EphB4 Promotes Drug Resistance With SDF1/CXCR4 Axsis Induced By Bone Marrow Microenvironment

BackgroundIn the last decade,with the widely use of Tyrosine Kinase Inhibitors(TKIs)represented by Imatinib,the treatment of Chronic Myeloid Leukemia(CML)has a big increase,the aim of therapy change from prolonging life to develop the quality of patients’ life.TKIs has not only improved the prognosis of CML patients but also make the research of anti-cancer targeted drugs blow-out.However,with the using of the first generation of TKI,Imatinib(IM),part of patients failed to the treatment of take place to drug resistance which make it became the hotspot to find out the mechanism of drug resistance.In the past,studies were focused on endogenous factors such like the mutation of BCR-ABL or the BCR-ABL kinase independent way.However,exogenous factors,the microenvironment of neoplasm,has became the researchful focus.In hematological tumors,The bone marrow microenvironment can supply the nomarl hematopoietic cells to grow or proliferation as well as tumour cells.Because of the commensalism of leukemia cells,the marrow stroma cells(MSC)develop some changes which is benefit to the growth of leukemia cells so that it can protect leukemia cells to escape from the apoptosis caused by chemotherapy drugs.The multiformity of bone marrow microenvironment makes it complicated to learn about the mechanism of drug resistance mediated by bone marrow microenvironment and the present studys showed that the following aspects may be the possible causes:drug resistance mediated by soluble factors secreted by MSCs,drug resistance mediated by enhancing cell adhesion of MSCs and tomor cells,MSCs up-regulating the drug resistance gene of tomor cells,MSCs regulating the metabolisim of leukemia cells and the change of tumor cell cycles.Erythropoietin producing human hepatocellular receptor(Eph)is the largest receptor tyrosine kinases.With the combination of its ligands called Ephrine,Eph/Ephrine axsises mediate physiological activities among systems in vivo and also show their espeical effects on solid tumors as well as hematological neoplasms for the oncogensesis,development and metastasis.EphB4/EphrinB2 axsis is the hot spot of Eph family.After their combinding and phosphorlating,it can activate downstream proteins so that it can take part in angiogenesis,cell migration,bone steady-state and hematopoiesis.At the same time,lots of researches showed that EphB4 is related to the rumorigenesis and drug resistance among solid tumors,but little was known in hemotologic tumors.Our early study showed that EphB4 is high expressed in the K562-R cell line which fail to Imatinib therapy,and EphB4 join in the migration,invasion and drug reststance in CML cells.Even though it is well known that the movement and adhesion of neoplasm is related to Eph/Ephrin axsises,and EphB4 can accommodate the interaction of hematopoietic stem cells and bone marrow stromal cells and also take part in the drug resistance by cell adhesion,the mechanism is not totally clear yet.Ombretta et al.showed that EphB4 can promote the expression of SDF1 on Human umbilical vein endothelial cells in vitro and cooperate the cell migration and angiogenesis mediated by SDF-1/CXCR4 axsis.Thao.M et al.also show that EphB4 can regulate hematopoiesis and promote secretion of SDF-1 by stroma cell by animal experiment.Stromal cell-derived factor 1(SDF1)is one of the key cell chemoattractant which can combine with C-X-C chemokine receptor type4 to form the SDF1/CXCR4 axsis that can play important roles in mediating inflammatory reaction,promoting the migrating and homing of hematopoietic cells.It can also mediated the proliferation,drug resistance,infiltration and metastasis of tumor especially for leukemia.The SDF-1 secreted by stroma cell can combine with the CXCR4 on leukemia cells,then activate the downstream signaling pathways so that it can inducing the homing of leukemia cells and by a role in adhesion,it can aggrandize the adhesion of CML cells to bone marrow cavity then protect leukemia cells from TKI effect.Now that there are some inhibitor of CXCR4 and one of them is call Plerixafor(AMD3100)which is used for Auto-HSCT.And it is clear that SDF1/CXCR4 axsis can protect leukemia cells from the damage of anti-tumor drugs by regulate the adhesion between cells.We hope to learn more about whether the drug resistance process that EphB4 joined mediated by cell adhesion in bone marrow microenvironment is related by SDF-1/CXCR4,and combination of IM and AMD3100 can inprove the therapy efficacy.ObjectWith the simulation of bone marrow microenvironment,we hope to find out whether EphB4 can promote SDF1/CXCR4 and what would happen if combination of AMD3100 and IM were used in CML treatment.methods1.Collect the bone marrow samples of patients who were CML onset,chronic phase,blastic phase and normal people,separate and gather the mononuclear cells and bone marrow stromal cells,4.2.Cell culturing:K562、K562-R、K562-R-EphB4-sh cell lines are offering by our lab and MSCs were collecting from healthy donors with agreement.K562、K562-R、K562-R-EphB4-sh cell lines were cultured with RPMI-1640 medium and MSCs were cultured with MSC-culturing-medium.3.Extract RNA and detect the concentration,use QT-PCR to test mRNA expression.4.Use westernblot to test the protein expression of interest protein during the experiment.5.CCK8 test:Culture 1×104 cells/well by 96-well plates with 90ul K562、K562-R.K562-R-EphB4-Sh cell lines,add 10ul IM which concentration among 0 to 64mmol/L and make the final concentration among 0 to 6.4mmol/L,incubate in the incubater for 72h.After incubation,add 10ul CCK8 to each well and incubate away from light for 4h,test the OD on 450nm with microplate reader.The inhibition rate is quantified as[(ODcontrol-ODtest)/(ODcontrol-ODblank)]×100%.The IC50 rate is the final concentration for further experiment.6.Use transwell chamber for migration assay,compare the migration changes before and after EphB4 stimulation.7.Dying the K562 cells with Calckin-AM and then add to the plate that has been inoculated with MSCs,test the OD by microplate reader and compare the changes.8.Co-culturing:Inoculate MSCs on plates for 24h,make sure the MSCs adhere to the plate by fusiformis and fill the plate,add K562、K562-R、K562-R-EphB4-Sh with the proportion of 1:5 and then culture with MSC-culturing-medium.9.Apoptosis test:Use the Annexin V apc/7aad double-dying kit to test the apoptosis among different groups,analyze the data by Flow Cytometer.10.Zoopery:Apply with 4 or 5-week NOG mouse for experiment.Subcutaneous injection or intrafemoral injection with K562-R or K562-R-EphB4-Sh cells,and right side were injected with K562-R + MSCs or K562-R-EphB4-Sh + MSCs.Extent the length and width of tumor and measure the weigh of mouse,record the time of tumorigenesis and the overall survival.11.Statistical analysis:Statistical analysises were performed with SPSS 13.0 software and all the experiments were repeated for 3 times.Measurement datas were described for Mean ± stan.dard deviation.One-way ANOVA and independent-samples T test was choosen as required.Results:1.High expression of EphB4 and CXCR4 in CML patients with IM resistance.With QT-PCR to test mRNA expression of EphB4 and CXCR4 of the CML patients during incipient,remission and resistance and the healthy donors,we figure out that the expression of EphB4 and CXCR4 show significant differences among different groups(F=23.294,p<0.001;F=11.110,p<0.001)and both of them highly express rather than other groups(EphB4:p<0.001,p<0.001,p<0.001;CXCR4:p<0.001,p<0.001,p<0.001).2.The different expression of EphB4 among K562、K562-R、K562-R-EphB4-sh cell lines.With QT-PCR and westernblot test,K562、K562-R、K562-R-EphB4-sh cell lines show different expression for EphB4(F=361.131,p<0.001).For mRNA expression,the expression of K562-R cells is higher than the others(p<0.001,p<0.001),and the expression of EphB4 on K562-R-EphB4-sh is the lowest(p<0.001,p<0.001).The same result was showed by westermblot test.3.K562、K562-R、K562-R-EphB4-sh cell lines show different response to IM.With CCK8 test,K562、K562-R、K562-R-EphB4-sh cell lines show different response to IM(F= 287.875,p<0.001)and the K562 cells has a good response to IM(p<0.001,p<0.001)while K562-R is the worst(p<0.001,p<0.001).4.EphrinB2 and SDF-1 expressed on MSCEphrinB2 and SDF-1 was tested on MSC by westernblot.We found it show different by different phrase with EphrinB2 and SDF-1 expression(F=126.733,p<0.001;F=34.136,p=0.001).EhprinB2 has a higer expression on CP rather than HC and BC patients(p=0.001,p<0.001)while SDF-1 showes higher expression on BC patients(p=0.001,p<0.001).5.Stimulating with EphB4-Fc,MSC expresses more SDF-1 tested by westernblot and RT-PCR compared with Leptin-Fc(t=-5.284,p=0.011).6.EphB4 can promote the migration and adhesion of K562 cells under the influence of bone marrow microenvironment and can be reverse by AMD3100With the simulation of bone marrow microenvironment,after the impact of EphB4,K562 cells up-regulate the migration(t=10.253,p=0.001)as well as the adhesion(t=7.797,p=0.001).It can also be reverse by AMD3100,the inhibitor of CXCR4(p<0.001,p<0.001)7.With the test of westernblot and RT-PCR,we found that CXCR4 expression is different before and after co-culturing(F=966,P<0.001),the CXCR4 expression were increasing(p<0.001)on K562-R cells in the co-cultured system.8.The co-cultured systems increased the drug resistance to IM.With co-culture systems to simulate the bone marrow microenvironment,Annexin V-APC/7-AAD apoptosis kit find it out that the apoptosis rate is decreasing among co-cultured groups compared to the single-cultured groups(p=0.031,p<0.001,p<0.001).And after the combination of AMD3100,the drug resistance some how can be reversed(p<0.001,p<0.001,p<0.001).9.When subcutaneous injected with tumor cells,different groups has different tumor growing time and tumor volume(F=8.767,p=0.007;F=368.01,p<0.001).It has a shorter time in K562-R+MSC or K562-R-EphB4-sh+MSC co-cultured groups than K562-R or K562-R-EphB4-sh groups(p=0.012,p=0.009)and large vulume(p<0.001,p=0,001).By IHC test,K562-R+MSC co-cultured group expresses more CXCR4(p<0.05).For therapy,after IM treatment,volume of tumor in all groups has increase and it achieve a better effect in single-cultured groups than in co-cultured groups(p<0.001,p<0.001).Also,the volume in K562-R single-cultured or co-cultured group is larger than in K562-R-EphB4-sh groups(p=0.031,p<0.001).And combination of IM and AMD3100 has a obvious effect in K562-R co-cultured group.10.When intrafemural injected with tumor cells,different groups has different tumor growing time and overall survival((F=81.698,p<0.001;F=20.286,p<0.001).K562-R co-cultured group has the shortest time of tumor formation(p<0.05)and the co-cultrued groups has a shorted life time than singel-cultured group(p<0.001,p<0.001).By IHC test,K562-R+MSC co-cultured group expresses more CXCR4(p<0.05).For therapy,IM has a worse effect in co-cultured groups(p<0.001,p<0.001)and the overall survival in K562-R single-cultured or co-cultured group is shorted than it in K562-R-EphB4-sh groups(p=0.009,p=0.013).And combination of IM and AMD3100 has theraputic action in co-cultured groups.Conclusion:1.The CML patients is BC highly expresses EphB4 and CXCR4;2.The expression of EphB4 is different among K562、K562-R、K562-R-EphB4-sh cell lines and the drug resistance is different;3.EphB4 can up regulate the secretion of SDF1 on MSCs under the bone marrow microenvironment,then increase the expression of CXCR4 on cancer cells,thus EphB4 can promote the SDF1/CXCR4 axsis to mediate drug resistance;4.Combination of AMD3100 and IM may be a new way to reverse IM resistance in CML treatment.