Clinical Study On The Correlation Of Peripheral Blood Circulating Tumor Cells And Their Chemokine Receptor CCR9 With Non-small Cell Lung Cancer

BackgroundMalignant tumor is the global public health problem which threates the human health from past to present.The World Health Organization reported that in 2018,there were 18.1 million newly increased malignant tumor cases in the world,and around 9.6 million people died from malignant tumors.The newly research report"Global Cancer Statistics 2018"declared that lung cancer was still the world’s leading malignancy with the incidence rate of 11.6%and the mortality rate of 18.4%.In China,lung cancer is also at the top of malignant tumors with the highest morbidity and mortality.There are about 80～85%non-small-cell lung cancer（NSCLC）of lung cancer cases.Circulating tumor cells（CTCs）detection is the"liquid biopsy"technology which has attracted much people,s attention,and it has been proved to have a positive clinical application value in the diagnosis,treatment and prognosis of lung cancer,breast cancer,colorectal cancer,prostate cancer and the other tumors.The chemokine receptor is a G protein coupled receptor with seven transmembrane structures.According to the different binding ligands,chemokine receptors are divided into four subfamilies:CCR,CXCR,XCR and CX3CR.Chemokines（CKs）and their specific receptors participate in a great diversity of pathological and physiological processes,such as adhesion,proliferation,anti-apoptosis and angiogenesis of various tumor cells.As a regulatory medium,chemokines and their receptors play the role in the formation,invasion and metastasis of tumors,and they are related to the poor prognosis of malignant tumours.The previous research had found that chemokine receptor-9（CCR9）could regulate the expression levels of matrix metalloproteinase（MMP2,MMP9）and tissue-specific inhibitors（TIMP-1,TIMP-2）of lung cancer cells through the CCR9-CCL25 axis and promote the invasion and metastasis of lung cancer cells,CCR9 was related to the prognosis of NSCLC patients.However,the research on the CCR9 expression levels of CTCs was still in the deep sea.Our study plans to investigate the clinical relevance of CTCs and NSCLC,the correlation between the CCR9 of CTCs and NSCLC,and the clinical value and mechanism of CTCs and CCR9 in NSCLC tumor cells metastasis.Methods1.A total of 62 NSCLC patients were admitted to our study from May 2018 to June 2019,all the cases were from the Clinical Oncology Center of the People’s Hospital of Guangxi Zhuang Autonomous Region.Firstly,we used the Canpatrol ^TMCTC technique to test the expression level of CTCs and CCR9 of CTCs in peripheral blood of patients,after that we analyzed the relationship between CTCs and the clinical and pathological characteristics of NSCLC patients,the correlation of CCR9 expression levels and the clinical and pathological characteristics of NSCLC patients.Besides,we also explored the clinical correlation of CTCs and CCR9 and NSCLC in the end.2.We constructed the lentivirus LV-CCR9 with overexpressing CCR9 and the negative control virus CON335.Then we used the lentivirus LV-CCR9 and negative control virus CON335 to infect the human lung adenocarcinoma cells,incluing A549 cells and PC-9 cells,in order to regulate the CCR9 expression of lung cancer cells.In our shudy,we set the OE-A549 and OE-PC9 which were infected with the LV-CCR9 as the experimental group,and the expression of CCR9 was up-regulated in the experimental group.The control group（NC-A549and NC-PC9）was infected by the negative control virus CON335,and the negative control virus CON335 had no effect on the expression of CCR9.3.Cell function experiments were carried out in the experimental group and the control group to investigate the effect of over-expressed CCR9 on migration and metastasis of A549 cells and PC-9 cells.Results1.The Canpatrol ^TMCTC technology test results showed that CTCs were detected in 56（90.3%）of 62 NSCLC patients.The CTC count was associated with the TNM stage,lymph node metastasis and distant metastasis of NSCLC（P<0.05）.The advanced NSCLC patients had higher CTC count than the early NSCLC patients（6.98±6.28/5m L VS 3.13±2.55/5m L,P<0.05）,the NSCLC patients with distant metastasis had higher CTC count than the non-metastasis NSCLC patients,and the CTC count was higher in the NSCLC patients whose tumor size was T3-T4 stage than the T1-T2 stage（7.08±6.47/5m L VS 3.83±3.39/5m L and 7.62±7.08/5m L VS 4.37±3.60/5m L,P<0.05）.2.In the clinical correlation analysis between CTC subtypes and NSCLC,we founded that the epithelial CTC count was related to the TNM stage and distant metastasis of NSCLC（r=0.296 and r=0.273,P<0.05）.And the epithelial/mesenchymal hybrid CTC count was also connected with NSCLC tumor metastasis（r=0.253,P=0.047）.3.In the clinical correlation analysis of CCR9 expression levels of CTCs and NSCLC,we founded that the positive rate of CCR9 of epithelial/mesenchymal hybrid CTC was associated with the distant metastasis of NSCLC（r=0.353,P=0.038）.4.The results of cell wound healing test and cell transwell migration assay attested that the migration ability of OE-A549 and OE-PC9 cell lines with over-expressed CCR9 was enhanced significantly compared to the control group（NC-A549 and NC-PC9 cell lines）.The in vitro experiments proved that overexpression of CCR9 could promote the migration and metastasis of human lung adenocarcinoma cells（A549 cells and PC-9 cells）.Conclusion1.The CTC count was associated with the TNM stage,lymph node metastasis and distant metastasis of NSCLC.2.The high expression of CCR9 promoted the migration and metastasis of NSCLC in vitro.3.The epithelial/mesenchymal hybrid CTC is the tumor cell with both epithelial and interstitial characteristics.CCR9 may be involved in the tumor cell epithelial-interstitial process（EMT）and the generation of epithelial/mesenchymal hybrid CTC,which may be the mechanism of tumor cells distant metastasis.4.The expression of CCR9 of CTCs is expected to be used as a biomarker to evaluate the risk of tumor metastasis in NSCLC.