1. Cell-free mitochondrial DNA is elevated in multiple myeloma.
Fig 1a： 为了定量MM患者的mtDAMP，作者首先取了MM患者的外周血和骨髓，检测了mtDNA。
Fig 1b-c： MM患者外周血的mtDNA显著升高，genomic DNA则没有明显变化。
Fig 1d： 对骨髓样本的检测发现，同一个患者的骨髓mtDNA显著高于外周血mtDNA，suggesting that the majority of cell-free mtDNA associated with MM is maintained within the BM microenvironment。
Fig 1e： Schematic of the cell-free media extraction process. DNA was extracted from cell-free media extracted from cells in culture and analysed via TaqMan qPCR
Fig 1f： 和CD34+单核相比，MM patient-derived cells和immortalised MM cell lines的mtDNA更高。
These data show that cell-free mtDNA is elevated in MM.
2. Increased mitochondrial DNA in MM originates from the malignant plasma cells
Fig 2a-b： 为了探究血浆中mtDNA的来源，作者使用immunocompromised NSG移植了immortalised human MM cell line MM1S。作者每周取一次小鼠外周血，检测了其中的鼠源和人源mtDNA含量。
Fig 2c： 结果显示病程中鼠源mtDNA没有变化，人源mtDNA在14天开始升高。
Fig 2d： Engraftment of the MM1S cells in mice was confirmed by measuring the percentage of human CD38+ cells in the BM by flow cytometry.
Fig 2e：而且在21天的时候，外周血和骨髓中的mtDNA都是升高的，而且骨髓中mtDNA的水平显著高于外周血。
Fig 2f-g：作者在另一个小鼠模型中验证了前面的结果
3. MM induces STING-mediated activation of macrophages via mtDAMPs
Fig 3a： 作者从5TGM1^(GFP+ LUCI+)^ engrafted KaLwRij mice中分离了巨噬细胞。
Fig 3b-c： Engraftment of 5TGM1 in the KaLwRij mice was determined by GFP expressing cells in the BM.
Fig 3d： RT-qPCR 结果显示 5TGM1 engrafted mice的BM macrophages中 GBP2, IFIT3 和 IRF7表达增加。
Fig 3e： 为了确定巨噬细胞中STING的激活是mtDNA的直接作用，作者使用了5TGM1条件培养基培养了KaLwRij鼠的BMDM。条件培养基首先使用离心去除了细胞和大的extracellular vesicles，然后使用100 kDa ultra-centrifugal filter过滤，去除小extracellular vesicles和残留的蛋白（培养基里只剩下mtDNA）。Removal of the EVs therefore allowed us to purely focus on the effect of cell free mtDNA.
Fig 3f： Results show that STING-pathway genes were upregulated in BMDM treated with conditioned media from 5TGM1.
Fig 3g： 作者从小鼠肝脏分离了mtDAMPs干预BMDM也得到了一致的结果。
Fig 3h： 而CpG ODN 1826 (a molecular mimic of mtDNA), failed to fully activate the STING-related genes with only Irf7 being significantly upregulated.
Fig 3i： 骨髓瘤患者的外周血单核细胞也存在着STING通路的激活（附件）。STING inhibitor H-151 inhibits mtDAMP-induced STING activation genes in BMDM.
The data suggest that mtDAMPs released from myeloma cells induce a STING response in BM macrophages.
4. MM progression is attenuated by STING inhibition
使用STING inhibitor H-151对骨髓瘤小鼠起到了很好的治疗效果。
5. MM-derived mtDAMPs Induce a migratory signature in BM macrophages
Fig 5a： To investigate the secretome changes in macrophages exposed to MM-derived mtDAMPs, we treated BMDMs with myeloma-derived mtDAMPs and assayed the cell supernatant using a Proteome Profiler Mouse Cytokine Array.
Fig 5b： 使用mtDAMPs刺激的BMDM的上清中，促炎的和与STING激活相关的细胞因子上调。趋化因子同样出现上调。因此作者推测，MM-derived mtDAMPs 可以通过激活巨噬细胞的STING通路促进malignant plasma cells in the BM在骨髓中的潴留。
Fig 5c： To address this, we first determined in vivo if these chemokines were upregulated in FACS-purified BM macrophages from 5TGM1 engrafted KaLwRij mice and whether this upregulation could be inhibited by H-151.
Fig 5d： 与 KaLwRij 鼠的对照M巨噬相比, 5TGM1 engrafted animals 的巨噬的 CCL5, CXCL2 和 CXCL10 mRNA表达显著上调。 Moreover, STING inhibition 显著降低了CXCL2 和 CCL5 的表达。
Fig 5e： 接着，作者使用 in vitro migration assay in which conditioned media from BMDM were treated with mtDAMPs with and without STING inhibition with H-151. 结果显示 conditioned media from mtDAMP treated macrophages increased the migration of 5TGM1 cells, whereas when STING signalling was inhibited cell migration was reduced.
Fig 5f： STING inhibition in 5TGM1 engrafted KaLwRij mice resulted in an increased frequency of 5TGM1 cells in the BM.
作者使用氯膦酸盐脂质体清除巨噬也得到了一致的结果（附件）。
Together, these data show that myeloma cells release mtDAMPs into the BM which activate resident macrophages to produce a chemotaxis signature resulting in myeloma BM retention.
