Myeloma-modified adipocytes exhibit a senescent-associated secretory phenotype

Bone marrow -adipocytes (BMAs) have recently been implicated in accelerating bone metastatic cancers such as AML and breast cancer. Importantly, bone marrow adipose tissue (BMAT) expands with aging and obesity- two key risk factors in multiple myeloma (MM) disease prevalence- suggesting that BMAs influence and are influenced by myeloma cells in the marrow. Here we examined how myeloma cells affect adipocytes and provide evidence that MM cells alter adipocyte gene expression and cytokine secretion profiles, creating a “MM-associated” adipocyte (MM-adipocyte) phenotype. Our findings indicate that: (1) Multiple myeloma cells decrease BM adiposity in vitro, in myeloma animal models, and in clinical samples, (2) myeloma induces widespread gene expression and phenotypic changes in adipocytes in vitro, most notable, the induction of a senescent-like phenotype in BMAs, (3) MM-adipocytes affect myeloma cell cycle, drug sensitivity, and aggressiveness, illuminating a new driver of MM cell evolution in a drug resistant clone. We demonstrate that myeloma cells exposed to MM-adipocytes are rescued from dexamethasone-induced cell cycle arrest and have increased expression of FKBP5, a potential drug resistance gene. Our findings in patients confirm that BMAs are dynamic during myeloma disease progression (decrease during MM initiation, recover during disease remission) and that the interactions between BMAs and MM cells have previously unappreciated implications in the understanding and treatment of myeloma. Primary human mesenchymal stromal cells (MSCs) were isolated from bone marrow samples received from deidentified male and female donors after total hip arthroplasty. MSCs were isolated by surface adherence, and seeded into 6-well plates at a density of 150,000 cells/well and grown until 80-90% confluent. To generate bone marrow adipocytes (BMAs), MSCs were differentiated for 21 days in hMSC adipogenic media consisting of DMEM/F12 supplemented with FBS, antibiotic/antimycotic, 500 µM IBMX, 50 µM indomethacin, 1 µM dexamethasone, and 1 µM insulin. After 21 days, cells were washed and myeloma cells (OPM-2, MM.1S, and RPMI-8226) were added via transwell co-culture (0.4 µm pores) at a density of 500,000 cells/well in RPMI-1640 media containing FBS and antibiotic/antimycotic for 72 hours. Following co-culture the individual cell types were then pooled (n=2 wells) prior to RNA extraction.

骨髓脂肪细胞（bone marrow-adipocytes, BMAs）近期被证实可加速急性髓系白血病（acute myeloid leukemia, AML）与乳腺癌等骨转移性癌症的进展。值得注意的是，骨髓脂肪组织（bone marrow adipose tissue, BMAT）会随衰老与肥胖发生扩张，而二者均为多发性骨髓瘤（multiple myeloma, MM）患病率上升的两大关键风险因素，这提示骨髓内的BMAs与骨髓瘤细胞存在相互调控关系。
本研究探讨了骨髓瘤细胞对脂肪细胞的调控作用，并证实多发性骨髓瘤细胞可改变脂肪细胞的基因表达与细胞因子分泌谱，进而形成"MM-associated"脂肪细胞（MM-adipocyte）表型。
本研究结果显示：① 多发性骨髓瘤细胞可在体外、骨髓瘤动物模型及临床样本中降低骨髓脂肪含量；② 骨髓瘤细胞可在体外诱导脂肪细胞发生广泛的基因表达与表型改变，其中最为显著的是诱导BMAs产生衰老样表型；③ MM-adipocytes可影响骨髓瘤细胞的细胞周期、药物敏感性与侵袭能力，为耐药克隆中骨髓瘤细胞进化的潜在驱动机制提供了新的阐释。
本研究证实，暴露于MM-adipocytes的骨髓瘤细胞可摆脱地塞米松诱导的细胞周期阻滞，且耐药相关潜在基因FKBP5的表达水平显著上调。
针对患者的研究结果证实，BMAs在骨髓瘤疾病进程中呈现动态变化（MM发病初期减少，疾病缓解期恢复），且BMAs与MM细胞间的相互作用此前未被充分认知，对骨髓瘤的诊疗具有重要启示意义。
本研究从接受全髋关节置换术后的匿名男女供者的骨髓样本中分离原代人骨髓间充质基质细胞（mesenchymal stromal cells, MSCs）。MSCs通过贴壁法分离，以150,000个细胞/孔的密度接种于6孔板中，培养至细胞汇合度达80%~90%。
为构建BMAs，将MSCs在人骨髓间充质干细胞成脂诱导培养基中分化21天，该培养基成分为添加了胎牛血清（FBS）、抗生素/抗真菌剂、500 μM 3-异丁基-1-甲基黄嘌呤（IBMX）、50 μM 吲哚美辛、1 μM 地塞米松及1 μM 胰岛素的DMEM/F12培养基。
分化21天后，清洗细胞，通过0.4 μm孔径的Transwell小室共培养体系加入骨髓瘤细胞（OPM-2、MM.1S及RPMI-8226），以500,000个细胞/孔的密度接种于含FBS与抗生素/抗真菌剂的RPMI-1640培养基中，共培养72小时。
共培养结束后，收集2个复孔的细胞进行混合（n=2），随后开展RNA提取实验。