第一部分 肿瘤性骨软化症患者的骨骼特征 【背景】 肿瘤性骨软化症（tumor-induced osteomalacia，TIO）是由于肿瘤组织分泌过量的成纤维细胞生长因子23（fibroblast growth factor 23，FGF23）所导致的副肿瘤综合征，是最常见的获得性低血磷性骨软化症。骨活检显示TIO患者存在骨骼矿化障碍，且由双能X线吸收仪（dual-energy X-ray absorptiometry，DXA）评估的面积骨密度（areal bone mineral density，aBMD）显著降低，但DXA无法评估骨骼的微结构。而高分辨外周定量计算机断层扫描（high-resolution peripheral quantitative computed tomography，HR-pQCT）和基于腰椎DXA影像的骨小梁分数（trabecular bone score，TBS）的出现，分别为外周骨和腰椎的骨微结构评估提供了可能。国内外文献中鲜见TIO患者的骨微结构相关研究。成功切除TIO肿瘤后，患者的生化指标及临床症状可逐步改善至恢复正常，但缺乏术后短期内BMD及骨微结构变化的相关研究。此外，TIO患者中身高变矮十分常见且多不可逆转。然而，与身高变矮密切相关的椎体形变，在TIO患者中尚无研究进行系统描述。鉴于骨微结构在多种疾病中与骨折密切相关，其与TIO患者中椎体形变的关系也值得进一步探索。 【目的】 1、通过横断面研究，在TIO队列中应用HR-pQCT和TBS分别评估TIO患者术前外周骨及中轴骨的骨微结构特征；分析骨微结构与临床特征、生化指标的相关性；探索HR-pQCT参数与TBS的相关性。 2、通过纵向研究，评估TIO患者术后短期内（术后3个月）外周骨及中轴骨的骨密度和骨微结构的变化。 3、通过横断面研究，明确TIO患者中椎体形变的发生率、分布、类型、与临床指标的相关性，尤其是与骨微结构受损的相关性。 【方法】 1、研究对象：2016年1月-2021年3月期间于北京协和医院就诊的术后血磷恢复且经病理证实的TIO患者。 （1）TIO患者骨微结构横断面研究：术前进行了HR-pQCT或者DXA扫描；按年龄、性别、体重指数匹配的健康对照用于HR-pQCT参数的比较。 （2）TIO患者肿瘤切除术后骨微结构变化纵向研究：术前及术后3个月进行了HR-pQCT及DXA扫描的患者； （3）TIO患者椎体形变及其与骨微结构的关系研究：术前进行了胸腰椎侧位片拍摄的患者。 2、资料收集：通过病历系统收集患者的一般情况、病史、生化检查、术后病理等信息。并通过酶联免疫吸附试验测定血清全段FGF23（intact FGF23，iFGF23）。 3、影像学检查：（1）DXA扫描腰椎及右侧髋部评估中轴骨aBMD，并分析TBS；（2）HR-pQCT扫描桡骨和胫骨远端评估外周骨体积骨密度（volumetric BMD, vBMD）、骨微结构、骨强度；（3）胸腰椎侧位X线片用于评估椎体形变的类型和分级。 【结果】 1、TIO患者骨微结构横断面研究 （1）临床特征：共纳入186例TIO患者，其中男性107例，女性79例。平均年龄为44.5岁，中位病程为42个月。多数患者出现身高变矮（中位数为3.0 cm）。110例患者（59.1%）存在活动能力受损，112例患者（60.2%）有骨折史。实验室检查显示TIO患者血磷降低、肾脏排磷增多。 （2）TIO患者与健康对照的HR-pQCT比较：共计113例TIO患者在术前进行了HR-pQCT扫描。骨几何学方面，患者桡骨皮质骨面积（p = 0.028）和胫骨皮质骨面积（p < 0.001）显著低于对照，且胫骨的小梁骨面积显著高于对照（p = 0.002）。体积骨密度方面，患者桡骨和胫骨的小梁骨体积骨密度和皮质骨体积骨密度均低于对照（所有p < 0.001）。小梁骨微结构方面，患者桡骨和胫骨的骨小梁数量和小梁骨体积分数显著低于对照，而骨小梁分离度显著高于对照（所有p < 0.001）。皮质骨微结构方面，患者桡骨（p = 0.004）和胫骨（p < 0.001）的皮质骨厚度显著低于对照，而桡骨（p < 0.001）和胫骨（p = 0.030）皮质骨孔隙度显著高于对照。骨强度方面，患者桡骨骨硬度（p = 0.007）和骨破坏载荷（p = 0.005），以及胫骨骨硬度和骨破坏载荷（p < 0.001），均显著低于对照。组间差异以胫骨更为明显。 （3）DXA：TIO患者的TBS（1.15 ± 0.16）低于正常界值（1.35）。根据TBS评判的骨微结构异常（TBS < 1.35）的比例显著高于根据L1-4 aBMD评判的骨量异常（Z值 ≤ -2.0）的比例（89.6% vs. 43.9%，p < 0.001）。 （4）骨微结构与临床指标的相关性：更高的iFGF23、甲状旁腺素（parathyroid hormone，PTH）、碱性磷酸酶（alkaline phosphatase，ALP）、I型胶原C末端肽交联（β-isomerized C-terminal telopeptide of type I collagen，β-CTX）水平，以及骨折和更差的活动能力，均与更差的HR-pQCT参数相关，但与TBS无显著相关性。此外，更长的病程、更明显的身高变矮均与更差的HR-pQCT参数和更低的TBS显著相关。（5）HR-pQCT参数与TBS的相关性：在99例术前进行了DXA和HR-pQCT扫描的患者中，TBS与桡骨和胫骨的HR-pQCT参数均显著相关。 2、TIO患者肿瘤切除术后骨微结构变化纵向研究 （1）临床特征：共有22例患者在术后3个月进行了HR-pQCT和DXA扫描。手术3个月后，所有患者的血磷均已恢复正常，ALP显著下降，但β-CTX显著升高（所有p < 0.001）。术后PTH水平趋于升高。 （2）术后aBMD和TBS的变化：股骨颈、全髋、L1-4的aBMD分别增加了21.6%、18.9%、29.5%（所有p < 0.001）。TBS增加了14.1%（p < 0.001）。 （3）术后HR-pQCT参数的变化：骨几何学方面，桡骨皮质骨面积显著降低（p = 0.045）。体积骨密度方面，桡骨小梁骨体积骨密度（p = 0.002）和胫骨皮质骨体积骨密度显著降低（p = 0.046）。骨微结构方面，桡骨小梁骨体积分数（p = 0.007）、骨小梁数量（p = 0.025）、皮质骨厚度（p = 0.045）显著降低，但骨小梁分离度显著升高（p = 0.011）。胫骨小梁骨体积分数（p = 0.006）、骨小梁数量（p = 0.013）显著降低，但骨小梁分离度（p = 0.024）和皮质骨孔隙度（p = 0.019）显著升高。桡骨和胫骨的骨强度均无显著变化。 （4）术后DXA参数变化与HR-pQCT参数变化的相关性：股骨颈aBMD的变化与桡骨小梁骨体积骨密度的变化及小梁骨体积分数的变化呈显著负相关，但与骨小梁分离度的变化呈正相关。全髋aBMD的变化与桡骨上述参数的变化也存在类似的相关性。 3、TIO患者椎体形变及其与骨微结构的关系研究 （1）临床特征：共纳入术前进行了胸腰椎侧位片拍摄的164例患者。中位年龄是45.0岁，中位病程为43个月。患者在病程中平均变矮3.0 cm。 （2）椎体形变概况：99例患者（60.4%）发生了椎体形变，总计517个椎体。形变椎体呈双峰模式分布（T7-9和T11-L1），双凹变是最常见的形变类型（267/517，51.6%）。 （3）发生椎体形变的TIO患者与未发生椎体形变的TIO患者的临床特征比较：椎体形变组的男性比例更高（p < 0.001）、病程更长（p = 0.002）、身高变矮更明显（p < 0.001）；血磷更低（p = 0.025）、骨转换标志物更高（p < 0.016）；股骨颈aBMD、全髋aBMD、L1-4 aBMD、TBS更低；外周骨vBMD更低、骨微结构更差（p < 0.05）。 （4）TIO患者椎体形变与骨微结构的相关性分析：TBS及HR-pQCT参数中部分小梁骨相关的参数与椎体形变显著相关，且独立于中轴骨aBMD。 （5）不同椎体形变类型组间比较：根据Genant半定量方法的结果将患者分为楔形变、压缩变、双凹变、混合形变、无形变组，对发生形变的椎体的数目进行校正后，绝大多数组间差异均不再显著。 （6）发生形变的椎体数目、椎体形变指数（spinal deformity index，SDI）与临床指标的相关性：形变椎体的数目及SDI与病程、身高变矮呈正相关；与血磷呈负相关，但与PTH、骨转换标志物、iFGF23呈正相关；与TBS、aBMD呈负相关，也与大部分的HR-pQCT参数呈显著相关。 【结论】 1、本研究在世界最大的TIO队列中，同时运用DXA和HR-pQCT揭示了TIO患者存在广泛的外周骨和中轴骨骨密度降低、骨微结构受损。骨微结构受损与病程、活动能力、骨折、生化指标密切相关。TIO患者中HR-pQCT参数与TBS显著相关。 2、术后短期内，TIO患者的中轴骨aBMD及TBS即可出现显著改善，但外周骨vBMD和骨微结构进一步恶化。髋部和股骨颈的改善与外周骨的恶化显著相关，提示术后可能存在外周骨向中轴骨的钙转移，需要进一步的研究证实该发现并探讨其潜在机制。 3、首次在TIO大队列中描绘了TIO患者椎体形变的概况，发现约60%的TIO患者发生了椎体形变。男性、病程长、身高变矮、血磷低、骨转换标志物高、骨损害与椎体形变相关。TBS低、HR-pQCT测得的小梁骨体积骨密度低及骨微结构受损是椎体形变的危险因素，且独立于中轴骨aBMD。临床表现、生化指标、骨骼受损情况均与发生形变的椎体数目及椎体形变程度密切相关，但与椎体形变类型的相关性较小。 【关键词】 肿瘤性骨软化症；骨微结构；高分辨外周定量计算机断层扫描；骨小梁分数；椎体形变 第二部分 肿瘤性骨软化症患者的血清代谢组学研究 【背景】 肿瘤性骨软化症（tumor-induced osteomalacia，TIO）是一种由于致病灶分泌过量的成纤维细胞因子23（fibroblast growth factor 23，FGF23）导致的十分罕见的代谢性骨病，以血磷降低为主要的生化表现。目前TIO患者的诊断困难，且致病机制不明。虽然代谢组学对于探索TIO的发病机制并发现早期诊断标志物可能存在重要意义，但TIO患者的代谢组学研究尚未开展。 【目的】 1、描绘TIO患者代谢轮廓，明确TIO患者代谢谱的变化。 2、鉴定诊断标志物。 3、对氧化脂质进行定量，为后续机制研究探寻思路。 【方法】 1、研究对象：纳入2018年1月-2019年1月期间于北京协和医院就诊的术后血磷恢复且经病理证实的TIO患者。年龄、性别匹配的健康对照源于我院健康体检中心。 2、资料收集：通过病历系统收集患者的一般情况、病史、生化检查、影像学检查、术后病理等信息。并通过酶联免疫吸附试验测定血清全段FGF23（intact FGF23，iFGF23）。 3、非靶向代谢组学：通过非靶向代谢组学，分析96份血清样本（32份TIO患者术前血清、32份对应的术后血清、32份匹配的健康对照血清）的代谢轮廓。通过主成分分析（PCA）、正交偏最小二乘法-判别分析（OPLS-DA）、稀疏偏最小二乘判别分析（sPLS-DA）判断模型对不同分组的区分度。对差异代谢产物进行通路富集寻找异常代谢通路。通过受试者操作特征（ROC）曲线寻找最具诊断价值的代谢物。 4、氧化脂质靶向代谢组学：通过靶向代谢组学，对36份血清样本（18份TIO患者术前血清、18份匹配的健康对照血清）中的氧化脂质进行绝对定量。通过PCA和OPLS-DA判断模型对患者和健康对照的区分度。比较并寻找患者与健康对照间差异显著的氧化脂质。 【结果】 1、非靶向代谢组学 OPLS-DA、sPLS-DA、聚类分析均显示TIO患者与健康对照间差异显著，但TIO患者术前与术后没有显著差异。对TIO患者与健康对照间的差异代谢物进行注释后，通路富集分析发现TIO患者中多条代谢途径受影响，其中以花生四烯酸（arachidonic acid，AA）代谢途径最为显著。基于ROC曲线，将曲线下面积（AUC）排名前五的氧化脂质（4-HDoHE、LTB4、5-HETE、17-HETE、9,10,13-TriHOME）组合成诊断panel，可有效区分TIO患者和健康对照（AUC = 0.951；95% CI，0.827-1）。 2、氧化脂质靶向代谢组学 对TIO患者和健康对照的血清氧化脂质进行靶向定量后，OPLS-DA显示两组间区分度较好。亚油酸（linoleic acid，LA）的代谢产物9,10-EpOME（p = 0.003）和12,13-EpOME（p = 0.011）在TIO组显著高于健康对照。AA的代谢产物6S-LXA4（p = 0.035）、19S-HETE（p = 0.003）、PGE2（p = 0.046）、PGD2（p = 0.024）在TIO组显著高于健康对照，而12S-HHTrE在TIO组显著低于健康对照（p = 0.035）。二十碳五烯酸（eicosapentaenoic acid，EPA）的代谢产物5S-HEPE在TIO组显著高于健康对照（p = 0.049）。二十二碳六烯酸（docosahexaenoic acid，DHA）的代谢产物4-HDoHE（p = 0.011）、19,20-EpDPE（p = 0.029）在TIO组显著高于健康对照组。 【结论】 1、TIO患者血清代谢谱与健康对照差异显著，其中花生四烯酸代谢途径异常最为显著。5种氧化脂质组合而成的诊断panel可有效识别TIO患者。 2、TIO患者血清氧化脂质与健康对照差异显著，其中PGE2升高在TIO过量分泌FGF23中的作用值得进一步探索。 【关键词】 肿瘤性骨软化症；代谢组学；花生四烯酸代谢途径；氧化脂质；PGE2 第三部分 PGE2通过EP4-ERK-GALNT3通路调控FGF23的机制研究 【背景】 成纤维细胞生长因子23（fibroblast growth factor 23，FGF23）是一种重要的调磷因子。在FGF23相关的低磷血症中，以因肿瘤分泌过量FGF23导致的肿瘤性骨软化症（tumor-induced osteomalacia，TIO）最为典型。目前认为炎症是FGF23的关键调控因素之一。我们前期的血清代谢组学研究发现TIO患者中花生四烯酸代谢途径过度活化，其下游代谢产物前列腺素E2（prostaglandin E2，PGE2）水平显著升高。作为促炎因子的PGE2对FGF23的作用尚不明确。探索PGE2对FGF23的调控机制，有望为TIO患者提供辅助治疗，特别是为难治性TIO患者提供新的治疗靶点。 【目的】 1、探索PGE2对FGF23的作用。 2、阐明PGE2对FGF23的调控机制。 【方法】 通过慢病毒感染构建稳定转染FGF23的MG63稳转株（MG63-FGF23-OE）及对照株（MG63-FGF23-NC）。对上述细胞进行PGE2干预，观察FGF23转录水平、胞内和分泌至培养基内的全段FGF23（intact FGF23，iFGF23）蛋白水平变化。通过麦胚凝集素亲和层析的方法富集发生O-糖基化修饰的FGF23蛋白（FGF23 with O-glycosylation，O-FGF23），观察PGE2干预后的变化。同时检测对FGF23进行O-糖基化修饰的多肽N-乙酰半乳糖氨基转移酶3（polypeptide N-acetylgalactosaminyltransferase 3，GALNT3）的转录及蛋白水平的变化，以及ERK和p-ERK蛋白水平变化。分别应用ERK激酶抑制剂（PD98059）、PGE2受体（EP4受体）拮抗剂预处理后，进行PGE2干预，观察是否削弱PGE2对FGF23的调控作用。在TIO肿瘤和瘤周组织中，通过定量PCR和免疫组化分别在转录水平和蛋白水平验证细胞实验中的发现。 【结果】 1、PGE2干预显著上调MG63-FGF23-OE细胞中FGF23的转录水平，但胞内FGF23蛋白水平变化不显著。PGE2干预可上调细核内缺氧诱导因子1A（hypoxia inducible factor 1 subunit alpha，HIF1A）蛋白水平。 2、PGE2干预可增加MG63-FGF23-OE细胞分泌至培养基中的iFGF23水平，但未在MG63-FGF23-NC中观察到该变化。同时，PGE2干预增加了MG63-FGF23-OE培养基中O-FGF23。此外，GALNT3的转录水平和蛋白水平，以及p-ERK蛋白水平均显著上调。 3、EP4受体拮抗剂预处理可削弱PGE2对MG63-FGF23-OE细胞中p-ERK、GALNT3的上调，和对培养基中iFGF23和O-FGF23的上调。 4、TIO肿瘤组织中FGF23和GALNT3的转录水平显著高于瘤周组织。 5、免疫组化显示FGF23、GALNT3、p-ERK，以及PGE2的上游合成酶（COX-1/2、mPGES-1/2）在TIO肿瘤组织中广泛表达，且表达水平高于瘤周。 【结论】 PGE2通过EP4受体激活下游ERK信号通路，从而上调GALNT3，增加FGF23的O-糖基化修饰，最终升高培养基中iFGF23水平。抑制PGE2生成有望成为TIO患者的辅助治疗选择。 

Part 1 Skeletal features of patients with tumor-induced osteomalacia 【Background】 Tumor-induced osteomalacia (TIO), the most prevalent form of acquired hypophosphatemic osteomalacia, is a rare paraneoplastic syndrome caused by excessive production of fibroblast growth factor 23 (FGF23) by a tumor. Biopsy studies have revealed generalized mineralization defects in TIO patients. Consistently, areal bone mineral density (aBMD), assessed by dual-energy X-ray absorptiometry (DXA) is reduced in most of them. However, DXA cannot evaluate bone microstructure. The emergence of high-resolution quantitative computed tomography (HR-pQCT) and trabecular bone score (TBS) based on the DXA image of lumbar spine made it possible to evaluate the bone microstructure of peripheral bone and lumbar spine, respectively. There are few studies focusing on the bone microstructure in TIO patients in the literature. After successful tumor resection, the biochemical indices and clinical symptoms could be obviously improved. However, there is a lack of research on short-term changes in BMD and bone microstructure after surgery. In addition, height loss is common and irreversible in TIO patients. However, vertebral deformity, which is closely related to height loss, has not been systematically described in TIO patients. Since bone microstructure closely relates to fractures in various diseases, the relationship between bone microstructure and vertebral deformity in TIO patients needs further exploration. 【Objectives】 1. To evaluate the bone microstructure of peripheral and axial bones in TIO patients by HR-pQCT and TBS; To analyze the correlation between bone microstructure and clinical parameters; To explore the correlation between HR-pQCT parameters and TBS. 2. To investigate the short-term changes in BMD and microstructure in both peripheral and axial bone after tumor resection in TIO patients. 3. To identify the prevalence, distribution, type and risk factors for the vertebral deformity in TIO patients. 【Methods】 1. Subjects: TIO patients who visited and underwent tumor excision surgery at Peking Union Medical College Hospital between January 1, 2016, and April 31, 2021, with recovery of serum phosphate and pathological confirmation after tumor resection. (1) Patients who underwent HR-pQCT scan or DXA scan before surgery were enrolled in the cross-sectional study of bone microstructure in TIO patients. Age-, sex-, and body mass index (BMI)-matched healthy control were also enrolled for the HR-pQCT study. (2) Patients who underwent HR-pQCT or DXA scan before surgery and 3 months after surgery were enrolled in the longitudinal study of changes in bone microstructure after tumor resection in TIO patients. (3) Patients who had lateral thoracolumbar spine radiographs before tumor resection were enrolled in the study of the relationship between vertebral deformity and bone microstructure in TIO patients. 2. Data collection: General condition, medical history, biochemical indices, and pathology of TIO patients were collected through the medical record system. Serum intact FGF23 (iFGF23) levels were measured by enzyme-linked immunosorbent assay. 3. Imaging examination: (1) DXA scans lumbar spine and right hip to evaluate aBMD in the axial bone and analyze TBS; (2) HR-pQCT scans distal radius and tibia to evaluate volumetric BMD (vBMD), microstructure and bone strength in the peripheral bone; (3) Lateral thoracolumbar spine radiographs were used to evaluate the type and grade of vertebral deformity. 【Results】 1. Cross-sectional study of bone microstructure in TIO patients (1) Clinical characteristics: A total of 186 adult TIO patients were enrolled, including 107 male and 79 female. Mean age was 44.5 years and median disease duration was 42 months. Patients experienced a median height loss of 3.0 cm. A total of 110 patients (59.1%) had impaired mobility, and 112 patients (60.2%) had a history of fracture. Laboratory investigation revealed hypophosphatemia with high urinary excretion of phosphate. (2) Comparison of HR-pQCT parameters between TIO patients and healthy controls: A total of 113 TIO patients underwent HR-pQCT scan before surgery. TIO patients had smaller cortical area than controls at the radius (p = 0.028) and tibia (p < 0.001), with larger trabecular area at the tibia (p = 0.002). Both cortical and trabecular vBMD were lower in TIO patients than in controls at two sites (all p < 0.001). TIO patients had lower trabecular bone volume ratio and trabecular number with higher separation than controls at both two sites (all p < 0.001). Both cortical thickness at the radius (p = 0.004) and tibia (p < 0.001) in TIO patients were lower than in controls, while cortical porosity at the radius (p < 0.001) and tibia (p = 0.030) were higher than in controls. Finally, stiffness (p = 0.007) and failure load (p = 0.005) at the radius, as well as tibia (p < 0.001), were lower in patients than in controls. Differences between patients and controls were more obvious at the tibia. (3) DXA: TBS in TIO patients (1.15 ± 0.16) was lower than the normal value (1.35). The proportion of patients with abnormal bone microstructure according to TBS (< 1.35) was higher than that with abnormal bone mass according to L1-4 aBMD (Z score ≤ -2.0) (43.9% vs. 89.6%, p < 0.001). (4) Correlation of bone microstructure with clinical indices: Higher iFGF23, parathyroid hormone (PTH), alkaline phosphatase (ALP), and β-isomerized C-terminal telopeptide of type I collagen (β-CTX) levels, worse mobility, and fracture were associated with poorer HR-pQCT parameters but not with lower TBS. Additionally, greater height loss and longer disease duration were correlated with worse HR-pQCT parameters and lower TBS. (5) Correlation of TBS and HR-pQCT parameters: In 99 TIO patients who had underwent HR-pQCT and DXA before tumor resection, TBS correlated with both trabecular and cortical HR-pQCT parameters. 2. Longitudinal study of changes in bone microstructure after tumor resection in TIO patients (1) Clinical characteristics: A total of 22 patients completed HR-pQCT and DXA scans at the time of 3-month follow-up. All patients recovered from hypophosphatemia. Although ALP declined, β-CTX increased significantly (all p < 0.001). PTH levels tended to increase. (2) Changes in aBMD and TBS: After surgery, aBMD increased by 21.6% in the femoral neck, by 18.9% in the total hip, and by 29.5% in the lumbar spine. TBS increased by 14.1% (all p < 0.001). (3) Changes in HR-pQCT parameters: After surgery, cortical area at the radius (p = 0.045), trabecular vBMD at the radius (p = 0.002), and cortical vBMD at the tibia (p = 0.046) decreased significantly. At the radius, trabecular bone volume ratio (p = 0.007), trabecular number (p = 0.025) and cortical thickness (p = 0.045) decreased, but trabecular separation increased (p = 0.011). At the tibia, trabecular bone volume ratio (p = 0.006) and trabecular number (p = 0.013) decreased, but trabecular separation (p = 0.024) and cortical porosity (p = 0.019) increased. Bone strength did not change significantly. (4) Correlation of changes in DXA parameters and HR-pQCT parameters: Changes in femoral neck aBMD were conversely associated with changes in trabecular vBMD and bone volume ratio, while positively correlated with change in trabecular separation at the distal radius. Similar pattern was observed between the changes in total hip aBMD and the above HR-pQCT parameters. 3. Study of the relationship between vertebral deformity and bone microstructure in TIO patients (1) Clinical characteristics: A total of 164 patients were enrolled. Median age was 45.0 years and median disease duration was 43 months. Patients experienced a mean height loss of 3.0 cm. (2) Outline of the vertebral deformity in TIO patients: 99 (60.4%) patients had 517 deformed vertebrae with a bimodal pattern of distribution (T7-9 and T11-L1). Biconcave deformity was the most common type (267/517, 51.6%). (3) Comparison between TIO patients with or without vertebral deformity: Patients with vertebral deformity had a higher male/female ratio (p < 0.001), longer disease duration (p = 0.002), more height loss (p < 0.001), lower serum phosphate (p = 0.025), higher bone turnover markers (all p < 0.02), lower aBMD and TBS, lower peripheral vBMD and worse microstructure (all p < 0.05). (4) Correlation of TBS and HR‑pQCT with vertebral deformity in TIO patients: TBS and partial trabecular HR-pQCT parameters were associated with vertebral deformity independently of aBMD. (5) Comparison among TIO patients with different types of vertebral deformity: After adjusting for the number of deformed vertebrae, most of the differences among patients with different types of vertebral deformity became unsignificant. (6) Correlation of number of deformed vertebrae and spinal deformity index (SDI) with clinical parameters in TIO patients: Number of deformed vertebrae and SDI were positively correlated with disease duration, height loss, PTH, bone turnover markers and iFGF23, but negatively correlated with serum phosphate, TBS and aBMD. They also significantly associated with most HR-pQCT parameters. 【Conclusion】 1. This study revealed extensive impairment in both the peripheral and axial skeleton by DXA and HR-pQCT in a large cohort of TIO patients. Bone microarchitecture impairment was correlated with disease duration, mobility, history of fracture, and biochemical indices. HR-pQCT parameters were correlated with TBS in TIO patients. 2. Although aBMD and microstructure in the axial bone were improved shortly after surgery, vBMD and microstructure in the peripheral bone were further impaired. Correlation of the improvement of hip and femoral neck with the deterioration of peripheral bone indicated shift in calcium from the peripheral bone to the axial bone in the short term after surgery, which needs further study to explore the potential mechanism. 3. We firstly described the vertebral deformity in a large cohort of TIO patients, and found 60% patients had vertebral deformity. Vertebral deformity was correlated with male sex, long disease duration, height loss, low serum phosphate, high bone turnover markers, and bone impairment. Lower vBMD and worse microstructure evaluated by HR-pQCT and lower TBS were associated with vertebral deformity independently of aBMD. Clinical manifestation, biochemical indices and bone impairment closely correlated with the number of deformed vertebrae and degree of vertebral deformity, but rarely correlated with the type of vertebral deformity. 【Keywords】 Tumor-induced osteomalacia; bone microstructure; high-resolution quantitative computed tomography; trabecular bone score; vertebral deformity Part 2 Serum metabolomics in patients with tumor-induced osteomalacia 【Background】 Tumor-induced osteomalacia (TIO), an extremely rare metabolic bone disease resulted from the excessive production of fibroblast growth factor 23 (FGF23) by the causative tumor, is characterized by hypophosphatemia. Currently, the diagnosis of TIO is difficult and the potential pathogenesis remains to be explored. Despite its importance to comprehensive understanding of pathogenesis and diagnosis, metabolomics analysis has not been conducted in TIO patients. 【Objectives】 1. To describe the metabolite profiling of TIO patients and characterize the metabolome alteration associated with TIO. 2. To identify diagnostic biomarker for TIO. 3. To quantify the oxylipins levels, providing clues for mechanism research. 【Methods】 1. Subjects: (1) TIO patients who visited and underwent tumor excision surgery at Peking Union Medical College Hospital between January 1, 2018, and January 1, 2019, with recovery of serum phosphate and pathological confirmation after tumor resection. (2) Age- and sex-matched healthy controls (HC) were recruited from Physical Examination Center. 2. Data collection: General condition, medical history, biochemical indices, and pathology of TIO patients were collected through the medical record system. Serum intact FGF23 (iFGF23) level was measured by enzyme-linked immunosorbent assay. 3. Untargeted metabolomics: By means of untargeted metabolomics, we analyzed the metabolite profiling of 96 serum samples (32 from TIO patients before surgery, 32 pairwise samples after surgery, and 32 from HC). Principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and sparse partial least squares discriminant analysis (sPLS-DA) were used to determine the discriminative power of the model for different groups. Pathway enrichment on differentially expressed metabolites (DEMs) was performed to find abnormal metabolic pathways. Receiver operating characteristic (ROC) curve was conducted to find the potential diagnostic markers. 4. Targeted metabolomics for oxylipins: By means of targeted metabolomics, we quantified the oxylipins levels in 36 serum samples (18 from TIO patients before surgery, and 18 from HC). PCA and OPLS-DA were used to determine the discriminative power of the model for different groups. Then, we compared oxylipins levels between TIO patients and HC to search for differentially expressed oxylipins. 【Results】 1. Untargeted metabolomics OPLS-DA, sPLS-DA, and cluster analysis revealed the significant differences between TIO patients and HC, while the TIO patients before and after surgery could not be well discriminated. Pathway enrichment on annotated DEMs revealed multiple affected metabolic pathways in TIO patients, especially the arachidonic acid (AA) metabolic pathway. Based on ROC curve, a diagnostic panel combining the top 5 oxylipins (4-HDoHE, LTB4, 5-HETE, 17-HETE, and 9,10,13-TriHOME) ranged by area under ROC curve (AUC) can effectively distinguish TIO patients from HC (AUC = 0.951; 95% CI, 0.827-1). 2. Targeted metabolomics for oxylipins After quantification of oxylipins in TIO patients and HC, OPLS-DA revealed good discrimination between two groups. The metabolites of linoleic acid (LA), including 9,10-EpOME (p = 0.003) and 12,13-EpOME (p = 0.011), were significantly higher in TIO patients than in HC. The metabolites of AA, including 6S-LXA4 (p = 0.035), 19S-HETE (p = 0.003), PGE2 (p = 0.046), and PGD2 (p = 0.024), were significantly higher in TIO patients than in HC, while 12S-HHTrE (p = 0.035) was lower in TIO patients. The metabolites of eicosapentaenoic acid (EPA), 5S-HEPE (p = 0.049), was significantly higher in TIO patients than in HC. The same pattern was observed among the metabolites of docosahexaenoic acid (DHA), including 4-HDoHE (p = 0.011) and 19,20-EpDPE (p = 0.029). 【Conclusion】 1. Metabolite profiling of TIO patients showed significant differences from HC. A diagnostic panel with 5 oxylipins could effectively discriminate TIO patients from HC. 2. Oxylipins levels in TIO patients showed significant differences from HC. Among then, function of high PGE2 levels in excessive production of FGF23 in TIO patients needs further study to explore. 【Keywords】 Tumor-induced osteomalacia; metabolomics; arachidonic acid metabolic pathway; oxylipins; PGE2 Part 3 Study on the mechanism of PGE2 regulating FGF23 via EP4-ERK-GALNT3 pathway 【Background】 Fibroblast growth factor 23 (FGF23) is an important regulator of phosphate. Among the FGF23 related hypophosphatemia, tumor induced osteomalacia (TIO) caused by excessive FGF23 secretion by the causative tumor is the most typical type. Currently, inflammation has been considered as one of the key regulators of FGF23. By serum metabolomics, we have found the arachidonic acid metabolic pathway was overactivated and prostaglandin E2 (PGE2), the downstream metabolite, increased significantly in TIO patients. As a pro-inflammatory factor, the effect of PGE2 on FGF23 is unclear. Exploring the regulatory mechanism of PGE2 on FGF23 is expected to provide adjuvant therapy for TIO patients, especially new therapeutic target for refractory TIO patients. 【Objectives】 1. To explore the effect of PGE2 on FGF23. 2. To clarify the regulatory mechanism of PGE2 on FGF23. 【Methods】 MG63 cell line stably transfected with FGF23 (MG63-FGF23-OE) and the negative control (MG63-FGF23-NC) were constructed by lentivirus infection. Cells were treated with PGE2 to observe the changes of FGF23 in the transcription level and protein level, including the intracellular and secreted intact FGF23 (iFGF23). The FGF23 protein with O-glycosylation （O-FGF23）was enriched by wheat germ agglutinin (WGA) affinity chromatography to observe the changes after PGE2 treatment. At the same time, we detected the changes in the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3), ERK, and p-ERK. After pretreatment with inhibitor of ERK kinase (PD98059) or antagonist of PGE2 receptor (EP4 receptor), PGE2 treatment was then performed to observe whether the effect of PGE2 on FGF23 was weakened by the above pretreatment. The findings in cell experiments were validated in TIO tumors and their surrounding tissues through quantitative PCR and immunohistochemistry at the transcriptional and protein levels, respectively. 【Results】 1. PGE2 treatment significantly upregulated the transcription level of FGF23 in MG63-FGF23-OE cells, but there was no significant change in intracellular FGF23 protein level. PGE2 treatment increased the level of hypoxia inducible factor 1A (HIF1A) protein in the nucleus. 2. PGE2 treatment increased the secreted iFGF23 protein in the medium of MG63-FGF23-OE cells, but not in the medium of MG63-FGF23-NC cells. At the same time, PGE2 treatment increased O-FGF23 in the medium of MG63-FGF23-OE cells. Additionally, transcription and protein levels of GALNT3, as well as protein level of p-ERK, were significantly upregulated. 3. Pretreatment with antagonist of EP4 receptor weakened the upregulation of p-ERK and GALNT3 by PGE2 in MG63-FGF23-OE cells, as well as the upregulation of iFGF23 and O-FGF23 in the medium. 4. The transcription levels of FGF23 and GALNT3 in TIO tumors were significantly higher than those in the surrounding tissue. 5. Immunohistochemistry showed that FGF23, GALNT3, p-ERK, and the upstream synthetases of PGE2 (COX-1/2, mPGES1/2) were widely expressed in tumor tissues, and their expression was higher than those in the surrounding tissues. 【Conclusion】 PGE2 activates the downstream ERK signaling pathway through the EP4 receptor, thereby upregulating GALNT3 which increases the O-glycosylation of FGF23, and ultimately increases the level of secreted iFGF23. Inhibiting PGE2 production is expected to become an adjuvant treatment option for TIO patients.