基于超高通量液滴微反应器的单细胞水平肿瘤异质性研究体系的建立

Tumor heterogeneity is now widely accepted as the most basic mechanism for many important clinical cancer phenotypes, such as invasion, metastasis, and drug resistance. The illumination of this issue will enable more specific and highly efficient cancer diagnosis and therapy. However, the separation, purification, and characterization of low-abundance heterogeneous cell populations in tumor tissues is still a bottleneck for the exploration of tumor heterogeneity. In this study, we propose an ultrahigh-throughput droplet-based microreactor system for studying tumor heterogeneity at the single-cell level, which will allow us to achieve fast, sensitive, and low-cost analysis of tumor heterogeneity. Using the droplet-based microreactor generated and manipulated in a microfluidic chip system, we, for the first time, develop a cellular functional phenotype-based cell sorting technique at the genomic level, including KRAS gene mutation [anti-epidermal growth factor receptor (EGFR) resistance] phenotype and P16 gene promoter methylation (malignant) phenotype, which overcome the dependence of cell sorting on cell surface molecular markers, and get access to more significant genomic information in terms of diagnosis and treatment. At the transcriptomic level, we will barcode the transcriptome of each cell in droplets, and perform deep sequencing together through a single run. Each barcodes are unique, and represent only one single cell, demonstrating the capability of comparing gene expression level and splicing mode among individual cancer cells.

肿瘤异质性被广泛认为是肿瘤侵袭、转移及耐药等重要临床表型的共同机制，阐明其特殊的生物学特征将在制定特异、高效的肿瘤诊治策略上取得重大突破。但目前对大量低丰度的异质性细胞进行分离、纯化和鉴定仍然是现今研究的技术瓶颈。我们在本课题中拟建立的"基于超高通量液滴微反应器的肿瘤异质性研究体系"将有望实现快速、灵敏和低成本的单细胞水平肿瘤异质性分析。结合微流控芯片超高通量液滴微反应器平台，在基因组水平，我们首次以KRAS基因突变(抗表皮生长因子靶向治疗耐药表型)和P16基因启动子甲基化(恶性表型)等细胞功能表型作为筛选单个靶细胞的标志并测序，克服了利用细胞表面分子进行细胞分选的诸多局限，并能获得更具诊断及治疗意义的基因组信息。在转录组水平，我们建立起基于转录组条形码标记的单细胞水平肿瘤异质性研究新体系，高通量地进行单个细胞转录水平的异质性分析，从而获得各细胞间基因表达水平和剪接模式的差异。

肿瘤异质性被广泛认为是肿瘤侵袭、转移及耐药等重要临床表型的共同机制，阐明 其特殊的生物学特征将在制定特异、高效的肿瘤诊治策略上取得重大突破。但目前对大量低丰度的异质性细胞进行分离、纯化和鉴定仍然是现今研究的技术瓶颈。我们在本课题中成功建立起了"基于超高通量液滴微反应器的肿瘤异质性研究体系"，为实现快速、灵敏和低成本的单细胞水平肿瘤异质性分析奠定了基础。结合微流控芯片超高通量液滴微反应器平台，在基因组水平，我们首次以KRAS基因突变和P16基因启动子甲基化等细胞功能表型作为筛选单个靶细胞的标志并测序，克服了利用细胞表面分子进行细胞分选的诸多局限，并能获得更具诊断及治疗意义的基因组信息。在转录组水平，我们建立起基于转录组条形码标记的单细胞水平肿瘤异质性研究新体系，高通量地进行单个细胞转录水平的异质性分析，从而获得各细胞间基因表达水平和剪接模式的差异，将对未来临床诊断与治疗产生深远影响。