State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education (Southeast University), School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, P. R. China.
School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, P. R. China.
Theranostics. 2018 Nov 10;8(20):5772-5783. doi: 10.7150/thno.28949. eCollection 2018.
Molecular subtyping of breast cancer is of considerable interest owing to its potential for personalized therapy and prognosis. However, current methodologies cannot be used for precise subtyping, thereby posing a challenge in clinical practice. The aim of the present study is to develop a cell-specific single-stranded DNA (ssDNA) aptamer-based fluorescence probe for molecular subtyping of breast cancer. Cell-SELEX method was utilized to select DNA aptamers. Flow cytometry and confocal microscopy were used to study the specificity, binding affinity, temperature effect on the binding ability and target type analysis of the aptamers. and fluorescence imaging were used to distinguish the molecular subtypes of breast cancer cells, tissue sections and tumor-bearing mice. Six SK-BR-3 breast cancer cell-specific ssDNA aptamers were evolved after successive selection over 21 rounds by Cell-SELEX. The Kd values of the selected aptamers were all in the low-nanomolar range, among which aptamer sk6 showed the lowest Kd of 0.61 ± 0.14 nM. Then, a truncated aptamer-based probe, sk6Ea, with only 53 nt and high specificity and binding affinity to the target cells was obtained. This aptamer-based probe was able to 1) differentiate SK-BR-3, MDA-MB-231, and MCF-7 breast cancer cells, as well as distinguish breast cancer cells from MCF-10A normal human mammary epithelial cells; 2) distinguish HER2-enriched breast cancer tissues from Luminal A, Luminal B, triple-negative breast cancer tissues, and adjacent normal breast tissues (ANBTs) ; and 3) distinguish xenografts of SK-BR-3 tumor-bearing mice from those of MDA-MB-231 and MCF-7 tumor-bearing mice within 30 min . The results suggest that the aptamer-based probe is a powerful tool for fast and highly sensitive subtyping of breast cancer both and and is also very promising for the identification, diagnosis, and targeted therapy of breast cancer molecular subtypes.
由于其在个性化治疗和预后方面的潜力，乳腺癌的分子亚型分类受到了极大的关注。然而，目前的方法无法用于精确的亚型分类，因此在临床实践中构成了挑战。本研究旨在开发一种基于细胞特异性单链 DNA（ssDNA）适体的荧光探针，用于乳腺癌的分子亚型分类。利用细胞 SELEX 方法筛选 DNA 适体。利用流式细胞术和共聚焦显微镜研究适体的特异性、结合亲和力、温度对结合能力的影响以及靶标类型分析。荧光成像用于区分乳腺癌细胞、组织切片和荷瘤小鼠的分子亚型。经过 21 轮连续筛选，通过 Cell-SELEX 从 SK-BR-3 乳腺癌细胞中筛选出 6 种特异性 ssDNA 适体。所选适体的 Kd 值均处于纳摩尔级低浓度范围内，其中适体 sk6 的 Kd 值最低，为 0.61±0.14 nM。然后，获得了一种基于截断适体的探针 sk6Ea，其仅含有 53 个核苷酸，对靶细胞具有高度特异性和结合亲和力。该基于适体的探针能够：1）区分 SK-BR-3、MDA-MB-231 和 MCF-7 乳腺癌细胞，并区分乳腺癌细胞与 MCF-10A 正常人类乳腺上皮细胞；2）区分 HER2 富集型乳腺癌组织与 Luminal A、Luminal B、三阴性乳腺癌组织和相邻正常乳腺组织（ANBTs）；3）在 30 分钟内区分 SK-BR-3 荷瘤小鼠的异种移植物与 MDA-MB-231 和 MCF-7 荷瘤小鼠的异种移植物。结果表明，基于适体的探针是一种快速、高灵敏度的乳腺癌亚型分类的有力工具，对于乳腺癌分子亚型的鉴定、诊断和靶向治疗也具有很大的应用前景。
