Abstract

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.

Highlights

  • The CD63 protein, the firstly characterized tetraspanin protein encoded on human chromosome12q13, is a type III lysosomal membrane protein and plays important roles in membrane transport, fusion, and protein kinase signalling [1]

  • The tertiary structure of certain domains of the natural protein may undergo substantial changes after the nickel-coated plate-based immobilization, which in the end detrimentally affect the application of the identified aptamers to bind to native human CD63 proteins [22]

  • To other target immobilization methods, during target/library incubation, the library sequences might non- bind to the plastic matrices of the wells and result in systematic evolution of ligands by exponential enrichment (SELEX) failure [6]

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Summary

Introduction

The CD63 protein, the firstly characterized tetraspanin protein encoded on human chromosome. It has been observed that certain protein markers that are commonly expressed on exosomes offer a chance for the development of immunoaffinity-mediated exosome separation strategy, via exploring the specific binding between such markers and their affinity agents (e.g., antibodies). Even though antibody-based immunoaffinity ensures high-purity exosomes, in addition to the expensive antibody applications, the non-neutral pH and non-physiological elution system (for exosome and antibody separation) used in this strategy detrimentally affects the natural structure and biological functions of the resulted exosomes, which are not favorable for exosome-mediated biomedical study and various therapeutic investigations [27]. Via introducing a competitive-SELEX procedure, accompanied by using NaCl as an elution system, we endeavor to develop aptamer sequences eligible for CD63 positive tumor detection, and for the development of novel immunoaffinity strategy, enabling the separation of natural state exosomes for various basic and applied applications

Results and Discussion
CD63-1 Aptamer Is Able to Specifically Recognize CD63 Positive Cells
CD63-1 Aptamer Held Potential for Cancer Detection
Cell Lines and Cell Culture
CD63 Protein Target Immobilization
SELEX Selection
Emulsion PCR
ELISA Assay
FACS Assay
Fluorescence Microscopy Imaging
Immunohistochemistry Assay
3.10. Construction of CD63 Aptamer-Based Exosome Immunoaffinity Bead System
3.13. Statistical Analysis

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