Molecular and functional characterization of the normal and malignant plasma cell – bone interface – from single cell mutual interaction analysis to clinical implications in myeloma bone disease and survival

多发性骨髓瘤是一种恶性浆细胞在骨髓中扩散和积聚的疾病，随后诱导骨病和血管生成。这些特征与其他恶性疾病相同。多发性骨髓瘤在两个方面不同：骨髓瘤细胞（MMC）扩散并生活在与其生理对应物骨髓浆细胞（BMPC）相同的“自然环境”中。此外，如我们所示，MMC在诱导血管生成和与骨转换相互作用时，至少部分依赖于正常浆细胞的恶性行为功能。恶性浆细胞与正常浆细胞一样，依赖于通过与骨髓中的其他细胞类型相互作用而获得生存支持，统称为“小生境”。在体外，我们已经表明，在骨髓瘤/间充质基质细胞（MSC）//成骨细胞共培养，这种相互作用是突出的物理相互作用，并导致在两种细胞类型的表达和行为的变化。我们假设，这种物理相互作用产生了壁龛，并激发了个体特征，这些特征加强了粘附，支持了肿瘤细胞，破坏了骨生成，在临床上导致了骨丢失和病理性骨折。小生境可能赋予骨髓瘤休眠或抵抗（微小残留病）特征。我们通过阻断骨细胞特异性wnt拮抗剂sclerostin，观察骨髓瘤小鼠成骨细胞数量和骨形成的增加，证明了这种分子串扰是可药物化的。我们还表明，接触诱导的KISS 1 R系统是一个假定的目标，作为一个治疗诊断PET示踪剂。在这个应用中，我们将集中在表面分子之间的正常和恶性浆细胞和MSC/成骨细胞的相互作用。我们将使用包括单细胞RNA-seq在内的表达分析来解决群体异质性，并通过原子力显微镜和CRISPR/CAS9筛选对相互作用进行功能评估，随后在骨髓瘤小鼠模型中进行体内协作验证。在第二步中，我们的体外研究结果将与大量分子特征的恶性浆细胞样本以及评估骨疾病、浆细胞浸润和血管生成的全身成像相关。我们已经表明，纳米/微米级数据与大规模临床数据集的关系可以作为相关性的过滤器，并将允许识别临床前适用的发现。第一个证据是硬化蛋白仅在骨细胞中表达，而不是来自630例MM患者的恶性浆细胞;第二个证据是我们共同开发的针对BMPC/MMC细胞表面分子BCMA的T细胞双特异性抗体EM 801/901在2018年第1季度进入临床试验，为开发治疗诊断BCMA-PET-示踪剂的单独应用奠定了基础。我们将在µBone-consortium内为骨髓瘤传播，骨疾病和血管生成相关研究问题的合作网络做出贡献。

Multiple Myeloma is a disease of malignant plasma cells spreading and accumulating in the bone marrow with subsequent induction of bone disease and angiogenesis. These features are in common with other malignant diseases. Multiple myeloma is different in two regards: Myeloma cells (MMC) spread to and live in the same “natural environment” as their physiological counterpart, bone marrow plasma cells (BMPC). Further, as shown by us, MMC rely at least partly on normal plasma cell functions for malignant behaviour when inducing angiogenesis and interacting with bone turnover. Malignant like normal plasma cells depend on receiving survival-support by interaction with other cell types in the bone marrow, collectively called “niche”. In vitro, we have shown in myeloma/ mesenchymal stromal cell (MSC)//osteoblast co-cultures that this interaction is prominently exerted by physical interaction and leads to changes in expression and behaviour in both cells types. We hypothesize that this physical interaction creates niches and elicits individual signatures that strengthen adhesion, support tumor cells, and disrupt osteogenesis, clinically leading to bone loss and pathological fractures. Niches may confer dormancy or resistance (minimal residual disease) features to myeloma. We have shown this molecular crosstalk being drugable exemplified by blocking the osteocyte-specific wnt antagonist sclerostin, observing increased osteoblast numbers and bone formation in myeloma bearing mice. We also showed that the contact-induced KISS1R system is a putative target as a theranostic PET tracer.In this application, we will focus on surface molecules featuring the interaction between normal and malignant plasma cells and MSC/osteoblasts. We will use expression analysis including single cell RNA-seq to address population heterogeneity, and functional assessment of interaction by atomic-force microscopy and CRISPR/CAS9 screen with subsequent collaborative in vivo validation in myeloma mouse models. In a second step, our in vitro findings will be related to a large cohort of molecularly characterized malignant plasma cell samples alongside whole-body imaging assessing bone disease, plasma cell infiltration and angiogenesis. We have shown that relation of data at nano-/micro- to large scale clinical data sets can act as a filter for relevance and will allow identification of pre-clinically applicable findings. The first is evidenced by sclerostin being expressed in osteocytes only, in contrast to malignant plasma cells from 630 MM patients; the second by our co-development of the T-cell bispecific antibody EM801/901 against the BMPC/MMC cell-surface molecule BCMA entering clinical trials in Q1/18, building the basis for a separate application for development of a theranostic BCMA-PET-Tracer. We will contribute to a collaborative network for myeloma dissemination, bone disease and angiogenesis related research questions within the µBone-consortium.