Characteristic Analysis Of The Circulating Tumor Cells And Its Clinical Application Value In Lung Cancer

Lung cancer is the leading cause of cancer-related death in the world,and approximately 85%of lung cancer cases are non-small cell lung cancer(NSCLC).Although systematic treatment has been improved,the overall 5-year survival rate is only 17%.The major cause of the low survival rate is tumor metastasis.In the metastatic cascade,circulating tumor cells(CTCs)have the capacity to spread into the blood stream and have been considered to be key participants in the formation of distant metastases.A number of studies in the past decade have indicated that CTCs can be used as a marker to predict disease progression and survival in metastatic and high CTC numbers correlate with aggressive disease,increased metastasis,and decreased time to relapse.Therefore,CTCs bear a tremendous potential to improve our understanding of steps involved in the metastatic cascade,starting from intravasation of tumor cells into the circulation until the formation of clinically detectable metastasis.In addition,the study of CTCs is not only instrumental as a basic research tool,but also allows the serial monitoring of tumor genotypes and may therefore provide predictive and prognostic biomarkers for clinicians.Despite this great potential,the use of CTCs faces many hurdles.One challenge for CTC research is posed by the rarity of tumour cells in blood.Hence the first step for CTCs research is to isolate CTCs.It has been considered that the detection methods for circulating tumor cells(CTCs)based on epithelial cell adhesion molecule(EpCAM)underestimate the number of CTCs and may miss a metastatic subpopulation without expression EpCAM(EpCAM-negative CTCs,E-CTCs).Until now,the clinical value and biology of E-CTCs has been unclear.Given the metastatic potential of E-CTCs,future studies should include the detection of E-CTCs.CTCs undergoing epithelial-mesenchymal transition(EMT)have been considered to play an important role in the formation of neoplasms.EMT can generate cells with stem-like properties.During EMT,the expression of EpCAM in tumor cells will be down-regulated.However,it remains unclear whether these(or any)CTCs with down-regulated EpCAM have an increased metastatic seeding potential or heightened resistance to systemic therapy and a greater prognostic value.The other challenge is that CTC has shown complex biological characteristics.With the development and application of the single cell analysis technology,a significant heterogeneity between CTCs has been proved.The tumour-initiating hypothesis posits that only a minority of cells with metastatic capability possess all the required adaptations to survive passage through the circulation and produce metastases.Therefore,not all of the CTCs have the clinical relevance.Thus,it is necessary to study the biological function of CTCs and to screen out the markers which can characterize the tumor-initiating subpopulations.In breast cancer,using flow cytometry analysis of blood from one patient whose CTCs formed in a mouse xenograft,a subpopulation of cells with the putative tumour-initiating phenotype was identified,and when transplanted into NSG mice,gave rise to metastases.At present,there are no relevant reports in NSCLC.It has been indicated that aberrant over expression of CXCR4 is associated with worse overall survival,adenocarcinoma histology,distant metastasis,lymph node involvement inNSCLC.We recently found that CXCR4-positive CTCs are more stem-like than other types of CTCs in NSCLC patients.Besides,the up-regulation of CXCR4 is functionally crucial for maintenance of sternness in drug-resistant NSCLC cells.Moreover,CXCR4 promotes EMT by activationof the Wnt/?-catenin signalling pathway in colorectal cancer and there is a crosstalk existed among the TGF-? and CXCR4 pathways in liver tumor.These studies suggest CXCR4 play an important role in the processes of EMT and forming CSCs.To explore the clinical value of CTCs classified with EpCAM in NSCLC.We collected peripheral blood samples(each 7.5ml)from 47 NSCLC patients,and blood samples were collected at the end of each periodin 3 index patients for longitudinal monitoring of CTCs.CTCs were enriched by the depletion of leucocytes using a magnetic bead separation technique,stained by EpCAM,cytokeratin 7/8and their isotypic control antibodies,respectively,and then identified and counted by multi-parameter flow cytometry.CTCs with EpCAM expression were detected in 9 of 14(64.3%,stage ?-?),4 of 10(40.0%,stage ?)and 10 of 23(43.5%,Stage ?)patients,and CTCs without EpCAM expression were detected in 11 of 14(78.6%,stage ?-?),9 of 10(90.0%,stage ?)and 21 of 23(91.3%,Stage ?)patients.These two types of CTCs were detected to be significantly different in stage ?(P=0.001).The number of CTCs without EpCAM expression was significantly higher than that of CTCs with EpCAM expression in all stages(P values were 0.010,0.001 and<0.001 for stage ?-?,? and ?,respectively).We calculated the percentage of the CTCs without EpCAM expression in both types of CTCs,and found that the frequency of the patients with the percentage more than 90%was significantly higher in stage ? than that in stage ?-?(P=0.030),while the frequency of the patients with that between 50?90%was significantly lower in stage IV than that in stage ?-?(P=0.001).The results of longitudinal monitoring of CTCs revealed that there would be invalid during treatment if the percentage of the CTCs without EpCAM expression more than 50%(P=0.033).High expression of Chemokine receptor 4(CXCR4)is important in tumor invasion,metastasis,drug-resistance and maintenance of sternness in non-small cell lung cancer(NSCLC).We therefore studied the molecular characteristics of drug-resistantCXCR4 positive cells on epithelial-mesenchymal transition(EMT)for the future identification of the tumor cells with the properties of both EMT and sternness.EMT RT2 Profier PCR Array was performed to determine the expression levels of mRNAgenes in A549 with TGF-?1 induced EMT(A549/TGF-(31)and gefitinib-resistant CXCR4 positive cells(A549/GR).TCGA database on the cBio Cancer GenomicsPortal website and Gene Network Central(GNC)Pro Tutorial were used to analyzetheir clinical relevance and pathway interactions.CXCR4 up-regulated both in TGF-? induced EMT and gefitinib-resistant cells.In 84 mRNA genes related to EMT,17 mRNA genes up-regulated in CXCR4 positive population of A549/GR whencompared those in CXCR4 negative fraction,while 66 mRNA genes up-regulated during TGF-? induced EMT.ITGA5,BMP7,MMP3,VIM,RGS2,ZEB2,TCF3,SNAI2,VCAN,PLEK2,WNT5A,COL3A1,SPARC and FOXC2 were double up-regulated in the two type of cells.Kaplan-Meier analysis indicated that the double up-regulated ITGA5,RGS2,SNAI2 and PLEK2 mRNA genes were related to pooroverall survival in lung adenocarcinoma patients(P=0.0000,0.0090,0.0000 and0.0013,respectively).In GNC analysis,SNAI2 mRNA gene was dependent on thesignaling pathway of CXCR4 but ITGA5,RGS2 and PLEK2 mRNA genes were relatively independent on that.In conclusion,classify CTCs by EpCAM is useful in evaluating the distant metastasis and treatment effect of NSCLC patients.Through analysis the molecular characteristics of EMT in drug-resistant CXCR4-positive cells,a series of candidate biomarkers with clinical value were obtained,which could be applied to characterize the tumor-initiating CTCs subpopulations in NSCLC.