An Aptamer-Based Probe for Molecular Subtyping of Breast Cancer
An Aptamer-Based Probe for Molecular Subtyping of Breast Cancer
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DOI：
10.7150/thno.28949
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发表时间：
2018-01-01
期刊：
影响因子：
12.4
通讯作者：
He, Nongyue
中科院分区：
文献类型：
--
作者：
Liu, Mei;Wang, Zhifei;He, Nongyue
乳腺癌的分子分型由于其个性化治疗和预后的潜力而引起了相当大的兴趣。然而，目前的方法不能用于精确的亚型，从而在临床实践中提出了挑战。本研究的目的是开发一种基于细胞特异性单链DNA （ssDNA）适配体的荧光探针，用于乳腺癌分子分型。方法：采用Cell-SELEX法筛选DNA适配体。利用流式细胞术和共聚焦显微镜研究了适体的特异性、结合亲和力、温度对其结合能力的影响以及靶型分析。采用体外和体内荧光成像技术区分乳腺癌细胞、组织切片和荷瘤小鼠的分子亚型。结果：通过Cell-SELEX连续21轮体外筛选，进化出6个SK-BR-3乳腺癌细胞特异性ssDNA适配体。所选适配体Kd值均在低纳摩尔范围内，其中适配体sk6 Kd值最低，为0.61 +/- 0.14 nM。然后，获得了一个截断的基于适配体的探针，sk6Ea，只有53 nt，对靶细胞具有高特异性和结合亲和力。这种基于适配体的探针能够区分SK-BR-3、MDA-MB-231和MCF-7乳腺癌细胞，并能区分乳腺癌细胞和MCF-10A正常人乳腺上皮细胞；2)体外区分her2富集乳腺癌组织与Luminal A、Luminal B、三阴性乳腺癌组织及邻近正常乳腺组织（anbt）；3)在体内30min内将SK-BR-3荷瘤小鼠与MDA-MB-231和MCF-7荷瘤小鼠的异种移植物区分开来。结论：基于适配体的探针是一种快速、高灵敏度的乳腺癌体外和体内分型的有力工具，在乳腺癌分子亚型的识别、诊断和靶向治疗方面具有广阔的应用前景。
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.Methods: 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. In vitro and in vivo fluorescence imaging were used to distinguish the molecular subtypes of breast cancer cells, tissue sections and tumor-bearing mice.Results: Six SK-BR-3 breast cancer cell-specific ssDNA aptamers were evolved after successive in vitro 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) in vitro; 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 in vivo.Conclusion: The results suggest that the aptamer-based probe is a powerful tool for fast and highly sensitive subtyping of breast cancer both in vitro and in vivo and is also very promising for the identification, diagnosis, and targeted therapy of breast cancer molecular subtypes.