| BackgroundThe morbidity and mortality rate of lung cancer has been increasing year by year,but the age of the patients is decreasing.In recent years,with the in-depth study of lung cancer and the improvement of the therapy,more patients get the better therapy.However,due to the metastases of lung cancer,the prognosis of the patients will be serious as long as the metastases happens.But the technologies have some limits in diagnosis,assessment of prognosis and instruction of therapy,and the patients usually lose the chance of getting the best therapy.Therefore,how to increase the detectable rate of lung cancer is the key for the clinic.In recent,to overcome the obstacles of the classic detection,there has been a more focus on the development of an efficient technique for detection of circulating tumor cells(CTCs),due to their significance in prognosis and therapy of metastatic cancer as a liqid pathology.As well,CTCs detection will be easily accepted for the benefits ofno-invasive,simple operation,easy to repetition.However,as the low count of CTCs,it has been a challenge to make a effective isolation.Nowadays,with the development of the microfluidic technology for cell biology,it is very suitable for the isolation of blood and tumor cells.And some improvements have been made in CTCs isolation by themicrofluidic,according to the principle for isolation,it will be dividied into twomethods,one is the isolation by the physical features,and the other is by the biologic features.The former is depended on the size,the density and the electrode of CTCs.And the advantages of it is simple,low cost and high throughput,but the disadvantages are the low capture rate and purity.The latter is depedent on the immunity,to isolate CTCs by the specific protein on it.Therefore,our study aims to develop a device for rapid and effective of CTCs.By the isolation of cell line,the patients blood sample and the comparsion with CellSearch System,the capture rate has been confirmed.Then the device is used to anlysis the relationship of CTC number with clinical stage,metastasis and therapy.At last,to further analysis the mechanism of metastasis,single cell will be captured and made transcriptome sequencing,to find the genes associated withmetastasis and make some testifition.Our study will be introduced as the four aspects below.Methods1.A development of the device for CTCs detection on microfluidicThe integrated microfluidic device for CTCs isolation has gathered up thebenefits of every single method and overcome the defects.And the device was made of three parts,the first part was the DLD isolating chip which was dependent on the size of the cells,the second part was a purifying structure which relied on the negative magnetic isolation and the CD45 protein expressed on WBCs,and the last part wasdeveloped on the pore plate coated with the rat-tail collange on which CTCs would be capture by the immunity reaction.2.The testification of the CTCs detection deviceThis part was devided into three parts,the cell line,the clinical patientsdetection and the CellSearch System comparsion.Cell line:to prepare 5 samples with the different dilution of H1299-GFP in the healthy peripheral blood which mimicked the clinical patients(10~1/mL-10~5/mL)and to detect the cell lines by the integrated device and calculate the capture rate and purity.Then the cell viability would be measured by the Hochest/PI for both the experiment group and the control.Clinical patients:to detect CTCs in the clinical patients by the integrate device,and to analysis the results with the patients condition as well.At last,the capture rate would be compared with theCellSearch System by testing 30 patients.3.The clinical application of the deviceThis part was aimed to anlysis the CTCs detection results of 55 patients,with the clinical information,it would be anlysised the relationship of CTCs number withclinical stage,metastasis and patients’condition.4.Single cell transcriptome sequencing analysis and screen the genes associated with lung cancer metastasisThis part was to analysis the different expression of genes in lung cancer cell line with high metastasis or low metastasis,to explore the genes associated with metastasis.Results1.The constribution of the integrated device on chipThe device for CTCs detection in this study was constitude of three parts.Firstly,depending on the size of different cells,we tried different parameters of the chip design.To find when the longth of the chamber was 68mm,width was 3.5mm,the height was50μm and the critical separation size was 6.75μm,the isolating rate of DLD would be th90%.Then depending on the negative magnetic isolating,the automatic purifyingdevice was built,when the reacting time for CD45 dynabeads and the WBCs was 20min,and the ratio of them was 10:1,the removal rate of WBCs would be more than 80%.At last,CTCs would be captured on the pore plate coated with rat-tail collagen,whereCTCs was indentified by the immunity reaction.2.The testification of the CTCs detection deviceThis part was to tetify the capture rate,capture purity and cell viability.Firstly,we mimicked the patients blood samples by adding the H1299-GFP cell line into thehealthy peripheral blood samples,to find that at the high throughput of 1mL/min,the capture rate would be 90%,and the capture purity be 50%.By Hochest/PI test,wfound that there was no difference of the cell viability between the experimental and the control groups which maintained 90%,and cell line could continue culturing,proliferating and passaging.At last,we tested CTCs from 15 lung cancer patients and 2healthy volunteers,to find that 12 in 15 patients was positive with CTCs isolation with the capture rate 80.0%,however,none was found in the healthy,which excluded thefalse positive results.At the same time,with the analysis of the patients’clinicalinformation,we found the results for CTCs detection were coincide with the patients’condition.At last,we compared the device with the Cellsearch System by detecting CTCs from clinical patients.Analysing the results by t test,we found p>0.05,which meaned no difference in capturing efficieny by the two methods.3.The clinical application of the deviceThis part was to anlysis the clinical use of the device by anlysis the results of 55patients,to explore the CTC number with clinical stage,metastasis and therapy.And in the early stage(I,II),the capture rate of CTCs was 7.1%.But it increased to 68.3%in late stage.And to the 29 positive patients,28 cases of them had been diagnosised asmetastasis.We also found the detection rate was positive when the patients were recurred or progressed,and negative when the patients were remitted or stable.4.Single cell transcriptome sequencing analysis and screen the genes associated with lung cancer metastasisTo further explore the mechanism of lung metastasis,we anlysed the single cell sequencing by RNA-seq.Firstly,by lung cancer cell line 95-D which had highmetastasis capability and SPCA-1 which had low metastasis,we got single cell by capillary tube absorbing.Then,the difference of genes between them was tested by RNA-seq.And TSPAN6、CFH、GCLC、HS3ST1 may have relationship with lung metastasis.Further,we would focous on the single CTC transcriptome sequencing of lung cancer patients.Conclusion:1.We have successfully developed an integrated device for CTCs detection in lung cancer on the microfluidic technology which has integrated the benefits of three single methods and achieve rapid and efficient detection.2.The effieiency of device is confirmed by the detection of the cell line,clinical patients’samples and the comparsion with CellSearch System.3.By the analysis of CTCs detection,we found the number of CTCs was related with clinical stage,metastasis and patients’condition.4.By single cell sequencing,we found some genes might have relationship withlung cancer metastasis,such as TSPAN6,CFH,GCLC and HS3ST1,more testification would be done in further.Also,based on the cell line experiment,we would focous on the single CTC transcriptom sequencing of lung cancer patients. |
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