Single Cell Technologies for Rapid Detection of Tumor Heterogeneity

 描述（由申请人提供）：目前，几乎没有方法可用于分析肿瘤异质性的演变;肿瘤的显微解剖仅提供关于主要种类的恶性细胞的信息，但无法检测具有再生肿瘤潜力的罕见治疗抗性亚克隆。通过单细胞分离和测序鉴定这些稀有细胞既耗时又昂贵。最近的下一代深度测序研究已经证明了个体癌症中克隆异质性的临床相关性，但目前尚不存在快速且具有成本效益的方法来测量和跟踪细胞群体中突变的共同发生率。因此，开发快速、灵活、单细胞技术，具有鉴定大量群体内单个细胞中多个基因的异质突变的能力，对于开发预防肿瘤复发的有效靶向疗法至关重要。为了克服这一挑战，我们建议采用新型纳米分子支架（称为“DNA折纸”）来捕获肿瘤细胞并捕获编码已知肿瘤抑制基因的mRNA。我们建议在乳腺肿瘤细胞系和原发性乳腺肿瘤中测试这些支架的特异性， p53、PTEN和PIK 3CA基因中的已知突变。这种方法将允许快速定量实体瘤细胞中的基因组多样性和进化顺序，以改善罕见恶性亚克隆的靶向。

 DESCRIPTION (provided by applicant): Currently, there are few methods available to analyze the evolution of tumor heterogeneity; micro-dissection of tumors only provides information on major species of malignant cells but is unable to detect rare therapy-resistant subclones that have the potential to regenerate tumors. Identification of these rare cells by single-cell isolatio and sequencing is both time-consuming and prohibitively expensive. Recent next-generation deep sequencing studies have demonstrated the clinical relevance of clonal heterogeneity within individual cancers, but currently rapid and cost-effective methods to measure and track the rates of co-occurrences of mutations in cell populations do not exist. Therefore, the development of rapid, flexible, single- cell technologies with the capacity to identify heterogeneous mutations of multiple genes in individual cells within bulk populations is critical for the development of effective targeted therapies that prevent tumor relapse. To overcome this challenge, we propose to adapt novel nanomolecular scaffolds (termed "DNA origami") to transfect tumor cells and capture mRNA encoding known-tumor suppressor genes. We propose to test the specificity of these scaffolds in breast tumor cell lines and primary breast tumor with known mutations in p53, PTEN, and PIK3CA genes. This approach will allow the rapid quantitation of genomic diversity and evolutionary order in cells from solid tumors for improved targeting of rare malignant subclones.