Effects Of Activin A On The Migration In Vitro And Metastasis In Vivo Of Mouse Breast Cancer 4T1 Cells

Breast cancer is one of the most common tumors that threaten women’s health.The main cause of death in breast cancer patients is tumor metastasis.Malignant tumor metastasis involves multiple links,such as primary cancer growth,tumor cell shedding,invasion of stroma or vascular system,formation of tumor thrombus,and localization and growth of secondary tissues and organs.Triple-negative breast cancer(TNBC)is a subtype of breast cancer with high metastasis,invasion and recurrence.About 10%~20%of breast cancer cases belong to the triple-negative phenotype.Therefore,the control of triple-negative breast cancer cell metastasis is the key to its treatment.Activin A(Act A)is a member of the transforming growth factorβ(TGF-β)superfamily and is involved in the previous study of the research group found that activin A can promote the migration of MDA-MB-231 cells in human triple-negative breast cancer cell line,but the effect of activin A on the growth and metastasis of triple-negative breast cancer in vivo has not been clarified.he regulation of proliferation,apoptosis,and migration of various tumor cells.Therefore,in this study,we selected mouse triple negative breast cancer cell line 4T1 cells and studied the effect of activin A on migration in vitro and metastasis in vivo of 4T1 cells in an orthotopic breast cancer transplanted tumor model mouse,providing a research basis for further searching for therapeutic targets to regulate breast cancer cell metastasis.1.METHODS(1)ELISA was used to detect the level of activin A in 4T1 cell culture supernatant.(2)Real-time cell analysis(RTCA)technology was used to detect the proliferation and adhesion of 4T1 cells.(3)Flow cytometry was used to detect 4T1 cell apoptosis.(4)Scratch migration assay was used to detect the wound healing ability of 4T1cells in vitro.(5)Microfluidic chip technology was used to detect the migration ability of 4T1cells in vitro.(6)Western blotting was used to detect the expression levels of EMT-related proteins(E-Cadherin,N-Cadherin,Vimentin andα-SAM)and activin signaling proteins(Smad3,p-Smad3,ERK,p-ERK,AKT,p-AKT and P38).(7)When the tumor volume reached 60 mm~3,the mice were randomly divided into the activin A treated group(ACT group)and the normal saline group(NS group).The ACT group was treated daily with 25μL of activin A(2μg/mL)for seven consecutive days by tail vein injection,and 25μL of normal saline as control.The mice were sacrificed day14 after the administration.The tumor,liver and lung of the mice were taken out,and then used for H&E.staining and detection of the tumor growth and metastasis in vivo.2.RESULT(1)Activin A was present in the 4T1 cell culture supernatant and increased with increasing culture time.(2)Activin A promotes 4T1 cell viability and inhibits 4T1 cell apoptosis.(3)Activin A promotes wound healing and cell adhesion of 4T1 cells.(4)The morphology of 4T1 cells cultured with activin A was significantly increased and irregular.(5)Activin A promoted the expression of interstitial marker proteins N-cadherin and Vimentin in 4T1 cells.(6)Activin A had no significant effect on the levels of activin classical signaling pathway proteins Smad3 and p-Smad3 in 4T1 cells,but promoted the increase of non-classical pathway related proteins p-ERK,P38 and p-AKT.(7)Activin A increase the tumor weight,tumor volume and decrease the survival rate significantly,compared with the normal saline group.3.CONCLUSIONIn summary,mouse breast cancer cell line 4T1 cells can secrete activin A,which promotes 4T1 cell viability,inhibits 4T1 cell apoptosis,promotes 4T1 cell migration in vitro and EMT.Activin A may regulate 4T1 cell activity through ERK/MAPK and AKT signaling pathways,rather than Smad-dependent signaling pathway.Further more,activin A can promote 4T1 cell tumor growth and tumor metastasis in vivo,and can reduce the survival time of tumor-bearing mice.Therefore,activin A secreted by triple-negative breast cancer cells may be essential for tumor metastasis and growth.It may be considered a therapeutic target in metastasis of triple-negative breast cancer.