Nanofluidic Wet-Cell for Genomic Analysis of Single Circulating Tumor Cells via Electron Microscopy

肿瘤内的遗传异质性是癌症治疗中的一个基本挑战。基因组变异可以在空间上发生 在给定的肿瘤内以及在身体不同区域建立的转移亚群之间。此外，癌症 随着治疗时间的推移而进化。为了选择最有效的治疗方法，需要能够 给出一幅病人癌症的“基因组图景”的全面图景，并衡量这幅图景是如何 不断进化。虽然重复组织活检是不切实际的，并且只提供局部信息，但循环中的肿瘤细胞(CTCs， 已经进入血流的癌细胞)可以提供患者癌症状态的微创图像。 然而，CTC既罕见又必然是异质性的：因此，以CTC为基础的基因组分析必须是 在单细胞水平上执行。在这项建议中，我们开发了一种直接分析单基因组的新方法 从单分子水平上从单CTCs中提取。我们的方法是基于开发一种纳米流体湿式电池 可以在电子显微镜内操作。完全密封的湿电池将允许DNA循环通过 位于电子透明膜(由薄的氮化硅或 原子薄的石墨烯薄膜)。DNA分子(专门用导电标记标记，而不是荧光标记) 将在通道中延伸，然后通过电子显微镜进行定位。该方法将产生一个全面的高分辨率 单个CTC的基因组图谱。

Intratumor genetic heterogeneity is a fundamental challenge in cancer treatment. Genomic variation can occur spatially within a given tumor and between metastatic sub-populations established in different regions of the body. In addition, cancer evolves in time in response to therapy. In order to choose the most effective therapy, there is need for technologies that can yield a comprehensive picture of the "genomic landscape" of a patient's cancer and a measure of how this landscape is evolving. While repeated tissue biopsies are impractical, and provide only local information, circulating tumor cells (CTCs, cancer cells that have entered the bloodstream) may provide a minimally invasive picture of a patient's cancer status. CTCs, however, are both rare and necessarily heterogeneous: CTC-based genomic analysis must thus necessarily be performed at the single cell level. In this proposal we develop a novel approach for direct analysis of single-genomes extracted from single-CTCs at the single-molecule level. Our approach is based on developing a nanofluidic wet-cell that can be operated inside an electron microscope. The wet-cell, which is completely sealed, will permit cycling of DNA through nanochannel arrays positioned below an electronically transparent membrane (formed from either thin silicon nitride or atomically thin graphene sheets). The DNA molecules (specifically labeled with conductive as opposed to fluorescent tags) will be extended in the channels and then mapped via electron microscopy. The approach will yield a comprehensive highresolution map of the genome of a single CTC.