Identification of the early footprint of myeloma bone disease: spatio-temporal mapping and quantifying of extracellular matrix biomarkers in murine model and human bone biopsies

多发性骨髓瘤(MM)导致骨组织中细胞外基质(ECM)的大量破坏，是最严重的骨病之一。损伤是通过刺激破骨细胞广泛的溶骨活性，同时阻止骨再生而引起的。骨细胞是适应性骨重建的协调者，刺激骨溶解，同时也是关键的骨形成信号通路的强有力的调节者，如Wnt和Notch信号。由于它们树枝状的相互连接，它们是成熟骨微结构建筑设计的中心。为了在最初阶段检测ECM和微结构的损伤并确定决定性的起始信号，我们需要新的用于骨ECM的分子和超微结构生物标志物和用于微结构成像的尖端方法。在前期工作中，我们发现小鼠骨细胞接种MM细胞后14天内ECM结构蛋白的表达减少。通过比较临床队列中人类多发性骨髓瘤细胞的表达数据集，我们证明了相同的ECM蛋白的表达减少与患者的生存不良有关。虽然骨细胞通过Notch信号通路直接与MM细胞相互作用，但我们已经证明Jagged1介导的Notch激活促进了MM细胞的增殖，而Notch抑制减少了MM骨病。目前尚不清楚骨细胞中的哪些信号通路控制着MM中ECM的破坏，我们假设在MM细胞扩散到骨髓并在骨髓中扩增后，骨细胞中的Notch信号通路在早期参与了ECM的改建。我们以我们的多发性骨髓瘤小鼠骨病模型为平台，比较骨细胞的分子变化和细胞外基质的超微结构降解。我们将在空间和时间上量化MM细胞接种后局部部位和MM细胞扩散时整个骨骼的早期ECM损伤演化。我们将应用适合骨结构表征的三维高分辨率材料科学技术。尤其是三维、亚毫米和亚微米同步加速器方法将根据空洞、矿物沉积和纤维取向来量化ECM结构损伤，我们将与MM骨病的进展和严重程度相关联。利用转录图谱，我们将随着时间的推移跟踪我们的候选途径的成分及其分子靶点，并识别骨细胞基因表达和途径活性的未知的非常早期的分子变化。小鼠模型中的结果将通过MM细胞和骨细胞的体外共培养来验证，并通过与人类骨活检和数据库中未确定意义的单克隆性伽马病、阴燃和症状性MM病例的比较来验证。我们相信，这将使我们能够在宿主和肿瘤中发现新的生物标志物和创新的治疗靶点。

Multiple myeloma (MM) causes massive destruction of the extracellular matrix (ECM) in bone, and is consequently one of the most serious bone diseases. Damage is induced by stimulating extensive osteolytic activity of osteoclasts while blocking bone regeneration. Osteocytes are the orchestrators of adaptive bone remodeling, stimulate osteolysis, and at the same time are potent modulators of key bone formation signaling pathways such as Wnt and Notch signaling. With their dendrite-like interconnectivity, they are at the center of the architectural design of the microstructure of mature bone. To detect damage to ECM and microstructure at the initial stage and to characterize decisive starter signals, we require new molecular and ultrastructural biomarkers for bone ECM and cutting edge methods for microstructure imaging. In preliminary work, we found decreased expression of structural proteins of the ECM in murine osteocytes within fourteen days after inoculation of MM cells into bone. By comparing expression data sets of human MM cells from clinical cohorts, we demonstrated that decreased expression of the same ECM proteins is associated with poor patient survival. While osteocytes interact directly with MM cells through the Notch signaling pathway, we have shown that Jagged1-mediated Notch activation drives MM cell proliferation and that Notch inhibition diminishes MM bone disease. It is not known which signaling pathways in osteocytes control ECM destruction in MM. We hypothesize that the Notch signaling pathway in osteocytes contributes early on to the altered remodeling of the ECM after MM cell spread to and expansion in the bone marrow. We use our murine model of MM bone disease as a platform to compare molecular changes in osteocytes and ultrastructural degradation of ECM. We will quantify in space and time, early ECM damage evolution at local sites after MM cell inoculation and throughout the skeleton as the MM cells spread. We will apply three-dimensional high-resolution material-science techniques suited for bone structural characterization. Especially three-dimensional, sub-millimeter and sub-micrometer synchrotron methods will quantify ECM structural damage in terms of voids, mineral deposits, and fiber orientation, in which we will correlate with the progression and severity of MM bone disease. Using transcriptomic profiling, we will track the components of our candidate pathway and its molecular targets over time and identify yet unknown very early molecular changes in osteocyte gene expression and pathway activity. Results in the mouse model will be validated with co-cultures of MM cells and osteocytes in-vitro and by comparison with human bone biopsies and databases in cases of monoclonal gammopathy of undetermined significance, smoldering and symptomatic MM. We are convinced that this will allow us to identify new biomarkers and innovative therapeutic targets in host and tumor.