Establishing a Single-Cell Proteomic Atlas for Normal and Osteoarthritic Articular Cartilage

摘要 尽管已经提出了多种途径和靶点用于OA治疗，但临床中的药物失败率仍然很高。 审判数量惊人地高。成功有限的原因包括疾病发现较晚 以及缺乏对患者之间分子异质性的理解。在本建议中，我们的目标是 利用新开发的单细胞蛋白质组学技术，质谱仪（CyTOF）， 在单个细胞中同时检测40-80种蛋白质，目的是鉴定不同的细胞 OA软骨中的亚群。虽然软骨是一种相对简单的组织， 包裹在其分泌的细胞外基质（ECM）中，与细胞外基质相关的变性程度不同。 每一位OA患者都认为，在单细胞水平上了解这种组织（和其他关节组织）可以提供 对OA病理学和患者异质性的新见解。这将有助于单细胞转录组学 数据，具有额外的优点，即蛋白质组快照还可以识别活性信号通路， 已识别的子群体。单细胞蛋白质组学方法在稳健地鉴定 罕见的细胞群体，难以从RNA测序数据中辨别。在这份提案中，我们将建立 使用稀土金属标记的精制板， Aim 1中的抗体，以鉴定OA软骨中的不同亚群。在目标2中，我们将测试 两个新发现的罕见亚群在创伤后OA小鼠模型中具有治疗作用， 追踪它们随疾病进展的动态变化在目标3中，我们将分析药物治疗如何影响软骨 不同患者的亚群及其串扰，特别是为了区分均匀或异质的 患者队列中的反应。总的来说，拟议的研究将在确定新的 再生和病理细胞群的OA和测试其调制的治疗潜力。

Abstract Although multiple pathways and targets have been proposed for OA treatment, the rate of drug failure in clinical trials has been astoundingly high. The reasons for the limited success include the late detection of the disease and a lack of understanding of the molecular heterogeneity between patients. In this proposal, we aim to capitalize on the newly developed single-cell proteomic technique, mass cytometry (CyTOF) that allows detection of 40-80 proteins simultaneously in single cells, with the aim of identifying the diverse cellular subpopulations in OA cartilage. Although cartilage is a relatively simple tissue, with a single cell type being encapsulated in its secreted extracellular matrix (ECM), the variable degree of degeneration associated with each OA patient suggests that understanding this tissue (and other joint tissues) at a single cell level can provide novel insights into both OA pathology and patient heterogeneity. This will compliment single-cell transcriptomic data, with the additional advantage that the proteomic snapshot can also identify active signaling pathways in the identified subpopulations. The single-cell proteomic approach is especially pertinent in robustly identifying rare cell populations that are difficult to discern from RNA-sequencing data. In this proposal, we will establish single cell profiles of a large cohort of OA cartilage samples using a refined panel of rare earth metal labeled antibodies in Aim1 to identify distinct subpopulations in OA cartilage. In aim 2, we will test if the modulation of two newly identified rare subpopulations would be therapeutic in a mouse model of post-traumatic OA as well as follow their dynamics with disease progression. In Aim 3, we will analyze how drug treatments affect the cartilage subpopulations and their crosstalk in different patients especially to discern between a uniform or heterogenous response among the patient cohort. Collectively, the proposed studies will be impactful in identifying novel regenerative and pathological cell populations in OA and testing the therapeutic potential of their modulation.