The Role Of Arginase Ⅰ In Breast Cancer Bone Metastasis And Its Mechanism

Breast cancer is the most common malignant tumor in women.65%-75% of advanced breast cancer patients will develop bone metastases,causing bone pain,pathological fractures,spinal cord compression and other skeletal-related events(SREs).The bone metastasis process of breast cancer and its mechanism are extremely complicated,and there is no clinically effective treatment method.Therefore,there is an urgent need to find a target to prevent and treat bone metastasis of breast cancer.Arginase I(Arginase I,Arg1),released by tumor-associated bone marrow cells in the tumor microenvironment,is an enzyme that can catalyze the hydrolysis of L-arginine into L-ornithine and urea.The expression level of Arg1 in breast cancer patients’ tumors and tumor-draining lymph nodes far exceeds the level in the blood.Even the early breast cancer patients also show changes in tumor-related Arg1 expression.Studies have shown that knocking out Arg1 in breast cancer cells MDA-MB-231 can significantly reduce tumor cell invasion,intravascular infiltration and spontaneous lung metastasis.However,the role of Arg1 in bone metastasis has not been reported yet.In this study,we aimed to explore the role of Arg1 in mediating the biological process of breast cancer bone metastasis and explore its potential mechanism,so as to provide theoretical basis and new ideas for the clinical treatment of breast cancer bone metastasis.This research is roughly carried out from the following aspects:1.Construction of bone metastasis cell line 4T1-luc-BM3.In this experiment,a highly metastatic mouse breast cancer cell line 4T1 was selected,and luciferase was transfected with lentivirus to construct a mouse breast cancer cell line 4T1-luc.We used intracardiac injection to inject100 ul of 4T1-luc cells(3000 cells/mouse)into the left ventricle of healthy female Balb/c mice,and used intravital imaging technology to evaluate the modeling situation 10-14 days after the injection.Subsequently,the mice were sacrificed,and the bone metastases of the mice were resected and the primary tumor cells were extracted.After cultured in vitro for 4-5 weeks,the generated cells were injected intracardiac again in new Balb/c mice.The above procedure was repeated three times to construct the mouse breast cancer bone metastasis strain 4T1-luc-BM3,which had experienced three bone metastases and had a tendency of bone metastasis.2.The expression of Arg1 in breast cancer bone metastasis.We injected 4T1-Luc-BM3 into the left ventricle of mice 14 days later to obtain tissue from the bone metastases.Histologically,HE staining showed that there was tumor cell infiltration in the bone marrow of the bone metastasis,and the results of immunohistochemical showed that Arg1 was positive in the mouse breast cancer bone metastasis samples.Meanwhile,we killed normal healthy female Balb/c mice and extracted normal femur tissue.RNA-seq analysis of bone metastasis tissue(bone metastasis + bone metastasis adjacent to cancer)and normal femur tissue showed that Arg1 was low expressed in normal femur tissue and high expressed in bone metastasis(normal femur vs bone metastasis: 5+1.732 vs 1708+1196,P<0.001).Respectively using q PCR and Western blot techniques to detect the expression of Arg1 in mouse bone marrow mesenchymal stem cell line BMSCs,mouse macrophage cell line Raw264.7,mouse breast cancer cell line 4T1-luc,and bone metastasis line 4T1-luc-BM3 from m RNA and protein levels,we found that Arg1 was highly expressed in 4T1-luc-BM3,4T1-luc-BM3 and Raw264.7(BMSCs vs 4T1-luc,P <0.001;BMSCs vs4T1-luc-BM3,P <0.001;BMSCs vs Raw264.7,P<0.001).Compared with breast cancer cell strains,the expression of Arg1 in bone metastasis cell strains was significantly increased(4T1-luc vs 4T1-luc-BM3,P<0.05).3.The study of Arg1 inhibitor(S)-(2-boronethyl)-L-cysteine HCl(BEC)on the proliferation and migration of bone metastasis cell strain4T1-luc-BM3 in vitro.CCK8 detection method and transwell experiment were used to study the effect of BEC on the proliferation and migration of mouse breast cell bone metastasis strain 4T1-luc-BM3 in vitro.The results showed that the BEC group(100u M)could inhibit the migration of4T1-luc-BM3 cells in vitro(control group vs.BEC group: 38.80+5.718 vs4.20+2.28,P<0.001),but did not affect their proliferation(P> 0.05).4.The in vivo study of Arg1 inhibitor BEC on the bone metastasis of breast cancer.We randomly divided 18 healthy female Balb/c mice into two groups: PBS control group and BEC treatment group.Both groups of mice were injected with 100 ul 4T1-luc cells(3000 cells/mouse)into the left ventricle for modeling.The BEC solution was prepared with PBS.The BEC treatment group was injected intraperitoneally with 100 ul BEC solution(300u M)immediately after modeling,and the PBS control group was injected intraperitoneally with 100 ul PBS solution immediately after modeling.The injection frequency was once every two days.After twelve days,we used in vivo imaging technology to evaluate the bone metastasis of the control group and the BEC group,and used 3D micro-computer tomography(u CT)to evaluate the bone destruction.The results showed that the luciferase signal of Balb/c mice in the BEC group was significantly reduced(control group vs.BEC group,P<0.001).Compared with the femur of normal mice,the control group and the BEC group intracardiacally injected with tumor showed different degrees of bone destruction.The bone destruction of the BEC group was generally milder than the control group.5.The potential effect and mechanism of Arg1 inhibitor BEC on the biological process of Arg1 mediated breast cancer bone metastasis.We co-cultured 4T1-luc-BM3 and RAW264.7 in transwell chamber,treated4T1-luc-BM3 with 100 u M BEC,and observed the in vitro migration of4T1-luc-BM3 and Raw264.7 cells in 24 hours,and explored the potential connection between BEC and RAW264.7.Transwell co-culture experiments found that BEC could significantly inhibit the promotion of4T1-Luc-BM3 cell migration in RAW264.7,which may be related to the inhibition of the migration of Raw264.7.The above results indicate that Arg1 is highly expressed in breast cancer and its expression is higher in bone metastasis tissues.The Arg1 inhibitor BEC contributes to inhibit the growth of breast cancer bone metastases in vivo,and reduce the bone destruction of bone metastases,and delay the progression of breast cancer bone metastases.BEC may inhibit the migration of 4T1-luc-BM3 cells in vitro by inhibiting the migration of macrophages Raw264.7.The mechanism may be related to the inhibition of the migration of Raw264.7.