Research On The Effect And Mechanism Of Myeloma Cell-derived Microvesicles On Tumor Growth And Angiogenesis In Tumor-bearing Mice

Objective: At present,effective clinical treatment strategies for multiple myeloma include radiotherapy and chemotherapy,autologous/ allogeneic stem cell transplantation and targeted drug therapy,etc.Despitethe use of ortezomib and lenalidomide as the representative of the new drug recently.The combination of transplantation programs significantly prolonged the survival of patients,but almost all patients still face disease progression and resistance.There have been many literatures that microvesicles derived from tumor cells play an important role in tumor growth,angiogenesis,immune escape,tumor metastasis and tumor resistance.The preoperative study of the myeloma cells derived from ZNF224 protein also showed that angiogenesis of endothelial cells in the myeloma cells under the condition of serum starvation stress was significantly enhanced.However,these are the results of in vitro experiments,the complexiy of the environment in the bodywhether the role of the environment and the impact of the various components play a role is not clear.This study intends to establish multiple myeloma tumor bearing mice model,and to study the effect and mechanism of myeloma cell-derived microvesicles on tumor growth and angiogenesis in tumor-bearing mice.Methods:RPMI8226 cells was inoculated on the out side of right upper limb of NOD/SCID mice to construct MM bearing mice..The mice were randomly divided into 2 groups when the tumor volume reached at 300mm3,namely the control group and the experimental group,which received tail vein injection of either PBS buffer or PKH26 staining myeloma cell-derived microvesicles70 ug respectively-every other day for 5 times.Survial status and diet of mice were observed and recorded and thevolume of transplanted tumor was measured every other day,drawing the volume change curve of the transplanted tumor.The transplanted tumor tissue samples were harvested at day 10,and every organ and sample were collected.Immunofluorescent staining was used to detect expression of endot helial cells marker CD31 in transplanted tumor tissue in order to compare tumor angiogenesis in transplanted tumor of mice in different groups.Frozen sections were made from transplanted tumor tissues and organs,fluorescent microscopy was used to detect and compare the distribution of microvesicles after PKH26 staining in tumor bearing mice in transplanted tumor and different organs through fluorescence intensity.Western blot was used to detect expression of NF-κB-pp65 and also the protein and mRNA level of a downstream target gene VEGF.Results:1 The distribution of MVs in vivo: RPMI8226 cells 3×107 was inoculated subcutaneously in the right upper limb of the NOD/SCID mice.Transplanted tumor was formed twenty days after inoculation of myeloma cells,and the tumor bearing mice model of MM was constructed sucessfully.Mice were divided into two groups randomly when the transplanted tumor volume reached 300mm3,designed as 0 days.At the time of day10,all the mice were killed to finish the observation,harvest all the different tissue samples including the transplanted tumor.PKH26 fluorescent dye labeled caudal vein injection of myeloma MVs,Detection of MVs distribution in tissue sections by fluorescent microscope showed the signal in liver(121.14±42.75)and spleen(101.63±18.92)were the highest followed by that of transplanted tumor(63.63±28.03)and Bone marrow(60.12±12.03),indicate its distribution is selective.2 The effect of injection myeloma MVs on the growth of transplanted tumor.in the multiple myeloma mice : the growth curve of transplanted tumor indicate that caudal vein injection of myeloma MVs may promotes the growth of transplanted tumor,the proliferation rate of mice in the experimental group was significantly higher than that in the control group(P<0.05).3 Immunofluorescent staining was used to detect expression of endot helial cells marker CD31 in transplanted tumor tissue in order to compare tumor angiogenesis in transplanted tumor of mice in different groups,and endothelial cell marker CD31 in the experimental group(100.14±1.13)showed higher expression in transplanted tumor tissue than that of the control group(88.84±2.7),the difference was statistically significant(P<0.01),which indicates caudal vein injection of myeloma MVs significantly promotes tumor angiogenesis growth in the transplanted tumor.4 The NF-κB pp65 and VEGF by western blot,and the results shows the expression of NF-κB pp65 in the transplanted tumor was significantly up-regulated in experimental group(2.01±0.06)than that of control group(1.40±0.19)(P<0.01),accordingly,VEGF protein(1.67±0.08)and mRNA levels(1.97±0.11)in the tumor tissues were elevated in the experimental group compares with VEGF protein(0.94±0.03)and mRNA levels(1.04±0.058)in the control group,the difference was statistically significant(P<0.01).Conclusion:Caudal vein injection of myeloma cell derived MVs selectively retained in the tumor tissue,significantly promots tumor or angiogenesis and transplanted tumor growth in MM tumor bearing mice through activated NF-κB singal pathway.