Biological Characteristics Of Circulating Breast Tumor Cells And The Survival Mechanism In Circulating

Tumor metastasis is the main cause of tumor related death,the cells detach from primary tumor tissue and the distant metastasis tissue,enter into blood,survive in blood and arrest in the target organ to form metastasis focuses,these cells were named circulating tumor cells(CTCs).Numbers of research have reported that CTCs play an important role in tumor metastasis.Hence,the identification and characteristics of CTCs is vital to investigate the molecule mechanism of tumor metastasis.AimIn our study,we established breast cancer CTCs and melanoma CTCs in mouse model,examined CTCs biological characteristics including:cell proliferation,migrate and invasive capacity,cell chemotherapeutic sensitivity and cell survival ability in vitro,tumor growth and metastasis capacity in vivo,also explored the mechanism of cell survival in circulating,tried to elucidate the mechanism of tumor hematogenous metastasis.Materials and methods1.Two CTCs lines were isolated by Ficoll density gradient centrifugation from orthotopic model of 4T1 breast cancer and experiment lung metastasis model of B16melanoma,named 4T1CTCs and B16 CTCs respectively.CTCs were identified by cell immunohistochemistry assay,RT-PCR and tumorigenic ability.The E-cadherin and Vimentin expression were examined by Western blot and flow cytometry assay.2.MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)proliferation assay and colony formation assay were used to examined cell proliferation and cell colony forming ability;flow cytometry assay analysis cell cycle distribution,cell cycle related protein CDK6 was determined by Western blot,tumor growth capacity was examined in vivo.Cell chemotherapeutic sensitivity was examined by MTT.3.Scratch assay and transwell migrate assay analysis cell migrate ability,transwell invasion assay analysis cell invasive ability.The F-actin distribution was determined by FITC-phalloidin staining and scanned by confocal laser scanning microscopy.Tumor lung metastasis ability was examined in vivo.4.The expression of EMT markers(E-cadherin,Ep CAM and Vimentin)were detected after suspension culture and fluid shear stress treatment.5.Quantitative iTRAQ proteomics was used to analyze the differential proteins in 4T1CTCs,and bioinformatics analysis was performed using GO database,KEGG database and String website.Kaplan-Merier Plotter database(http://kmplot.com/analysis)analyzes the relationship between MT2 and the prognosis of breast cancer patients;String database analyzes the interaction between MT2 and apoptosis and autophagy-related proteins online,and Gene MANIA database analyzes MT2 interactions with proteins related to apoptosis-related signaling pathways.6.Cell anoikis and fluid shear stress resistance ability were examined by flow cytometry,chemotactic peripheral blood mononuclear cells(PBMC)assay was used to examine the chemotactic ability of CTCs to CD8T cells and CD4T cells,and expression of CD47 and PD-L1 on CTCs were detected by flow cytometry.7.Western blot and cell immunohistochemical methods were used to detect MT2expression level in CTCs.We used sh RNA virus to decrease MT2 proteins,the transfection efficiency of sh RNA-MT2 virus,caspase-3,LC3I and LC3II were determined by Westernblot,and the percentage of PI positive cells was detected by flow cytometry.Results1.Isolation and identification of 4T1CTCs with different EMT phenotypes:We established 4T1 breast cancer orthotopic tumor model for 30 days.After collecting blood through cardiac,Ficoll density gradient centrifugation was used to separate CTCs in the blood,4T1CTCs were isolated from the blood of 18 mice.In this experiment,4T1CTC-1 was used as the representative of epithelial morphology CTCs,and 4T1CTC-2 was used as the representative of mesenchymal morphology CTCs.(1)4T1CTCs were identified by cell markers:The percentage of Ep CAM~+Vimenin~+cells in 4T1CTC-1 was 85.8%,the percentage of Ep CAM~-Vimenin~+cells in 4T1CTC-2 was 69.4%;The epithelial markers including E-cadherin,Ep CAM and CK were highly expressed in 4T1CTC-1(p<0.05,p<0.001),but 4T1CTC-2 expressed low level of above epithelial markers(p<0.001).Consisitent with the above results,4T1CTC-1 cells expressed high E-cadherin and Vimentin(p<0.05),whereas 4T1CTC-2 cells expressed a low level of E-cadherin and a high level of Vimentin(p<0.001),as compared to the 4T1 primary cells.(2)Identification of 4T1CTCs by subcutaneous tumor formation ability:In vivo tumorigenesis experiments show that 4T1CTC-1 and 4T1CTC-2 could form tumors,the nuclei in the tumor tissues formed by 4T1CTC-1 and 4T1CTC-2 were large and deeply stained,and nucleus division increased.Subcutaneous tumorigenesis experiments showed that 4T1CTCs have tumorigenic ability,indicating that4T1CTC-1 and 4T1CTC-2 are tumor-derived cells.2.Isolation and identification of B16CTCs:Cells were isolated from experiment lung metastasis model of B16 melanoma by density gradient centrifugation,and HMB-45 staining was positive in these cells,and these cells have tumorigenic ability.The above results indicated B16CTCs were tumor-derived cells.3.4T1CTCs were resistant to the Epirubicin(p<0.01,p<0.001),B16CTCs were resistant to the cisplatinum(p<0.05,p<0.01,p<0.001).4.CTCs exhibited low proliferation ability in vitro and growth in vivo:For4T1CTCs models,4T1CTCs exhibited low proliferation,colony ability and tumor growth rate compared with 4T1 primary cells(p<0.01,p<0.001),the G1 phase cell distribution in 4T1CTC-1 was higher than 4T1 primary cells(p<0.05),CDK6expression in 4T1CTC-1 was lower than 4T1 primary cells(p<0.001),4T1CTC-2exhibited low proliferation and colony formation ability compared with4T1CTC-1(p<0.05,p<0.01,p<0.001),the tumor growth rate of 4T1CTC-2 group was lower than 4T1CTC-1 significantly(p<0.05).For B16CTCs models,B16CTCs exhibited low proliferation and colony ability compared with B16 parental cells(p<0.05),the G1 phase cell distribution in B16CTCs was higher than B16 parental cells(p<0.05),CDK6 expression in B16CTCs was lower than B16 parental cells(p<0.001),and the tumor growth rate of B16CTCs was lower than B16 parental cells significantly(p<0.05).5.CTCs exhibited low migrate and invasive capacity in vitro,whereas had a capacity for tumor lung metastasis.For 4T1CTCs models,the migrate and invasive ability of 4T1CTCs was lower than 4T1 primary cells(p<0.01,p<0.001),the lung metastasis capacity of 4T1CTCs was lower than 4T1primary cells(p<0.05).4T1CTC-2 showed higher migrative and invasive ability than 4T1CTC-1 in vitro,and4T1CTC-2 exhibited higher lung metastasis than 4T1CTC-1(p<0.01,p<0.001).For B16CTCs models,the migrate and invasive ability of B16CTCs was lower compared with B16 parental cells(p<0.001).6.Suspension culture had no significant effect on the EMT markers(p>0.05),but fluid shear stress decreased epithelial marker including E-cadherin and Ep CAM(p<0.01,p<0.001),increased mesenchymal markers Vimentin(p<0.05).7.We detected 1,483 proteins downregulated and 791 proteins upregulated in4T1CTC-1 cells,compared to the 4T1 primary cells(fold-change<-1.5 or>1.5,p<0.05).In contrast,4T1CTC-2 cells had 179 proteins downregulated and 233upregulated,comparing the 4T1primary cells.4T1CTC-1 cells had 1473 proteins were downregulated and 1006 upregulated,as compared to 4T1CTC-2 cells.GO(biological process)in intermediate CTCs phenotypes is mainly enriched in positive regulation of defense response and positive regulation of innate immune response.GO(biological process)in mesenchymal CTC phenotypes is mainly enriched in epithelial cell differentiation and cellular response to cytokines stimulus.8.CTCs exhibited higher survival ability in circulating:The survival number of4T1CTCs was higher than 4T1 primary cells and 4T1 lung metastasis cells(p<0.05).The anoikis apoptosis assay showed that the apoptosis cells in 4T1CTCs were lower than 4T1 primary cells(p<0.01).Cells were treated by 15dyne/cm~2fluid shear stress,the cell apoptosis rate in 4T1CTCs was lower than 4T1 primary cells(p<0.01).9.CTCs exhibited an immune escaped phenotype:The chemotactic ability of CTCs to CD4T cells was higher than 4T1 primary cells(p<0.05).The chemotactic ability of CTCs to CD8 T cells was lower than 4T1 primary cells and 4T1 lung metastasis cells(p<0.05).The CD47 expression level of 4T1CTCs was higher than4T1 primary cells and 4T1 lung metastasis cells(p<0.001).10.4T1CTCs showed higher basal autophagy level and lower apoptosis level(p<0.05,p<0.01),and inhibiting autophagy level increased apoptosis rate in CTCs(p<0.05).4T1CTCs expressed high levels of MT2(p<0.01),inhibition of MT2decreased the autophagy level of 4T1CTC-1(p<0.05),increased the level of cell apoptosis(p<0.05),and inhibited cell survival ability(p<0.001);Inhibition of MT2combined with inhibition of autophagy significantly inhibited cell survival(p<0.001).Conclusion1.We successfully established B16CTCs and 4T1CTCs.2.4T1CTCs exhibited different EMT phenotypes,suggesting that 4T1 CTCs are heterogeneous.3.CTCs exhibited low proliferation ability in vitro,low tumor growth rate in vivo and resistant to Cisplatinum and Epirubicin,CTCs exhibited low migrate and invasion capacity in vitro,and low lung metastasis ability,but exhibited high survival ability in circulating.4.Fluid shear stress could incuce the EMT of CTCs.5.ITRAQ quantitative proteomics analysis showed that there are significant differences in protein levels as well as the function of enrichment in different EMT phenotype of CTCs.6.CTCs exhibited stronger survival ability in circulating,which may be related to immune escaped phenotype,stronger anoikis and fluid shear stress resistant ability,MT2 played a vital role in mediating CTCs survive through autophagy and apoptosis.