When the specificity was set at 90%,the sensitivity of the detection method was 77.1%in identifying BC patientsSecondly,to purify urine samples a single-membrane microfluidic device was developed to filter urine samples,then free nucleic acids in urine samples was extracted and detected.This method mainly detected cell-free DNA content in urine samples filtered by microfluidic device and compared DNA integrity amplification.The results showed that the cell-free DNA integrity test and abundance analysis could effectively preliminarily distinguish bladder cancer patients from healthy people.The sensitivity of the method was 88.6%when the specificity was set at 90%in identifying BC patientsThirdly,we developed an integrated double-filtration microfluidic device that isolated and enriched EVs with a size range of 30-200 nm from urine,and subsequently quantified the EVs via a microchip ELISA.Furthermore,the isolated exosomes were characterized by transmission electron microscopy,fluorescence staining,nanoparticle size analysis and dynamic light scattering analysis,and then quantitatively analyzed the number of urinary exosomes by microchip ELISA.Our results showed that the concentration of urinary EVs was significantly elevated in bladder cancer patients(n=16)compared to healthy controls(n=8).Receiver operating characteristic(ROC)analysis demonstrated that this integrated EV double-filtration device had a sensitivity of 81.3%at a specificity of 90%(16 bladder cancer patients and 8 healthy controls).Thus,this integrated device has great potential to be used to improve clinical diagnosis of bladder cancer in clinics and at point-of-care(POC)settings.Finally,we developed "Esophageal cancer-on-chip" and "Breast cancer-on-chip".The exosomes derived from natural killer cells(NK)were co-cultured with tumor cells in two-dimensional(2D)and three-dimensional(3D)dynamic conditions,respectively.Meanwhile,the cells were stained using live/dead kit and CCK8 analysis was used to detect the cell viability.Cytotoxicity test and flow cytometry were used to characterize the killing effect of NK cell-derived exosomes on tumor cells.The results showed that NK cell-derived exosomes had a certain killing effect on esophageal cancer cells and breast cancer cells in both 2D and 3D dynamic conditions.Compared with 2D conditions,NK cell-derived exosomes in 3D dynamic environment have more obvious killing effect on tumor cells.