Integrated Microfluidic Devices For Circulating Tumor Cell Isolation And Their Clinical Application In Lung Cancer Patients

Background:Tumor is a chronic disease that seriously endangers human health.According to the most recent data from World Health Organization,the incidence of tumor has been increasing during the past decade.Lung cancer is a common malignant tumor in our country,although its morbidity rate has been effectively reduced with the strengthening of tobacco control and the promotion of low-dose CT screening,its mortality rate is still highest among all malignant tumors.Due to the characteristics of insidious onset and early metastasis of lung cancer,coupled with the limitations of currently used diagnostic techniques,patients often miss the best chance for therapy when diagnosed.Therefore,how to diagnose lung cancer and control metastasis early is an urgent clinical problem.Liquid biopsy is an emerging technology for the early screening,molecular typing,tumor dynamics profiling and prognostic assessment of tumor,which has the advantages of less trauma,convenient sampling,and real-time dynamic detection compared with traditional tissue biopsy.Circulating tumor cell(CTC)is one of the most mature biomarkers in liquid biopsy,whose clinical application is greatly limited by its scarcity.In recent years,the development of microfluidics has promoted the innovation of CTC detection techniques.In this study,we established two integrated microfluidic platforms for rapid and effective CTC detection and single cell isolation and carried out preliminary clinical applications.The efficiency and reliability of the two devices has been confirmed by using lung cancer cell line and blood samples of patients.Then the devices were used to detect and analyze the relationship between CTC count of peripheral blood samples with pathological type,tumor stage,metastasis and treatment response.At last,single CTCs,matched tissue and other liquid biopsy samples,including circulating tumor DNA(ct DNA)and pleural effusion exfoliated cells were collected from 2 representative cancer patients and sequenced,so as to profile and compare genetic variations between different biological samples.Our study will be introduced from the following four aspects.Methods:1.Construction of the integrated microfluidic CTC detection devicesTwo integrated microfluidic devices have been established in this study.The first device was made of three parts.The first part was a CTC detection chip based on a deterministic lateral displacement(DLD)structure and a magnetic purifying chip.The second part was a CTC identification chip for immunofluorescence staining of captured CTC and residual white blood cells(WBCs).The third part was a single-CTC isolation chip for picking single CTCs manually.The second device consisted of two parts.One was a filtration system based on a packaged micropore-arrayed membrane,another was the magnetic microfluidic chip used in the first device.2.Validation of the integrated microfluidic devicesThe performance of these two devices was tested by using lung cancer cell line and blood samples of patients.For cell line,PC9-GFP cells was spiked into blood samples from healthy donors at different concentrations(10~1-10~4 cells/ml)to mimic CTCs,with blood samples without tumor cells as negative control.The efficiency and purity of CTC capture and single cell isolation,and the capability of WBCs clearance was calculated respectively.For clinical validation,2-4ml peripheral blood was drawn from36 tumor patients,while blood samples of 4 non-tumor patients were also collected as control to exclude false positives.3.Clinical application of the integrated microfluidic devicesIn this part,with the clinical basic information,the relationship between CTC detection results of 76 lung cancer patients with their pathological type,tumor stage,metastasis and conditions was analyzed.4.Single cell isolation and high-throughput sequencingSingle CTCs were isolated from blood samples of 2 representative cancer patients,while matched tumor tissue and invading tumor cells from pleural effusion were also collected.Then,DNA sequencing were performed to profile the genetic variations in different samples,while mutated genes possibly associated with tumorigenesis and metastasis were identified.Results:1.The development of the integrated microfluidic CTC detection devicesWe constructed two CTC detection platforms in this study.The first was an integrated microfluidic device for CTC detection and single-cell isolation,which consisted of three parts.The first part was named CTC detection chip,which included a DLD chip and a magnetic purifying unit.The second part was called CTC identification chip,with a reaction chamber where antibodies would be added to distinguish CTCs.The third part was a CTC isolation chip which contained a round chamber for isolating stained single CTCs by manual cell picking.The second platform was made of a filtration system and a magnetic microfluidic structure.The core of the filtration system was a micropore-arrayed membrane with a diameter of 10?m.Whole blood was allowed to be filtered by this membrane with high-throughput without any extra pressure.The magnetic microfluidic chip was the same with the first platform.After purification,captured cells would be transferred into other platforms for further identification or single cell isolation.2.The performance validation of the integrated microfluidic devicesWe tested the performance of the two devices on lung cancer cell line and blood samples of patients.Firstly,we spiked GFP labeled PC9 cells into healthy blood samples at concentrations of 10~1-10~4 cells/ml,with blood samples without tumor cells as negative control.For the first device,the efficiency of single-cell isolation was above90%and the purity was approximately 90%.For the second device,the CTC capture rate and purity were 85%and 60.4%respectively.And we found no CTC in any negative control group.Then,we conducted CTC detection on tumor and non-tumor patients.For the first device,we found CTCs in 15 of 20 patients,with a detectable rate of 75.0%.Additionally,the change of CTC counts was in line with the treatment response of these patients.For the second device,we enrolled 16 lung cancer patients and 4 non-tumor patients.CTCs were found in 14 of 16 lung cancer patients(87.5%detectable rate),while all non-tumor patients showed negative results.3.The clinical application of the microfluidic devices106 patients were recruited in this study in total,including 76 lung cancer patients and 30 non-tumor patients.50 of 76 lung cancer patients showed positive results in CTC detection,with a detectable rate of 65.8%,while false positive result showed in 2non-tumor patients.There were no significant differences between CTC detection results and pathological type or tumor stage.Although the CTC detection rate of patients with metastasis was higher than that in patients without metastasis,the difference is not statistically significant.Additionally,the change of CTC counts in peripheral blood could reflect patients'condition.The number of CTCs would decrease,even disappear when the patients were stable or remitted.However,when the patients progressed or recurred,CTC count would increase.4.Single CTCs isolation and DNA sequencing analysisTo further investigate the mechanism of tumorigenesis and metastasis,we isolated single CTCs from 2 representative tumor patients,including 1 lung cancer patients and a gastric cancer patient with lung metastases.DNA sequencing and comparative analysis were performed on single CTCs,together with matched tumor tissue and tumor cells from pleural effusion.The tumor heterogeneity was described at single-cell resolution and the consistency of genetic variations between liquid biopsy and tissue was confirmed.6 new gene mutations were found both in single CTC and primary tumor of an early lung cancer patient.Moreover,3 high frequency gene mutations with prognostic significance,HGF,QKI and RARA,were identified.In future studies,we will put more efforts to explore the function of these mutated genes in tumor metastasis.Conclusions:1.We have successfully established two integrated CTC detection platforms based on microfluidics,which achieved high-throughput,low-damage CTC capture and high-efficiency,high-purity single cell isolation.2.The performance and reliability of the two devices were validated by detecting lung cancer cell line and peripheral blood samples of patients.3.There was no significant difference in CTC detection results between lung cancer patients with different pathological types,tumor stages and metastases,while the absence or change of CTC number could reflect patients'condition and treatment response.4.The consistency of liquid biopsy and tissue biopsy was observed by single CTC isolation and single-cell sequencing,while mutated genes that may play a role in the occurrence and metastasis of lung cancer and gastric cancer were identified.