Abstract

Conventional approaches for studying and molecular typing of tumors include PCR, blotting, omics, immunocytochemistry, and immunohistochemistry. The last two methods are the most used, as they enable detecting both tumor protein markers and their localizations within the cells. In this study, we have investigated a possibility of using RNA aptamers, in particular, 2′-F-pyrimidyl-RNA aptamer ME07 (48 nucleotides long), specific to the receptor of epidermal growth factor (EGFR, ErbB1, Her1), as an alternative to monoclonal antibodies for aptacytochemistry and aptahistochemistry for human glioblastoma multiforme (GBM). A specificity of binding of FAM-ME07 to the receptor on the tumor cells has been demonstrated by flow cytometry; an apparent dissociation constant for the complex of aptamer – EGFR on the cell has been determined; a number of EGFR molecules has been semi-quantitatively estimated for the tumor cell lines having different amount of EGFR: A431 (10⁶ copies per cell), U87 (10⁴ copies per cell), MCF7 (10³ copies per cell), and ROZH, primary GBM cell culture derived from patient (10⁴ copies per cell). According to fluorescence microscopy, FAM-ME07 interacts directly with the receptors on A431 cells, followed by its internalization into the cytoplasm and translocation to the nucleolus; this finding opens a possibility of ME07 application as an escort aptamer for a delivery of therapeutic agents into tumor cells. FAM-ME07 efficiently stains sections of GBM clinical specimens, which enables an identification of EGFR-positive clones within a heterogeneous tumor; and providing a potential for further studying animal models of GBM.

中文翻译：

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用于人脑胶质母细胞瘤治疗诊断的 RNA 适体

摘要

肿瘤研究和分子分型的常规方法包括 PCR、印迹、组学、免疫细胞化学和免疫组织化学。最后两种方法是最常用的，因为它们能够检测肿瘤蛋白标记物及其在细胞内的定位。在这项研究中，我们研究了使用 RNA 适配体的可能性，特别是 2'-F-嘧啶基-RNA 适配体 ME07（48 个核苷酸长），特异于表皮生长因子受体（EGFR、ErbB1、Her1），如用于人类多形性胶质母细胞瘤 (GBM) 适体细胞化学和适体组织化学的单克隆抗体的替代品。流式细胞术已证明 FAM-ME07 与肿瘤细胞受体的特异性结合；已确定细胞上适体-EGFR 复合物的表观解离常数；每个细胞^{6 个}拷贝）、U87（每个细胞10 ^{4 个}拷贝）、MCF7（每个细胞10 ^{3 个}拷贝）和 ROZH，源自患者的原代 GBM 细胞培养物（每个细胞10 ^{4 个}拷贝）。根据荧光显微镜观察，FAM-ME07 直接与 A431 细胞上的受体相互作用，然后内化到细胞质中并易位到核仁；这一发现开启了 ME07 应用作为将治疗剂递送到肿瘤细胞中的护送适体的可能性。FAM-ME07 可有效染色 GBM 临床标本的切片，从而能够识别异质肿瘤内的 EGFR 阳性克隆；并为进一步研究 GBM 的动物模型提供了潜力。