Effects Of Tumor-associated Bone Marrow Microenvironment Re-modeling On Metastatic Breast Cancer Cells

Objective: To investigate: 1.The effects of high molecular weight HA(HMW-HA)on breast cancer cell dormancy;2.Abnormal changes of HA in tumor-associated bone marrow microenvironment and regulation to breast cancer cell growth.Methods: First,the effect of original HMW-HA on the growth of breast cancer cells.MDA-MB-231,MDA-MB-231 BO,4T1 and MCF7 breast cancer cells were selected.After HMW-HA treatment: 1.CCK-8 method to detect growth,immunofluorescence and flow cytometry method to analyze the expression of Ki-67;2.flow cytometry method to detect G0 / G1 phase of cell cycle;3.western blot and flow cytometry method to observe the levels of P38,ERK1/2,Cyclin D1 and P21cip1;4.Immunofluorescence method to detect the expression of Ki-67 and P21cip1 in 3D cell culture.Second,the effect of metabolically altered HA on the growth of breast cancer cells: 1.bone marrow stromal cells HS5 and MDA-MB-231 BO cells co-culture;2.chemiluminescence method to detect HA content;3.through 4-MU,detection the correlation between HA and the growth of MDA-MB-231 BO cell;4.si RNA HAS2 decreased HA level of HS5 cells,observed the growth of MDA-MB-231 BO cells,determined the source of HA in co-culture;5.sh RNA down-regulated the CD44 expression of MDA-MB-231 BO cells,observed the interaction of HA-CD44;6.Flow cytometry method to sort MDA-MB-231 BO cells,western blot method was used to detect the expression of PI3 K,Cyclin D1 and CDK4;7.Agarose gel electrophoresis was used to detect the molecular weight of HA.Third,in vivo experiments: nude mice model 1.Left ventricular injection,observed MDA-MB-231 BO cell metastatic bone destruction;2.Before and after CRISPR knockout CD44 of MDA-MB-231 BO cells.Tibia injection to observe the interaction of HA-CD44.Results: First,original HMW-HA: 1.Inhibited the growth of MDA-MB-231,MDA-MB-231 BO and 4T1 cells,maintain the slow growth state of MCF7 cells;2.Extended MDA-MB-231 BO,MDA-MB-231 and 4T1 cell cycle G0/G1 phase;3.Reduced the expression level of Ki-67 in MDA-MB-231 BO cells;4.Increased the ratio of P-P38/P-ERK1/2 in MDA-MB-231 BO cells;5.decreased the expression of Cyclin D1 and elevated the level of P21cip1;6.Inhibited the expression of Ki-67 and elevated the level of P21cip1 in 3D cell culture environment.Second: 1.MDA-MB-231 BO cells undergo osteolytic bone destruction;2.HS5 cells promoted MDA-MB-231 BO cell growth through PI3 K,Cyclin D1 and CDK4;3.The HA level of cell co-culture was significantly higher than that of HS5 and MA-MB-231 BO cells alone;4.4-MU inhibited the growth-promoting effect of cell co-culture environment HA on MDA-MB-231 BO cells;5.HA mainly came from HS5 cells;6.In vivo and in vitro experiments,HA promoted the growth of MDA-MB-231 BO cells through CD44;7.HA in the cell co-culture supernatant was composed of large,medium and small molecular weight.Conclusion: 1.HMW-HA regulated the cell cycle of breast cancer cells and mediated the growth inhibition of breast cancer cells.2.Tumor-associated bone marrow stromal cells microenvironment HA was a mixed molecular weight,and HA level was abnormally increased,suggesting that medium and small molecular weight HA provide a microenvironment for growth of bone metastatic breast cancer cells.To breast cancer cell bone metastasis treatment,HA re-modeling may be a new target.