Study On Formation And Immune Evasion Of Invasive Circulating Tumor Cells Promoted By BCAR1

Background and ObjectiveCurrently,lung cancer（LC）is the top cancer killer,and surgery is still the most important treatment for early lung adenocarcinoma（LUAD）.However,there are still numerous patients with postoperative recurrence and metastasis.Circulating tumor cells（CTCs）are shed from the primary sites and have the ability to develop distant metastasis.Studies have found that CTCs with epithelial-mesenchymal transition（EMT）are more prone to metastasis and invasion.Moreover,the expression of programmed death-ligand 1（PD-L1/CD274）in CTCs can help them escape the killing of T cells and survive in peripheral blood.However,the underlying mechanisms regarding the trigger of EMT process and CD274 expression in CTCs remain unclear and need further exploration.Our research found that Breast cancer antiestrogen resistance protein 1（BCAR1）,namely p130cas,is highly expressed in CTCs showing an potentially important biological role.Intriguingly,it has been proved that BCAR1 can promote EMT through various signaling pathways.In addition,our previous study found that bromodomain-containing protein 4（BRD4）,which is the transcription promoter of CD274,may interact with BCAR1.Therefore,we speculate that the high expression of BCAR1 in CTCs enhances EMT process to generate highly invasive CTCs.Meanwhile,BCAR1 can up-regulate the expression of CD274 through BRD4,to develop immune evasion of CTCs.To verify our speculation,this study focused on the prognostic value of BCAR1expression in CTCs in LUAD patients,the relationship between BCAR1 expression and the EMT process and CD274 co-expression in CTCs,to prove that BCAR1 promotes the formation of invasive circulating tumor cells and immune evasion.Study Methods1.Canpatrol^TM was used to detect CTCs and and the expressions of associating markers including EMT BCAR1 and CD274.Cytoplo Rare was used as validation.Immunohistochemistry（IHC）and Western blot（WB）are used to detect the expressions of BCAR1,CD274 and BRD4 in LC tissues.2.BCAR1-Knock Out（KO）in H1975 and H1299 cells and BCAR1-Over Expression（OE）in A549 cells were constructed.The expression of CD274 was detected by and WB,Quantitative-Polymerase chain reaction（Q-PCR）,respectively.BRD4 expression in total protein and nucleus in LUAD tissues or cells was detected by WB.Co-immunoprecipitation（CO-IP）was used to analyze the interaction between BCAR1and BRD4,as well as BCAR1 and CD274.3.CCK8 cell proliferation and clonony formation experiments were conducted.The subcutaneous tumor xenograft animal models by BCAR1-KO cells were constructed.4.ESTIMATE was used to calculate the immune score of LUAD tissues of The Cancer Genome Atlas（TCGA）.The relationship between BCAR1 expression and immune cells infiltration was analyzed by TIMER.5.COX model was used for survival analysis.Study Results1.Both Canpatrol^TM and Cytoplo Rare confirmed that the levels of BCAR1-positive CTCs in LUAD cases were significantly higher than those in healthy controls.Patients with highly expressed BCAR1 in CTCs had a poor prognosis.BCAR1 was more likely to be expressed in CTCs with both epithelial and mesenchymal markers.BCAR1 and CD274expression in CTCs was significantly correlated.2.The expression of CD274 m RNA and protein decreased after BCAR1-KO,while CD274 m RNA expression increased significantly after BCAR1-OE.CO-IP confirmed the protein interaction between BCAR1 and BRD4,rather than between BCAR1 and CD274.The BRD4 in nucleus can be divided into subtypes long（BRD4-L）and short（BRD4-S）.The expression of BRD4-L and BRD4-S was increased and decreased respectively after BCAR1-KO.And vice versa after BCAR1-OE.In LUAD tissues,high and low expression of BRD4-S and BRD4-L is associated with high CD274 expression.3.BCAR1-KO and-OE inhibited and promoted cell proliferation,as well as clone formation,respectively.Subcutaneous tumor xenograft was significantly inhibited after BCAR1-KO.4.The high expression of BCAR1 in LUAD tissues is correlated to a low immune score.BCAR1 expression was significantly positively correlated with the number of CD4+T cells,but negatively correlated with the number of CD8+T cells.And patients with high BCAR1expression and low immune score were most frequently in stage III&IV,with the worst prognosis.Study Conclusions1.BCAR1 expression promotes EMT of CTCs,leading to characteristics of mesenchymal cells and more invasiveness.2.In CTCs,BCAR1 may up-regulate the expression of CD274 by BRD4 transformation,leading to immune evasion of CTCs in the peripheral blood.3.BCAR1 play an important role in cell proliferation and growth,and inhibitition of immune cells infiltration.4.Preoperative detection of CTCs and BCAR1 expression can predict the high risk of recurrence and metastasis in patients with early LC.