CCR9 Promotes Migration And Invasion Of Lung Adenocarcinoma Cancer Stem Cells

Background and Objective Lung cancer is the most common cancer in humans,and its morbidity and mortality rank first among all cancers.Non-small cell lung cancer(NSCLC)accounts for about 83% of lung cancer.Lung adenocarcinoma is the most common subtype of NSCLC and also one of the most serious life-threatening diseases.Its morbidity is increasing year by year.Chemokine receptor 9(CCR9)is the only specific receptor for chemokine CCL25(CC chemokine ligand 25,CCL25).Increased expression of CCR9 have been detected in various solid tumors.It plays an important role in cancer development and metastasis.A lot of researches confirm that cancer stem cells are the source of tumor metastasis and recurrence.ALDH1A1(Aldehyde dehydrogena 1 family,member A1)is a member of the ALDH(Aldehyde dehydrogenase)superfamily.It is a biomarker of CSCs(Cancer stem cells).The expression of CCR9 in lung adenocarcinoma tissue is significantly increased.The role and mechanism of CCR9 in promoting distant metastasis of lung adenocarcinoma are still unknown.In order to clarify its role and potential mechanism,we studied the correlation between CCR9 and ALDHhigh CSCs,and the effect of CCR9/CCL25 signal transduction pathway on the migration and invasion of lung adenocarcinoma CSCs.Methods 1.The expression levels of CCR9 and ALDH1A1 in lung adenocarcinoma tissues were detected by clinical tissue samples,and their correlations with clinicopathological parameters and prognosis of lung adenocarcinoma were studied.The pathological paraffin sections of 76 lung adenocarcinoma patients diagnosed by pathology after operation in Guangzhou First People’s Hospital from 2007 to 2016.The expression levels of CCR9 and ALDH1A1 in patients’ tumor tissues were detected byimmunohistochemistry.According to the immunohistochemical score,the patients were divided into high expression group and low expression group.The relationship between the expression level of CCR9 and ALDH1A1 and the clinicopathological parameters of lung adenocarcinoma was studied,and the correlation between them and distant metastasis was studied.Kaplan-Meier survival curve was used to analyze the effect of CCR9 and ALDH1A1 on overall survival.Cox proportional hazards regression model was used to analyze the influencing factors of patients’ survival prognosis.2.Differential expression of CCR9 in lung adenocarcinoma ALDHhigh cells(ALDHhigh CSCs)and ALDHlow cells.ALDFLUOR kit and flow cytometry were used to sort out ALDHhigh cell subsets and ALDHlow cell subsets.Flow cytometry were used to detect the expression of CCR9 on ALDHhigh cells and ALDHlow cells.The real-time quantitative PCR were used to verify the difference expression of CCR9 m RNA between ALDHhigh cells and ALDHlow cells.3.The effect of CCR9/CCL25 signal transduction pathway on the migration and invasion of lung adenocarcinoma cells Transwell assay was conducted to study the effects of exogenous CCL25 and anti-CCR9 on migration and invasion of lung adenocarcinoma CSCs.Results 1.There are differences of CCR9 expression between lung adenocarcinoma patients with distant metastasis or not.The expression of CCR9 was positively correlated with distant metastasis.Patients with high CCR9 expression were more likely to have distant metastasis.The overall survival of patients with high CCR9 expression was lower than that of patients with low CCR9 expression.2.There are differences of ALDH1A1 expression between lung adenocarcinoma patients with distant metastasis or not.The expression of ALDH1A1 was positively correlated with distant metastasis.Patients with high ALDH1A1 expression were more likely to have distant metastasis.The overall survival of patients with high ALDH1A1 expression was lower than that of patients with ALDH1A1.3.Univariate analysis showed that CCR9 expression,ALDH1A1 expression,metastasis and TNM staging could predict the survival of lung adenocarcinoma patients.Multivariate analysis showed that ALDH1A1 expression and distant metastasis were independent risk factors affecting overall survival of patients with lung adenocarcinoma.4.In the same tumor microenvironment,the expression of CCR9 and ALDH1A1 waspositively correlated.Compared with ALDHlow cells,the expression of CCR9 in ALDHhigh cells was enhanced.Further transwell assays demonstrated that the numbers of CSCs migrated or invaded in response to CCL25 were more than that without CCL25 stimulation.Additional application of anti-CCR9 reversed the CCL25-induced migration and invasion of CSCs.Conclusion High expression of CCR9 and ALDH1A1 in lung adenocarcinoma is closely related to distant metastasis and poor prognosis.ALDH1A1 high expression and distant metastasis are independent risk factors affecting the overall survival of lung adenocarcinoma.The expression of CCR9 and ALDH1A1 in lung adenocarcinoma are positively correlated.The expression of CCR9 in ALDHhigh CSCs is significantly increased.CCL25/CCR9 can significantly promote the migration and invasion of CSCs.Through this study,we have revealed a new mechanism of distant metastasis and poor prognosis of lung adenocarcinoma,and also provide evidence for CCR9/CCL25 as a new target for the treatment of lung adenocarcinoma.