Effect Of Tumor Associated Macrophages On Angiogenesis In Multiple Myeloma Xenograft

Background and objectiveMultiple myeloma(MM)is a malignant proliferative disease of plasma cells common in middle-aged and elderly people,and ranks second among common hematological malignancies.The peak age of MM in china is 65-74 years old.In recent years,the incidence of patients has been increasing year by year.More and more medical researchers are engaged in the research and prevention of MM.Tumor metastasis and recurrence is one of the main causes of death in patients with advanced MM,and it is also a difficult problem in clinical treatment of MM.Studies have shown that the abnormal increase of tumor blood vessels plays an important role in the development and metastasis of tumors.Tumor-associated macrophages(TAMs)are macrophages evolved from peripheral blood mononuclear cells infiltrating into solid tumor tissues,and are an important part of tumor stromal cells.The infiltration of TAMs is closely related to tumor cell proliferation,migration,drug resistance and tumor angiogenesis.TAMs are mainly divided into two subtypes:classically activated M1 type TAMs and alternatively activated M2 type TAMs.Generally,M1 type TAMs is considered to promote inflammation and anti-tumor effects,and M2 type TAMs is considered to promote tumor occurrence and development,and can participate in the process of angiogenesis and cancer cell metastasis.Therefore,exploring the role of different subtypes of TAMs in the growth of MM transplanted tumors is of great significance for studying the impact of TAMs on MM patients.Removal of macrophages is an important method to study their function in the body.Clodronate liposomes is a highly effective macrophage scavenger,which has been widely used in tumor research in recent years.Vascular endothelial growth factor(VEGF)family proteins include VEGFA,VEGFB,VEGFC and other subtypes.Among them,VEGFA has the closest relationship with angiogenesis,and it can promote the proliferation and metastasis of vascular endothelial cells,thereby regulating angiogenesis.It has been reported that simply knocking down the expression of VEGFA in MM cells cannot significantly reduce angiogenesis.Therefore,we speculate that other cells or factors in the tumor microenvironment(TME)also participate in and significantly affect the angiogenesis process.In our previous in vitro experiments,M1 and M2 TAMs have opposite effects in the process of angiogenesis,and MM cells and M2 TAMs can synergistically promote tumor angiogenesis.Compared with in vitro experiments,it is not yet known whether ideal conclusions can be drawn in animal models with complex physiological and biochemical environments.In this study,we aimed to investigate the effects of simultaneously silencing VEGFA expression while clearing TAMs and reinfusing different subtypes of TAMs,respectively,on graft tumor growth and angiogenesis in mm nude mice,and we hope that this study will provide a new approach for the treatment of mm and provide a theoretical basis and experimental basis for clinically blocking the infiltration,metastasis,and improving the therapeutic efficacy and prognosis of patients with MM and other malignancies.Methods1.Induction and characterization of M1/M2 type TAMs in vitroRAW264.7 cells were cultured in complete medium containing 100ng/ml LPS and 50ng/ml IFN-γfor 48 h to obtain M1 macrophages,and in complete medium containing 50ng/ml IL-4 for 48 h to obtain M2 macrophages.Immunocytochemistry was performed to detect CD16/CD32 expression(to mark M1 type TAMs)and CD163 expression(to mark M2 type TAMs).2.Construction and transfection of VEGFA siRNAThree VEGFA siRNA sequences were constructed,and MM cell line RPMI8226 cells were transfected with these sequences,and the interfering sequences with the best silencing effect were detected and selected by qRT-PCR for subsequent experiments.3.Construct a nude mouse xenograft model of multiple myeloma.RPMI8226 cells in the logarithmic growth phase were resuspended in PBS∶Matrigel(1∶1)mixture and injected into the shoulder and back of Balb/c nude mice to observe and record the tumor volume changes.When the subcutaneous tumor volume reaches 100mm~3,it can be think that the tumor is successful.Use immunohistochemistry to detect the expression of CD38,CD138,Kappa,and Lambda to identify MM xenografts.4.Effects of clearing TAMs and silencing VEGFA expression on MM graft tumor growth and angiogenesisA total of 15 tumor bearing successful nude mice were randomly selected and divided into three groups,including NC-1 group,Clo group and Clo+siGroup.The groups were treated as follows:NC-1 group:tail vein and intratumoral injection of normal saline at the same time;Clo group:tail vein injection of clodronate liposomes with intratumoral injection of normal saline;Clo+siGroup:tail vein injection of clodronate liposomes with intratumoral injection of VEGFA siRNA and in vivo transfection reagent mixture.Dosing frequency:2 times/w;treatment duration was 2w.5.Effects of reinfusion of different subtypes of TAMs on MM graft tumor growth and angiogenesis.The remaining 15 nude mice bearing tumors were randomly divided into 3groups,namely NC-2 group,Clo+M1 group and Clo+M2 group.Each group was treated as follows:NC-2 group:Two weeks after tail vein injection of clodronate liposomes,normal saline was injected into the tail vein;Clo+M1 group:Clo+M1group:Two weeks after tail vein injection of clodronate liposomes,M1 macrophages(induced by RAW264.7 cells)were infused into the tail vein;Clo+M2 group:Two weeks after tail vein injection of clodronate liposomes,M2 macrophages(induced by RAW264.7 cells)were infused into the tail vein.Dosing frequency:2 times/w;duration of treatment:injection of Clodronate liposomes in the tail vein for 2w,infusion of normal saline and macrophages for 2w.6.Detect the related indexes of each group(1)CD38,CD138,Kappa,Lambda,CD31(to mark angiogenesis),CD163(to mark M2 type TAMs),VEGFA expression were assessed by immunohistochemistry in tumor xenografts from nude mice in each group;(2)The levels of VEGFA m RNA in transplanted tumor tissues from nude mice in each group were measured by qRT PCR;(3)The expression levels of VEGFA protein in transplanted tumor tissues of nude mice in each group were detected by Western Blot;(4)The levels of VEGFA protein in the serum of nude mice in each group were measured by ELISA.7.Statistical analysisThe experimental data was analyzed using SPSS 21.0 software,expressed as mean±standard deviation(X±S),and differences between samples were analyzed by LSD tests.The test level isα=0.05.All the experiments were carried out independently for 3 times.Results1.Successful induction of M1 type and M2 type TAMs.Among them,M1TAMs showed positive expression of CD16/CD32 and negative expression of CD163,while M2 TAMs showed positive expression of CD163 and negative expression of CD16/CD32.2.Successfully screen out the siRNA sequence with the best silencing effect.Compared with the control,all three VEGFA siRNA sequences could effectively silence the gene expression of VEGFA,of which S1 transfected cells had the most significant silencing effect(P<0.01),so S1 was used in subsequent experiments.3.The MM nude mice subcutaneous xenograft tumor model was successfully constructed.Immunohistochemistry showed that CD38,CD138 and kappa were positively expressed,but the expression of lambda was negative,which could confirm the successful construction of the subcutaneous xenografts in nude mice with MM.4.Experiment of clearing TAMs and silencing VEGFA expression on MM transplanted tumor growth,nude mice were sacrificed to obtain tumor body and tumor volume and weight were measured.The results showed that compared with the NC-1 group,the Clo+siGroup and the Clo+sigroup both significantly inhibited the growth of tumor(P<0.01),and the Clo+sigroup showed the most significant inhibition effect.The tumor weight inhibition rates of the Clo group and Clo+sigroup were 45.58%and 78.28%,respectively.5.In experiments on the effect of clearing TAMs and silencing VEGFA expression on angiogenesis of MM transplanted tumors,after nude mice were sacrificed and tumor bodies were harvested,angiogenesis and M2 type TAMs infiltration were detected immunohistochemically.The results showed that compared with the NC-1 group,both the Clo+siGroup and the Clo+sigroup could significantly inhibit angiogenesis(P<0.01)and the expression of the M2 type TAM marker CD163(P<0.01),and it is worth mentioning that the Clo+sigroup had the most significant effect in inhibiting angiogenesis(P<0.01)and M2 type TAM infiltration(P<0.05).6.For experiments on the effects of clearing TAMs and silencing VEGFA expression on angiogenesis of MM transplanted tumors,after nude mice were sacrificed and tumors were harvested,VEGFA expression was assessed by immunohistochemistry,qRT-PCR,Western Blot and ELISA.The q R-PCR results showed that the m RNA expression level of VEGFA in the tissues of NC-1 group was significantly higher than that in the Clo group(P<0.01)and the Clo+siGroup(P<0.01),and the expression level in the Clo group was higher than that in the Clo+siGroup(P<0.05).Immunohistochemical,Western blot and ELISA methods were used to detect the level of VEGFA protein expression also showed the same trend(P<0.05)7.Effect of reinfusion of different subtypes of macrophages on the growth of MM transplanted tumors in the experiment,after the nude mice were sacrificed and the tumors were harvested,the tumor volume and weight were measured,and the results showed that compared with the NC-2 group,the growth of tumors in the Clo+M1 group was significantly inhibited(P<0.05),and the growth of tumors in the Clo+M2 group was significantly promoted(P<0.01).The tumor repopulation rate of the Clo+M1 group was 65.29%.8.Experiments on the effect of reinfusion of different subtypes of macrophages on the angiogenesis of MM transplanted tumors after the nude mice were sacrificed and the tumor bodies were harvested,angiogenesis and M2 type TAMs infiltration were detected immunohistochemically.The results showed that compared with the NC-2 group,angiogenesis was inhibited in the Clo+M1 group(P<0.05)and significantly promoted in the Clo+M2 group(P<0.01).CD163 was expressed with Clo+M2>NC-2>Clo+M1(P<0.05),it is worth mentioning that M2 type TAMs infiltration was suppressed in the Clo+M1 group compared with the NC-2 group,so we speculated that the large presence of M1 type TAMs would inhibit M2 type TAMs infiltration.9.Effect of reinfusion of different subtypes of macrophages on growth and angiogenesis of MM transplanted tumors after the nude mice were sacrificed and the tumor bodies were harvested,the expression of VEGFA was detected by immunohistochemistry,qRT-PCR,Western Blot and ELISA.The qRT-PCR results showed that the m RNA expression level of VEGFA in the tissues of the NC-2 group was significantly higher than that of the Clo+M1 group(P<0.01),but lower than that of the Clo+M2 group(P<0.01).Immunohistochemical,Western Blot and ELISA methods were used to detect the VEGFA protein expression levels also showed the same trend(P<0.05).Conclusions1.Depletion of macrophages and simultaneous silencing the expression of VEGFA in tumors significantly inhibited graft tumor growth and angiogenesis in MM nude mice in vivo.2.M1 type TAMs can inhibit MM transplanted tumor growth and tumor angiogenesis in nude mice,and M2 type TAMs can promote MM transplanted tumor growth and tumor angiogenesis in nude mice.