CD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of various types of cancers such as melanoma and breast cancer and serves as a potential marker for cancer detection. Recently, its important role as a classic exosome marker was also emphasized. In this work, via using a magnetic bead-based competitive SELEX (systematic evolution of ligands by exponential enrichment) procedure and introducing a 0.5 M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein (Kd = 38.71 nM and 78.43, respectively). Furthermore, CD63-1 was found to be efficient in binding CD63 positive cells, including breast cancer MDA-MB-231 cells and CD63-overexpressed HEK293T cells, with a medium binding affinity (Kd ~ 100 nM) as assessed by flow cytometry. When immunostaining assay was performed using clinical breast cancer biopsy, the CD63-1 aptamer demonstrated a comparable diagnostic efficacy for CD63 positive breast cancer with commercial antibodies. After developing a magnetic bead-based exosome immunoaffinity separation system using CD63-1 aptamer, it was found that this bead-based system could effectively isolate exosomes from both MDA-MB-231 and HT29 cell culture medium. Importantly, the introduction of the NaCl elution in this work enabled the isolation of native exosomes via a simple 0.5M NaCl incubation step. Based on these results, we firmly believe that the developed aptamers could be useful towards efficient isolation of native state exosomes from clinical samples and various theranostic applications for CD63-positive cancers.

中文翻译：

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用于高效癌症免疫化学和基于免疫亲和的外泌体分离的 CD63 适体的开发

CD63 是四跨膜蛋白跨膜 4 超家族的成员，也是高度 N 糖基化的 III 型溶酶体膜蛋白，已知可调节各种癌症（如黑色素瘤和乳腺癌）的恶性程度，并作为癌症检测的潜在标志物. 最近，还强调了其作为经典外泌体标记物的重要作用。在这项工作中，通过使用基于磁珠的竞争性 SELEX（通过指数富集系统进化配体）程序并引入 0.5 M NaCl 作为洗脱缓冲液，我们鉴定了两种具有高亲和力的 DNA 适配体（CD63-1 和 CD63-2）和对 CD63 蛋白的特异性（Kd 分别为 38.71 nM 和 78.43）。此外，发现 CD63-1 可有效结合 CD63 阳性细胞，包括乳腺癌 MDA-MB-231 细胞和 CD63 过表达的 HEK293T 细胞，通过流式细胞术评估具有中等结合亲和力 (Kd ~ 100 nM)。当使用临床乳腺癌活检进行免疫染色测定时，CD63-1 适体证明了与商业抗体对 CD63 阳性乳腺癌的诊断功效相当。在使用 CD63-1 适配体开发基于磁珠的外泌体免疫亲和分离系统后，发现这种基于磁珠的系统可以有效地从 MDA-MB-231 和 HT29 细胞培养基中分离外泌体。重要的是，在这项工作中引入 NaCl 洗脱可以通过简单的 0.5M NaCl 孵育步骤分离天然外泌体。基于这些结果，