The Role Of Bone Marrow Small Adipocytes In Acute Myeloid Leukemia And Its Forming Mechanism

Objective:We retrospectively analyzed the morphological characteristics of the adipocytes in the biopsy sections from primary AML patients,definited the correlationship between small marrow adipocytes and the prognosis of AML patients and preliminarily explored the effect of leukemia cells on the generation of small marrow adipocytes.Methods:1.The diameter and area of marrow adipocytes were counted and compared between AML patients and the controls.Cox regression analysis was used to analyze the prognostic factors of AML patients.Kaplan-Meier survival analysis was used to analyze the effect of the content of small adipocytes on the survival of AML patients.2.Immunohistochemistry was used to detect the expression of UCP-1 and PGC1αin the bone marrow biopsy sections of AML patients and the controls.The morphology,phenotype and adipogenetic ability of MSCs from these two groups were compared.After co-cultured with leukemia cell lines by transwell,the morphology of adipocytes was detected by oil red staining and immunofluorescence and the expression of adipogenic genes was detected by qPCR.3.The expression of TGF-β1,CTGF,GDF6 and GDF15 in leukemia cells was detected by RT-PCR and qPCR.The effect of GDF15 on mature adipocytes was observed by exogenously adding human recombinant GDF15 factor or neutralizing antibody of GDF15.4.The expression of GDF15 in bone marrow mononuclear cells was detected by qPCR and Western Blot in AML patients and the controls.The expression of GDF15 in bone marrow sections was detected by immunofluorescence.The correlation between the content of GDF15 in bone marrow aspirates and the content of small marrow adipocytes was analyzed by linear regression.Results:1.The diameter and area of adipocytes in AML patients was 38.28±14.69um and 559.36±271.93um~2,respectively.It was significantly smaller than that in the controls(P<0.001).Linear analysis showed that neither the diameter nor the area of adipocytes was related with the amount of bone marrow blasts(P=0.21;P=0.245).The percentage of small adipocytes in AML patients was significantly higher than that in the controls(45.7%vs 25%,P=0.005).2.The number of small adipocytes in the refractory group was 31.56±11.72/mm~2,and the volume of small adipocytes was 6.98±3.18%,they were significantly higher than that in the the remission group(P<0.001).Cox regression analysis showed that the number and the volume of small adipocytes were the risk factors for the survival of AML patients.The more the number or the volume of small adipocytes was,the shorter the overall survival or the relapse free survival was.3.Both in AML patients and the controls,the lipid droplets of bone marrow sections were always unilocular.The marrow adipocytes negatively expressed UCP-1 and PGC1αin the serial sections.Similar to the controls,MSCs from AML patients highly expressed CD73,CD105 and CD90 and negatively expressed CD34,CD14 and CD45.The morphology of MSCs or MSC-derived adipocytes from these two groups was similar.There was no difference was observed in the adipogenic genes from the two groups.After co-culturing with leukemia cell lines,the lipid droplets of adipocytes significantly decreased,the size of adipocytes dramatically reduced and the levels of adipogenic genes were significantly reduced.4.Leukemia cell lines highly expressed GDF15.Adding rhGDF15 into the culture medium of mature adipocytes,both the size of the lipid droplets and the expression of adipogenic genes obviously decreased.In contrast,neutralizing anti-GDF15 antibody partly attenuated the effect of the GDF15 on the generation of small adipocytes.5.The mononuclear cells of bone marrow from AML patients exhibited higher expression of GDF15 than the controls.GDF15 was highly expressed in CD34+cells from BM sections,which were regarded as leukemia cells.The linear regression analysis indicated that the levels of GDF15 were positively correlated with the content of small adipocytes in the bone marrow sections.Conclusion:1.The marrow adipocytes in AML patients was less and smaller,it may be not due to the extensive infiltration of leukemic blasts.The proportion of small adipocytes in AML patients increased.The increase of the number or the volume of small adipocytes was poor for the prognosis of AML patients.2.The generation of small adipocytes in the bone marrow of AML patients may not be related with the browning of adipocytes or abnormal MSCs.3.GDF15 secreted by leukemia cells is involved in the formation of small adipocytes.AML patients highly expressed GDF15 in bone marrow and the level of GDF15 was correlative with the content of small adipocytes.