Kolovskaya Olga S, Zyuzyukina Alena V, Dassie Justin P, Zamay Galina S, Zamay Tatiana N, Boyakova Nina V, Khorzhevskii Vladimir A, Kirichenko Daria A, Lapin Ivan N, Shchugoreva Irina A, Artyushenko Polina V, Tomilin Felix N, Veprintsev Dmitry V, Glazyrin Yury E, Minic Zoran, Bozhenko Vladimir K, Kudinova Elena A, Kiseleva Yana Y, Krat Alexey V, Slepov Eugene V, Bukatin Anton S, Zukov Ruslan A, Shesternya Pavel A, Berezovski Maxim V, Giangrande Paloma H, Kichkailo Anna S
Laboratory for Biomolecular and Medical Technologies, Prof. V.F. Voino-Yasenetsky Krasnoyarsk State Medical University, Krasnoyarsk, Russia.
Laboratory for Digital Controlled Drugs and Theranostics, Federal Research Center "Krasnoyarsk Science Center of the Siberian Branch of the Russian Academy of Science", Krasnoyarsk, Russia.
Front Mol Biosci. 2023 Jun 8;10:1184285. doi: 10.3389/fmolb.2023.1184285. eCollection 2023.
Breast cancer (BC) diagnostics lack noninvasive methods and procedures for screening and monitoring disease dynamics. Admitted CellSearch is used for fluid biopsy and capture of circulating tumor cells of only epithelial origin. Here we describe an RNA aptamer (MDA231) for detecting BC cells in clinical samples, including blood. The MDA231 aptamer was originally selected against triple-negative breast cancer cell line MDA-MB-231 using cell-SELEX. The aptamer structure in solution was predicted using mFold program and molecular dynamic simulations. The affinity and specificity of the evolved aptamers were evaluated by flow cytometry and laser scanning microscopy on clinical tissues from breast cancer patients. CTCs were isolated form the patients' blood using the developed method of aptamer-based magnetic separation. Breast cancer origin of CTCs was confirmed by cytological, RT-qPCR and Immunocytochemical analyses. MDA231 can specifically recognize breast cancer cells in surgically resected tissues from patients with different molecular subtypes: triple-negative, Luminal A, and Luminal B, but not in benign tumors, lung cancer, glial tumor and healthy epithelial from lungs and breast. This RNA aptamer can identify cancer cells in complex cellular environments, including tumor biopsies (e.g., tumor tissues vs. margins) and clinical blood samples (e.g., circulating tumor cells). Breast cancer origin of the aptamer-based magnetically separated CTCs has been proved by immunocytochemistry and mammaglobin mRNA expression. We suggest a simple, minimally-invasive breast cancer diagnostic method based on non-epithelial MDA231 aptamer-specific magnetic isolation of circulating tumor cells. Isolated cells are intact and can be utilized for molecular diagnostics purposes.
乳腺癌（BC）诊断缺乏用于筛查和监测疾病动态的非侵入性方法和程序。公认的CellSearch用于液体活检和仅捕获上皮来源的循环肿瘤细胞。在此，我们描述了一种用于检测临床样本（包括血液）中乳腺癌细胞的RNA适配体（MDA231）。MDA231适配体最初是使用细胞SELEX技术针对三阴性乳腺癌细胞系MDA-MB-231筛选得到的。利用mFold程序和分子动力学模拟预测了该适配体在溶液中的结构。通过流式细胞术和激光扫描显微镜对乳腺癌患者的临床组织评估了进化后适配体的亲和力和特异性。使用基于适配体的磁分离开发方法从患者血液中分离循环肿瘤细胞（CTCs）。通过细胞学、逆转录定量聚合酶链反应（RT-qPCR）和免疫细胞化学分析确认了CTCs的乳腺癌来源。MDA231可以特异性识别来自不同分子亚型患者（三阴性、Luminal A和Luminal B）手术切除组织中的乳腺癌细胞，但不能识别良性肿瘤、肺癌、神经胶质瘤以及肺和乳腺的健康上皮细胞。这种RNA适配体可以在复杂的细胞环境中识别癌细胞，包括肿瘤活检样本（例如肿瘤组织与切缘）和临床血液样本（例如循环肿瘤细胞）。基于适配体的磁分离CTCs的乳腺癌来源已通过免疫细胞化学和乳珠蛋白mRNA表达得到证实。我们提出了一种基于非上皮MDA231适配体特异性磁分离循环肿瘤细胞的简单、微创乳腺癌诊断方法。分离出的细胞是完整的，可用于分子诊断目的。
