Advanced Cancer Classification via Single-Cell Electrophoretic Cytopathology

摘要 最近的努力，以确定和规避耐药的机制点的作用，HER 2截短 同种型，其缺乏曲妥珠单抗靶向的全长蛋白质的胞外结构域。理解 HER 2亚型的生物学对于开发针对耐药个体的个性化新药至关重要。 曲妥珠单抗治疗。然而，在石蜡包埋的组织切片（FFPE）中检测这些异构体是困难的。 由于缺乏同种型特异性抗体，无法通过常规方法（免疫组织化学，IHC） 以及在组织处理过程中表位完整性的丧失。为了克服这些障碍，本项目的目标是 是开发一种强大的工具，既可以对HER 2亚型进行回顾性研究， 以患者为基础进行亚型筛查，以定制乳腺癌治疗。 我们的努力填补了癌症研究中的一个主要空白：对组织切片进行蛋白质靶点检查的能力有限 缺乏高度特异性的抗体探针（分析）或在组织处理过程中失去表位完整性（预处理）， 分析的）。我们引入了全组织蛋白质印迹（wtWestern），可创建新型3D蛋白质“印记” 用于稳定储存和下游抗体探测。独特的双重功能 wtWestern电泳解析蛋白质（从2D组织切片中提取），沿着深度（z轴） 支撑光活性水凝胶层。暴露于UV光将蛋白质共价连接到水凝胶。我们 对生物样本库适用性的无固定剂印迹进行基准测试，将wtWestern应用于蛋白质测量 （包括同种型），并通过实现检测来最大化组织效用 20多种蛋白质用于癌症亚型分类。这种创新的wtWestern有望最大限度地提高质量 生物样本用于下游分析的实用性-对于形成 癌症诊断/诊断学和个性化医疗的基础。

ABSTRACT Recent efforts to identify and circumvent mechanisms of drug resistance point to the role of HER2 truncated isoforms, which lack the extracellular domain of the full-length protein targeted by trastuzumab. Understanding the biology of HER2 isoforms is crucial to developing new drugs personalized for individuals resistant to trastuzumab therapy. However, detection of these isoforms in paraffin-embedded tissue sections (FFPE) is impossible by conventional methods (Immunohistochemistry, IHC) due to the lack of isoform-specific antibodies and the loss of epitope integrity during tissue processing. To overcome these barriers, the goal of this project is to develop a robust tool that will both allow retrospective studies on HER2 isoforms and enable isoform screening on a patient-to-patient basis to tailor breast cancer treatment. Our efforts fill a major gap in cancer research: the limited ability to scrutinize tissue sections for protein targets that either lack highly specific antibody probes (analytical) or lose epitope integrity during tissue processing (pre- analytical). We introduce a whole-tissue western blot (wtWestern) that creates a novel 3D protein ‘imprint’ of the tissue section for stable storage and downstream antibody probing. The unique dual-functionality wtWestern electrophoretically resolves proteins (extracted from a 2D tissue section) along the depth (z-axis) of a supporting photoactive hydrogel layer. Exposure to UV light covalently links protein to the hydrogel. We benchmark the fixative-free imprint for biobanking suitability, apply the wtWestern to measurement of proteins (including isoforms) in archived frozen or FFPE tissue sections, and maximize tissue utility by enabling detection of 20+ proteins for cancer sub-type classification. This innovative wtWestern is expected to maximize the quality and utility of biospecimens for downstream analyses – critically important to retrospective studies that form the basis for both cancer diagnostics/prognostics and personalized medicine.