骨肉瘤（Osteosarcoma，OS）是一种高度恶性的原发性骨肿瘤，尽管“新辅助化疗+手术+术后辅助化疗”模式提高了患者的五年生存率，但整体治疗进展仍然有限。骨肉瘤的主要特征为染色体不稳定性（Chromosomal Instability，CIN），表现为染色体畸变、致癌基因扩增和核型异常，导致肿瘤异质性增加，进一步加大了治疗难度。近年来，单细胞转录组测序（Single-cell RNA-Seq，scRNA-seq）及空间转录组学（Spatial Transcriptomics，ST）等高通量技术的发展，为解析骨肉瘤的分子病理特征及肿瘤微环境提供了新的可能性。PRC1作为胞质分裂过程中的关键蛋白，其在骨肉瘤发生发展中的具体作用、表达异质性及与肿瘤微环境相互作用的机制尚不明确。本研究旨在针对骨肉瘤高度异质性和缺乏有效治疗方案的现状，综合运用scRNA-seq、转录组测序和ST等多组学技术，系统解析PRC1基因在骨肉瘤细胞增殖及染色体畸变中的关键作用，并结合高通量筛选发现高效的协同致死治疗策略，为骨肉瘤的精准诊疗提供新的思路与依据。

本研究首先从cBioPortal数据库获取了三个队列总计719例骨肉瘤患者的全基因组测序数据，初步分析了骨肉瘤的致癌机理。随后，我们对两个队列的骨肉瘤患者的骨肉瘤组织微阵列（Tissue Microarray，TMA）进行免疫组化染色，检测并量化PRC1的表达水平，再结合Cox生存分析及机器学习算法评估其在预后预测中的重要性。我们对14例骨肉瘤患者的肿瘤组织进行scRNA-seq，运用CIBERSORTx、CellChat、pySCENIC、Metaprogram、irGSEA、Numbat和scTour等多种分析工具，综合解析骨肉瘤的肿瘤微环境特征，从分子功能、肿瘤异质性、细胞互作网络及体细胞拷贝数变异（Somatic Copy Number Alteration，SCNA）等多个层面系统探究PRC1在骨肉瘤中的生物学作用。此外，结合ST技术进一步评估了骨肉瘤的空间异质性及PRC1与肿瘤微环境的相互作用特征。在功能验证方面，我们采用siRNA敲降PRC1，并通过克隆形成实验及分子生物学检测验证PRC1作为治疗靶点的潜力。我们进一步利用高通量药物筛选平台，评估PRC1敲降与靶向细胞周期检查点或DNA损伤应答（DNA Damage Response，DDR）相关药物的协同治疗效果，以筛选出最佳协同致死组合方案。最后，通过裸鼠成瘤模型验证筛选获得的高效治疗策略的体内疗效，为骨肉瘤的个性化治疗提供实验依据。

本研究通过多维度多组学分析系统阐明了CIN作为骨肉瘤的关键分子特征，并首次揭示了PRC1在骨肉瘤致癌机制中的重要作用及其作为潜在治疗靶点的可行性。通过cBioPortal数据库对骨肉瘤基因组突变的分析发现，骨肉瘤患者的基因组异常主要集中于细胞周期（如TP53、CDK4、RB1）和基因组稳定性（如MYC）相关基因，进一步明确了细胞周期失调与DDR缺陷在骨肉瘤致癌过程中的核心地位。此外，利用Numbat算法在单细胞水平对SCNA进行深入分析，揭示了骨肉瘤细胞群体内部明显的SCNA异质性及高度的CIN，这种广泛存在的CIN不仅推动了肿瘤细胞的克隆进化，也为骨肉瘤的侵袭、转移和耐药提供了分子基础，进一步强调了CIN在骨肉瘤发生发展中的核心驱动作用。免疫组化分析显示，PRC1的高表达与骨肉瘤患者的不良预后密切相关，进一步的Cox回归分析证实，PRC1高表达是骨肉瘤患者预后的独立危险因素。基于scRNA-seq数据的分析结合多种生物信息学工具及算法，发现PRC1不仅在骨肉瘤细胞周期和染色体倍数变化中发挥关键作用，还与肿瘤微环境中的免疫细胞群体，尤其是成骨细胞（Osteoblastic）和破骨细胞（OC）群体的相互作用密切相关。通过Metaprogram分析，我们进一步揭示了骨肉瘤细胞群体的异质性，特别是在细胞周期、应激反应、代谢调节等方面的功能分化。scTour算法分析显示，PRC1低表达的细胞呈现向高迁移性和侵袭性的状态转变，证明PRC1在细胞迁移和分化中的调控作用。功能验证方面，PRC1敲降与RAD51抑制剂联合使用在高通量药物筛选中表现出显著的协同致死效应，抑制了肿瘤细胞增殖并诱导DDR。裸鼠成瘤模型结果进一步支持了这一协同治疗策略的体内疗效，显著抑制了肿瘤的生长，表明PRC1与RAD51联合靶向治疗在骨肉瘤中具有潜在的临床应用价值。

本研究系统揭示了骨肉瘤中PRC1基因在染色体畸变、异常核型维持以及肿瘤细胞增殖过程中的关键作用。通过多组学整合分析和功能验证，不仅发现了PRC1高表达与患者预后不良的密切关联，而且确定了联合敲降PRC1与抑制RAD51的协同致死策略。该研究为进一步深入理解骨肉瘤的分子病理学机制，并为精准化的治疗干预及个性化药物筛选提供了全新的思路与证据。

Osteosarcoma (OS) is a highly malignant primary bone tumor. Although the "neoadjuvant chemotherapy + surgery + adjuvant chemotherapy" treatment strategy has improved the five-year survival rate, overall treatment progress remains limited. A prominent characteristic of OS is chromosomal instability (CIN), manifested by chromosomal aberrations, oncogene amplification, and abnormal karyotypes, which increase tumor heterogeneity and complicate treatment. Recent advancements in high-throughput technologies, such as single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST), have provided new opportunities to analyze the molecular pathological characteristics and tumor microenvironment (TME) of OS. PRC1, a key protein involved in cytokinesis, has an unclear role regarding its specific functions, expression heterogeneity, and interactions within the OS microenvironment. Given the high heterogeneity of OS and the lack of effective treatment strategies, this study systematically utilized multi-omics techniques, including scRNA-seq, transcriptomics sequencing, and ST, to investigate PRC1’s critical role in OS cell proliferation and chromosomal aberrations. Additionally, high-throughput drug screening was employed to identify effective synthetic lethality strategies, offering novel insights and evidence for precise diagnosis and treatment of OS.

Initially, whole-genome sequencing data from 719 OS patients across three cohorts from the cBioPortal database were analyzed to preliminarily elucidate OS tumorigenesis mechanisms. Subsequently, immunohistochemical staining on tissue microarray (TMA) from two OS patient cohorts was conducted to quantify PRC1 expression, combined with Cox survival analysis and machine learning algorithms to evaluate its prognostic significance. scRNA-seq was performed on tumor tissues from 14 OS patients, and comprehensive analysis using various bioinformatics tools, including CIBERSORTx, CellChat, pySCENIC, Metaprogram, irGSEA, Numbat, and scTour, was conducted to investigate OS TME characteristics. These analyses systematically explored PRC1’s biological role from multiple aspects such as molecular function, tumor heterogeneity, cellular interaction networks, and somatic copy number alterations (SCNAs). ST was further utilized to evaluate OS spatial heterogeneity and PRC1 interactions within the TME. Functionally, PRC1 knockdown using siRNA was validated as a potential therapeutic target through colony formation assays and molecular biology tests. High-throughput screening further evaluated the synthetic lethality effects of combining PRC1 knockdown with cell cycle checkpoint inhibitors or DNA damage response (DDR)-related drugs, identifying optimal synthetic lethality combinations. Ultimately, the efficacy of the selected therapeutic strategy was validated in xenograft mouse models, providing experimental support for personalized OS treatment.

Through multidimensional multi-omics analyses, this study systematically elucidated CIN as a key molecular feature of OS and revealed for the first time the significant role of PRC1 in OS tumorigenesis and its potential as a therapeutic target. Genomic analysis from the cBioPortal database showed that OS genome abnormalities mainly involved genes related to cell cycle regulation (e.g., TP53, CDK4, RB1) and genomic stability (e.g., MYC), further highlighting the central roles of cell cycle dysregulation and DDR defects in OS tumorigenesis. Additionally, single-cell analysis using Numbat demonstrated significant SCNA heterogeneity within OS cell populations and highlighted widespread CIN, driving tumor cell clonal evolution, invasion, metastasis, and drug resistance. Immunohistochemistry indicated that high PRC1 expression closely correlated with poor OS prognosis, confirmed by Cox regression analysis as an independent prognostic factor. Further scRNA-seq analysis using multiple bioinformatics tools revealed that PRC1 not only plays a crucial role in OS cell cycle and chromosomal ploidy alterations but also significantly interacts with immune cell populations in the TME, particularly osteoblastic and osteoclastic cells. Metaprogram analysis revealed functional heterogeneity within OS cell populations, especially in cell cycle regulation, stress responses, and metabolic processes. scTour analysis further indicated that cells with low PRC1 expression exhibited enhanced migratory and invasive phenotypes, underscoring PRC1's regulatory roles in cell migration and differentiation. Functionally, combining PRC1 knockdown with RAD51 inhibitors demonstrated significant synthetic lethality effects, inhibiting tumor cell proliferation and inducing DDR. The xenograft model results further supported this combination therapy's efficacy, significantly suppressing tumor growth, and demonstrating its clinical potential for OS.

Further scRNA-seq analysis using multiple bioinformatics tools revealed that PRC1 not only plays a crucial role in OS cell cycle and chromosomal ploidy alterations but also significantly interacts with immune cell populations in the TME, particularly osteoblastic and osteoclastic cells. Metaprogram analysis revealed functional heterogeneity within OS cell populations, especially in cell cycle regulation, stress responses, and metabolic processes. scTour analysis further indicated that cells with low PRC1 expression exhibited enhanced migratory and invasive phenotypes, underscoring PRC1's regulatory roles in cell migration and differentiation. Functionally, combining PRC1 knockdown with RAD51 inhibitors demonstrated significant synthetic lethality effects, inhibiting tumor cell proliferation and inducing DDR. The xenograft model results further supported this combination therapy's efficacy, significantly suppressing tumor growth, and demonstrating its clinical potential for OS.