1. Cell-free mitochondrial DNA is elevated in multiple myeloma.
Fig 1a： 為了定量MM患者的mtDAMP同规，作者首先取了MM患者的外周血和骨髓券勺，檢測了mtDNA关炼。
Fig 1b-c： MM患者外周血的mtDNA顯著升高匣吊，genomic DNA則沒有明顯變化。
Fig 1d： 對骨髓樣本的檢測發(fā)現(xiàn)社痛，同一個患者的骨髓mtDNA顯著高于外周血mtDNA命雀，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+單核相比，MM patient-derived cells和immortalised MM cell lines的mtDNA更高狐血。
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： 為了探究血漿中mtDNA的來源，作者使用immunocompromised NSG移植了immortalised human MM cell line MM1S房匆。作者每周取一次小鼠外周血，檢測了其中的鼠源和人源mtDNA含量浴鸿。
Fig 2c： 結果顯示病程中鼠源mtDNA沒有變化，人源mtDNA在14天開始升高岳链。
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天的時候，外周血和骨髓中的mtDNA都是升高的约急，而且骨髓中mtDNA的水平顯著高于外周血苗分。
Fig 2f-g：作者在另一個小鼠模型中驗證了前面的結果
3. MM induces STING-mediated activation of macrophages via mtDAMPs
Fig 3a： 作者從5TGM1^(GFP+ LUCI+)^ engrafted KaLwRij mice中分離了巨噬細胞。
Fig 3b-c： Engraftment of 5TGM1 in the KaLwRij mice was determined by GFP expressing cells in the BM.
Fig 3d： RT-qPCR 結果顯示 5TGM1 engrafted mice的BM macrophages中 GBP2, IFIT3 和 IRF7表達增加奴饮。
Fig 3e： 為了確定巨噬細胞中STING的激活是mtDNA的直接作用戴卜，作者使用了5TGM1條件培養(yǎng)基培養(yǎng)了KaLwRij鼠的BMDM琢岩。條件培養(yǎng)基首先使用離心去除了細胞和大的extracellular vesicles，然后使用100 kDa ultra-centrifugal filter過濾担孔，去除小extracellular vesicles和殘留的蛋白（培養(yǎng)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： 作者從小鼠肝臟分離了mtDAMPs干預BMDM也得到了一致的結果。
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單核細胞也存在著STING通路的激活（附件）娩缰。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對骨髓瘤小鼠起到了很好的治療效果拼坎。
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的上清中泰鸡，促炎的和與STING激活相關的細胞因子上調。趨化因子同樣出現(xiàn)上調盛龄。因此作者推測，MM-derived mtDAMPs 可以通過激活巨噬細胞的STING通路促進malignant plasma cells in the BM在骨髓中的潴留啊鸭。
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： 與 KaLwRij 鼠的對照M巨噬相比, 5TGM1 engrafted animals 的巨噬的 CCL5, CXCL2 和 CXCL10 mRNA表達顯著上調赠制。 Moreover, STING inhibition 顯著降低了CXCL2 和 CCL5 的表達。
Fig 5e： 接著钟些，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. 結果顯示 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.
作者使用氯膦酸鹽脂質體清除巨噬也得到了一致的結果（附件）政恍。
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.
