The Effect Of Macrophages In DNA Damage Response Of Multiple Myeloma Cells

BackgroundMultiple myeloma(MM)is a hematological malignancy of B cells in the terminal differentiation stage,characterized by clonal proliferation of malignant plasma cells.The clonal plasma cells secrete a large amount of monoclonal immunoglobulin(M protein),which causes damage to the terminal organs and result in clinical symptoms.Although the development of treatment methods now has greatly improved the curative effect,MM is still an incurable hematological malignant tumor,and the mechanism of MM development needs to be further explored.When normal plasma cells are stimulated by infection and inflammation,DNA damage and genomic instability occur,and by the accumulation of genetic events,they gradually develop into MM cells(MMCs).As the disease progresses,new cytogenetic events continue to emerge,and the accumulation of events such as chromosomal translocation,gene copy number variation and somatic single nucleotide variation promotes the clonal evolution of MM.The occurrence of genetic events is closely related to the genome instability of MMCs,and the abnormality of DNA repair is the chief reason.The most serious of DNA damage is DNA double strand break(DSB).The cells mainly adopt homologous recombination(HR)and non-homologous end joining(NHEJ)to repair the DSBs.Moreover,the survival of MM is inseparable from the bone marrow(BM)microenvirorunent,especially,the macrophages(MΦs)in BM are closely related to the development of MM.The previous researches also showed that MΦ5 are important for the survival,proliferation and drug resistance of MMCs.Based on the characteristics of high heterogeneity and genomic instability of MM,and the protective effect of MΦs on MMCs,our study intends to further clarify whether MΦs affect MMCs DNA damage response(DDR)and DNA repair,and the relationship between MΦs and genomic instability of MMCs.Objective1.To explore the relationship between MΦs and patient FISH results.2.To culture the M-CSF induced macrophages(MΦs)and identifiy their phenotypes,and to determine the role of MΦs in the instability of MMCs genome.3.To investigate the effects of MΦs on MMCs DDR and DNA repair in vitro and in vivo,and to elucidate the role of HR and NHEJ in the repair process in MMCs.Methods1.Immunohistochemistry was used to detect the content of CD68 in bone marrow biopsy specimens of patients.2.We detect the expression of MΦs’ CD80,CD86,CD163 and CD206 in vitro by Flow cytometry(FCM),and identified the phenotype.3.Experiments in vitro are mainly divided into MMCs cultUred-alone-group and MΦs co-cultured-group;experiments in vivo are mainly to observe the difference between MMCs group and MΦs inj ection group.4.The apoptosis and cell cycle of ARP-1,OPM2,MM.1S and CAG were absorved by Flow cytometry.5.Western blot was used to test γH2AX and Ras51 expression in MMCs.6.Immunofluorescence was used to detect the expression of MMCs’ γH2AX in vitro and in vivo,and the number of MMCs and γH2AX foci were identified by high-content cell imaging system with AI calculation.7.The comet assay was used to observe the DNA damage of MMCs in vitro and in vivo,and the comet pattern was read by CASP software.8.HR repair level was measured by U2OS-HR cells loaded with the SCR reporter system;while NHEJ repair level was detected by U2OS-NHEJ cells loaded with the vGEJ reporter system.9.gRNA CRISPR/Cas9 was used to induce DSB in AAVS1 and HBB respectly to cause translocation,and was used to detect MMCs9 endogenous NHEJ in vitro and in vivo.AAVS1 and HBB were selected as the detected genes,and the paired gRNA were screened in HEK293T cells.The sequence in the AAVS1 and HBB interface context were tested by the next generation sequencing(NGS).Results1.As FISH results,in the IgH translocation,D13S319 locus deletion and RB1 deletion,the content of MΦs was correlated with the positive rate.Among them,the positive rate of IgH translocation in patients with MΦs content of grade 2 and grade 3 were higher than that of grade 1(48.1%vs 86.7%,P=0.014;48.1%vs 83.3%,P＝0.017);the positive rate of D13S319 deletion in patients with MΦs content of grade 3 was higher than that of grade 1 patients(37.0%vs 72.2%,P=0.021).Among the above five FISH results,≥3 positive results’ patients were considered to be complex genetic abnormalities.The results showed that the complex genetic abnormality rates in MΦs content grade 1,grade 2 and grade 3 were respectively 37.0%,46.7%and 72.2%;and the positive rate of complex genetic abnormality in grade 3 MΦs was higher than that of grade 1 group with statistically significant(P=0.021).2.Flow cytometry was used to detect M-CSF induced macrophages(MΦs)in vitro,and CD163 and CD206 were highly expressed in MΦs which imply that MΦs tend to be M2 type.MΦs reduced the apoptosis of MMCs caused by BTZ,MPL and DEX.3.Western blot results showed that MΦs reduced γH2AX at the baseline of MMCs.After bleomycin caused MMCs’ DSBs,MΦs still reduced γH2AX of MMCs.yH2AX of MMCs was detected at 2h,4h,8h,16h,24h,etc.Western blot and immunofluorescence consistently demonstrated that MΦs accelerates DDR of MMCs.4.The comet assay again verified that MΦs promote DNA repair of MMCs and reduces the amount of damaged DNA.4h after irradiation,the damaged DNA content of ARP-1,MM.IS and CAG cells cultured alone and co-cultured with MΦs was respectily.5.HR repair was measured in U20S-HR cells loaded with SCR reporter(HR reporter),however MΦs did not significantly increase HR levels of U20S-HR.Western blot indicated that MΦs promoted the expression of Rad51(HR-related protein)in MMCs;FCM shows that MΦs promoted MMCs’ G2/M phase(the cycle when HR repair was active).6.NHEJ repair was measured in U20S-NHEJ cells loaded with vGEJ reporter(NHEJ Reporter).The results manifested that the NHEJ rate in the MΦs-separated culture group was significantly higher than that in the control group(34.4%±0.31%vs 37.03±0.18%,P<0.001).Using the paired gRNA-CRISPR/Cas9 method,AAVS1 and HBB were used as detection genes to detect the NHEJ of MMCs’ endogenous gene,and the sequence of about 250 bp near the NHEJ interface was sequenced by NGS.The results proclaimed that the MΦs co-culture group significantly increased the efficiency of NHEJ(AAVS1 locus:1.51 ± 0.25 vs 1.82 ± 0.32,P=0.024;HBB locus:1.81 ± 0.44 vs 2.25 ± 0.82,P=0.014),and decreased proportion of accurate NHEJ repair(AAVS1 locus:73.34%± 9.30%vs 68.57%±7.62%,P=0.016;HBB locus:37.31%±8.91%vs 32.15%±8.12%,P=0.046).Furthermore,analysis of the length of base sequence loss showed that the probability of base loss>3 bp in the MΦs co-culture group was higher than that in the culture group alone(AAVS1 site:48.72%vs 50.86%,P<0.001;HBB site:14.47%vs 21.22%,P<0.001).Moreover,in the HBB site,MΦs prolonged the average length of base loss in NHEJ(3.73 ± 0.08 bp vs 4.90 ± 0.13 bp,P<0.001).7.Our study used gRNA-CRISPR/Cas9 technology to cause fixed-point cleavage in AAVS1 and HBB respectively,and use PCR and NGS to detect translocation.The results show that MΦs can promote the probability of chromosomal translocation of MMCs.8.The NOD-SCID mouse was used as animal model.Western blot and immunofluorescence experiments confirmed that the yH2AX of the MΦs group was significantly lower than that of the irradiation only group(the ratio of yH2AX focus>7 cells:28.47%±3.67%vs 11.96%±7.04%,P<0.001).What’s more,in the immunofluorescent slides of the MΦs group,the cell with yH2AX focus>7 in ARP-1 directly adjacent to MΦs was significantly lower than that of the entire field(11.1%±6.64%vs.6.61%±4.37%,P=0.0096).9.The comet assay of MMCs in vivo directly proved that the tail of the comet pattern in MΦs group was significantly shorter than that of the irradiation only group,and the content of damaged DNA was less than that of control(19.55%±4.65%vs 7.59%±2.61%,P<0.001).10.We construct animal model to detect MMCs’ endogenous NHEJ repair in vivo.The subcutaneous tumor in the gene editing group(Ctr.)and gene editing group after injection of MΦs group of mice were ground and screened by CD138 magnetic beads sorting to obtain ARP-1 single cell suspension,and the about 250bp sequence near NHEJ interface were sequenced by NGS.The results indicated that the corrected NHEJ level in MΦs group was higher than in ctr.(1.47±0.03 vs 1.65±0.08,P=0.095),unfortunately the P value was>0.05;the precision repair was significantly lower than that in ctr.group(80.40%±3.56%vs 72.16%±1.97%,P=0.025).The sequence content of base loss>3 bp was significantly higher than that of ctr.group(54.12%vs 46.56%,P<0.001),and the average lost base length increased(7.78±0.39 vs 10.01±0.64,P=0.0029).Conclusions1.The content of MΦ5 in patients in bone marrow is related to the results of cytogenetics(FISH).The higher the content of MΦs,the more complicated the cytogenetic abnormalities of patients.2.MΦs reduces the baseline γH2AX of MMCs,contributing to MMCs survive in the case of genomic instability.3.In the case of severe injury of MMCs’ DNA,MΦs promote the of MMCs DDR and DNA damage repair.4.MΦs promotes S phase(in vivo)/G2 phase(in vitro)of MMCs,providing longer time and probability for HR repair,and significantiy reciucing the bias of LTGC in HR.5.MΦs significantly promoted the NHEJ in MMCs,and reduced accuracy of NHEJ repair,and increased the length of base loss in NHEJ meanwhile promoted genomic instability of MMCs.6.In the case of artificially induced DSB,MΦs can increase the probability of chromosomal translocation of MMCs.