BackgroundHER2 is considered as one of the most important, predictive biomarkers in oncology. The diagnosis of HER2 positive cancer types such as breast- and gastric cancer is usually based on immunohistochemical HER2 staining of tumour tissue. However, the current immunohistochemical methods do not provide localized information about HER2’s functional state. In order to generate signals leading to cell growth and proliferation, the receptor spontaneously forms homodimers, a process that can differ between individual cancer cells.Materials and methodsHER2 overexpressing tumour cells were dissociated from formalin-fixed paraffin-embedded (FFPE) patient’s biopsy sections, subjected to a heat-induced antigen retrieval procedure, and immobilized on microchips. HER2 was specifically labelled via a two-step protocol involving the incubation with an Affibody-biotin compound followed by the binding of a streptavidin coated quantum dot (QD) nanoparticle. Cells with membrane bound HER2 were identified using fluorescence microscopy, coated with graphene to preserve their hydrated state, and subsequently examined by scanning transmission electron microscopy (STEM) to obtain the locations at the single molecule level. Label position data was statistically analysed via the pair correlation function, yielding information about the presence of HER2 homodimers.ResultsTumour cells from two biopsies, scored HER2 3+, and a HER2 negative control sample were examined. The specific labelling protocol was first tested for a sectioned tissue sample of HER2-overexpressing tumour. Subsequently, a protocol was optimized to study HER2 homodimerization in single cells dissociated from the tissue section. Electron microscopy data showed membrane bound HER2 in average densities of 201–689 proteins/μm2. An automated, statistical analysis of well over 200,000 of measured protein positions revealed the presence of HER2 homodimers in 33 and 55% of the analysed images for patient 1 and 2, respectively.ConclusionsWe introduced an electron microscopy method capable of measuring the positions of individually labelled HER2 proteins in patient tumour cells from which information about the functional status of the receptor was derived. This method could take HER2 testing a step further by examining HER2 homodimerization directly out of tumour tissue and may become important for adjusting a personalized antibody-based drug therapy.

中文翻译：

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HER2 过表达原代福尔马林固定石蜡包埋肿瘤组织单细胞中 HER2 同源二聚体的可视化

背景HER2被认为是肿瘤学中最重要的预测性生物标志物之一。HER2 阳性癌症类型（如乳腺癌和胃癌）的诊断通常基于肿瘤组织的免疫组织化学 HER2 染色。然而，目前的免疫组织化学方法不能提供有关 HER2 功能状态的局部信息。为了产生导致细胞生长和增殖的信号，受体自发形成同源二聚体，这一过程在个体癌细胞之间可能有所不同。材料和方法 HER2 过表达的肿瘤细胞与福尔马林固定石蜡包埋 (FFPE) 患者的活检切片分离，经受热诱导抗原修复程序，并固定在微芯片上。HER2 是通过两步协议进行专门标记的，该协议涉及与 Affibody-生物素化合物一起孵育，然后结合链霉亲和素包被的量子点 (QD) 纳米颗粒。使用荧光显微镜鉴定具有膜结合 HER2 的细胞，涂上石墨烯以保持其水合状态，然后通过扫描透射电子显微镜 (STEM) 进行检查以获得单分子水平的位置。通过配对相关函数对标记位置数据进行统计分析，产生关于 HER2 同源二聚体存在的信息。结果 来自两次活检的肿瘤细胞评分为 HER2 3+，并检查了 HER2 阴性对照样品。首先针对 HER2 过表达肿瘤的切片组织样本测试了特定的标记方案。随后，优化方案以研究从组织切片分离的单细胞中的 HER2 同源二聚化。电子显微镜数据显示膜结合 HER2 的平均密度为 201–689 个蛋白质/μm2。对超过 200,000 个测量蛋白质位置的自动统计分析显示，患者 1 和 2 的分析图像中分别有 33% 和 55% 存在 HER2 同源二聚体。结论我们引入了一种能够测量单独标记位置的电子显微镜方法患者肿瘤细胞中的 HER2 蛋白，从中可以得出有关受体功能状态的信息。这种方法可以通过直接从肿瘤组织中检测 HER2 同源二聚化来进一步检测 HER2，并且对于调整基于抗体的个性化药物治疗可能变得重要。