| BackgroundAcute lymphoblastic leukemia(ALL)is a hematopoietic malignancy that originates from lymphocytes.Abnormally proliferated B-or T-primary/naive lymphocytes accumulated in the bone marrow and inhibits the normal hematopoietic functions.They can also invade other organizations besides the bone marrow,such as meninges,lymph nodes,gonads,etc.ALL accounts for more than 70%of children’s leukemias and about 20%of adults’leukemia.With more understanding and researches of these diseases,the therapeutic regimens has been continuously improved and standardized.About 80%of children’s ALL can obtain long-term disease-free survival(DFS),compared with only 30-40%of adults’.The presences of minimal residual diseases(MRD)and multidrug resistance(MDR)is the major cause of relapse and treatment failure in leukemia.Therefore,it is urgent to improve treatment strategies based on current regimens to sweep off MRD and improve the efficacy and prognosis of adults’ALL.More and more evidences had shown that the resistance of patients with ALL,especially those with relapsed and refractory ALL,is closely related to the bone marrow microenvironment.Traditional chemotherapy usually achieves therapeutic goals by clearing a large number of clonal cells.However,ALL cells secreted in the bone marrow niche can escape the killing of chemotherapy drugs,due to the reception of survival and drug resistance signals from stromal cells in the bone marrow.The CXCR4/CXCL12 signaling axis plays a key role in the residing of leukemia cells in the bone marrow to escape the toxicity of chemotherapeutic drugs.Activation of this axis may activate complex signaling factor networks in the bone marrow microenvironment and participate in the homing of leukemia cells,the survival and proliferation,extramedullary infiltration and other important processes,thus promoting the occurrence,development and even deterioration of the disease.Targeting the CXCR4/CXCL12 signaling axis to break the interactions between ALL cells and bone marrow stroma,prompting ALL cells to detach from the protective state of the bone marrow microenvironment and enhancing the killing of chemotherapeutic drugs,which may reduce the occurrence of MDR and clear the MRD.The purpose of this study is to investigate the reversal effects of targeting the CXCR4/CXCL12 signaling axis in ALL drug resistance and induced cell apoptosis,also to provide theoretical basis and experimental data for the development of more small molecule inhibitors and related targeted therapeutics.Thus,guiding the conversion of ALL treatment models,from single treatment focusing on malignant tumor cells to a combination of microenvironment treatment where ALL cells survived.Methods1.Cell isolation and culture:10mL of bone marrow from 5 relapsed and refractory B-ALL patients(primary cell count≥90%and peripheral blood leukocyte count≥10.0×10~9/L)in the Department of Hematology of the Affiliated Cancer Hospital of Zhengzhou University.The lymphocytes were separated using lymphocyte separation medium and cultured in RPMI 1640 medium containing 20%fetal bovine serum(FBS),culture time was less than 24 hours.Umbilical cord mesenchymal stem cells(UC-MSCs)were purchased from Shandong Cell-Tissue Bank and cultured in DMEM/F12(1:1)medium containing 10%FBS and changed the medium once every 48-72 hours.After the cells grew into fusion,digested the cells with 0.25%trypsin containing EDTA,and the passage number≤5.2.Establishment of co-culture system with ALL primary cells and UC-MSCs:1×10~5 UC-MSCs were inoculated into each well of a 6-well culture plate.After approximately 24 hours of culture,using the microscope to observe the degree of fusion,when it reached 90%,unattached cells were washed away with PBS,as well as the residual culture fluid.Then,1×10~6 ALL primary cells were added to each well to build the co-culture system,using RPMI 1640 medium containing 10%FBS to continue the culture.3.Using flow cytometry to detect the CXCR4 expression on the surface of ALL primary cells:The experiment was divided into two groups:the primary ALL cells culture group and the primary ALL cells with UC-MSCs co-culture group;they were separately divided into 4 groups:the control group was added with RPMI 1640medium containing 10%FBS,AMD3100(Plerixafor)single drug group(concentration was 5μg/mL,the drug concentration was determined according to relevant publications),VCR single drug group(concentration was 1μmol/l,the drug concentration was determined according to relevant publications),VCR+AMD3100double drugs group,a total of 8 groups,each group has 2 additional holes.After 24hours,ALL cells in the supernatant were collected,and the expression of CXCR4 on the surface of ALL cells was examined by flow cytometry.4.Using ELISA to detect the concentration of CXCL12:the supernatant of each experimental group mentioned above was collected,and the concentration of CXCL12 secreted by UC-MSCs in the supernatant was detected by human CXCL12ELISA kit.5.Using Annexin V-FITC/PI double staining to detect the apoptosis ALL primary cells:groups and drug doses were the same as CXCR4 expression detection group.After 24 hours,ALL cells in the supernatant were collected,and the apoptosis rate of primary ALL cells in each group was detected by flow cytometry.6.The expression of apoptosis-related protein in ALL primary cells was detected by Western blot:The experiment was divided into 6 groups:ALL primary cell culture group,ALL cells+VCR group(1μmol/l,the drug concentration was determined according to relevant publications),ALL cells and UC-MSCs co-culture group,co-culture+AMD3100(Plerixafor)group(5μg/mL,the drug concentration was determined according to relevant publications),co-culture+VCR+AMD3100 group,at least 5.0×10~6 ALL cells were collected each group after dosing 24h,and the expression of Bcl-2 and Bax was detected by Western blot.7.Statistical analysis of all the experimental data,using IBM SPSS Statistics24.0 software.The measurement data is represented by?X±S;when comparing the mean of two samples,independent sample T-test was used,P<0.05 was considered statistically significant.Results1 Expression of CXCR4 on the surface of primary ALL cells in the experimental groupThe mean fluorescence intensity(MFI)of CXCR4 was(335.24±12.85)in the control group,and(199.83±40.23)in the AMD3100 group which is lower than the control group(P=0.001);the MFI in the VCR group has increased to(475.22±69.17)compared to the control group(P=0.007);the MFI of VCR+AMD3100 group was(330.13±42.82),which is lower than VCR group(P=0.012).In the co-cultured group:the MFI of control group was(261.87±91.07),and the MFI of AMD3100 group was(134.71±21.78),which is lower than the control group(P=0.035);the MFI of VCR group is(426.00±13.66),has increased compared to the control group(P=0.012);VCR+AMD3100 group decreased to(275.78±54.20)compared with VCR group(P=0.002).2 The concentration of CXCL12 secreted by UC-MSCs in the experimental groupThe concentration of CXCL12 in the ALL cells mono-culture group was beyond the detection range.In the co-culture group:the concentrations of CXCL12 in control group and AMD3100 group were(0.698±0.088)ng/mL and(0.751±0.063)ng/mL respectively;there was no significant difference(P=0.307);the concentration of VCR group was(1.351±0.044)ng/mL,which was significantly higher than control group(P=0);the concentration in VCR+AMD3100 group was(1.108±0.041)ng/mL,which was lower than VCR group(P=0).3 The apoptosis rate of primary ALL cells in the experimental groupIn the mono-culture group:the apoptosis rate in the VCR group increased from(64.76±2.37)%in the control group to(84.04±1.16)%(P=0);the apoptosis rate in the VCR+AMD3100 group was(81.13±2.71)%,compared with VCR group,there was no significant difference(P=0.058).In the co-culture group:the apoptosis rate of ALL cells in the control group was(57.06±3.41)%,and the apoptosis rate in the AMD3100 group was(73.39±3.16)%,which was significantly higher than the control group(P=0);the rate of apoptotic cells in VCR+AMD3100 group increased from(76.6±2.93)%to(82.47±1.03)%compared with VCR group(P=0.003).Compare the mono-culture group with the co-cultured group:the apoptosis rate of mono-culture ALL cells has decreased from(64.76±2.37)%to(57.06±3.41)%after co-cultured with UC-MSCs(P=0.003);mono-culture and co-culture VCR group,the apoptotic rates were(84.04±1.16)%and(76.6±2.93)%respectively,and the apoptotic rate of the co-culture group decreased significantly(P=0.001).4 The apoptosis-related proteins expression in the experimental groupIn the co-culture group:compared with control group,the expression of the pro-apoptotic protein Bax was up-regulated and the anti-apoptotic protein Bcl-2 was down-regulated in the AMD3100 group;the expression of the pro-apoptotic protein Bax was not significantly changed in the VCR+AMD3100 group compared with the VCR group.Anti-apoptotic protein Bcl-2 expression was down-regulated.In the control group,ALL cells were co-cultured with UC-MSCs,the expression of pro-apoptotic protein Bax was down-regulated and the anti-apoptotic protein Bcl-2was up-regulated in the control group.In the VCR group,apoptosis was promoted after co-culture.The expression of protein Bax was down-regulated compared with the single group,and the expression of anti-apoptotic protein Bcl-2 was up-regulated compared with the single group.Conclusions1.UC-MSCs protecting ALL cells against apoptosis and drug resistance through the CXCR4/CXCL12 signaling axis.2.The CXCR4 antagonist(AMD3100)inhibits the interactions between ALL cells and the microenvironment by blocking the CXCR4/CXCL12 axis,thereby reversing the protective effect of UC-MSCs on ALL cells and the promotion of drug resistance. |
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