Advanced Cancer Classification via Single-Cell Electrophoretic Cytopathology

摘要 最近对识别和规避耐药机制的努力表明，HER2被截短的作用 缺乏曲妥珠单抗靶向的全长蛋白的胞外结构域的异构体。理解 HER2亚型的生物学对于开发针对耐药个体的新药至关重要。 曲妥珠单抗疗法。然而，在石蜡包埋组织切片(FFPE)中检测这些亚型是 常规方法(免疫组织化学，IHC)不可能，因为缺乏异构体特异性抗体 以及在组织处理过程中表位完整性的损失。为了克服这些障碍，这个项目的目标是 是开发一种强大的工具，既可以对HER2亚型进行回顾性研究，又可以使 根据患者的不同情况进行异构体筛查，以量身定制乳腺癌治疗。 我们的努力填补了癌症研究中的一个主要空白：仔细检查组织切片寻找蛋白质靶点的能力有限 缺乏高度特异的抗体探针(分析性)或在组织处理过程中失去表位完整性(前 分析)。我们介绍了一种全组织蛋白质印迹(WtWestern)，它能产生一种新的3D蛋白质“印记”。 用于稳定储存和下游抗体探测的组织切片。独一无二的双功能 Wt Western电泳沿深度(z轴)解析蛋白质(从2D组织切片中提取) 支持光活性水凝胶层。暴露在紫外光下，蛋白质与水凝胶共价连接。我们 对不含固定剂的印迹进行生物库适宜性基准测试，将wt Western应用于蛋白质测量 (包括异构体)保存在存档的冰冻切片或FFPE组织切片中，并通过启用检测来最大限度地提高组织利用率 20+个蛋白质用于癌症亚型分类。这一创新的wtWestern有望最大限度地提高质量 用于下游分析的生物标记物的用途--对形成 癌症诊断/预后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.