A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

Summary

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein. 

Abstract

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer.

This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps.

The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

Introduction

Cancer is still one of the leading causes of mortality worldwide¹. Despite the significant improvement in cancer treatment in recent decades, anticancer drug development is still a highly debated topic. This is because chemotherapy, as the mainstay of cancer treatment, is accompanied by serious side effects that limit patient compliance with the treatment. Moreover, chemotherapy-induced cancer resistance to treatment has restricted its application as the sole choice of medical intervention. The application of monoclonal antibodies (mAbs) introduced an enhanced response to cancer treatments². The rationale of using mAbs w....

Protocol

NOTE: Prior to starting the experiment, wear personal protective equipment, including a lab coat, gloves, and goggles. See the Table of Materials for details about materials, reagents, equipment, and software used in this protocol.

1. Buffers required for the assay

1. Prepare the buffers required for this experiment-the SELEX buffer required for aptamer folding, Blocking Buffer (BB), Wash Buffer (WB), and Binding Buffer (BiB) (Table 1)-freshly on the day of the experiment and keep them on ice or at 4 °C.
   NOTE: Each aptamer requires a unique folding condition. This includes the....

Results

An important aspect of new drug discovery and development is assuring the selectivity and specificity of the drug candidate. This means that the drug candidate should be able to discriminate between different cells and only affect the cell population of interest (selectivity). Selectivity is studied using cell lines that differ in terms of expression of the protein of interest. In this study, MDA-MB-231 and HEK 293T cell lines were chosen as EpCAM-positive and -negative cells. Specificity is another determinant that show.......

Discussion

The key challenge with developing new aptamers is the lack of standard guidelines that applies to different steps of this process. McKeague et al. have recently demonstrated some of the associated challenges, which lead to unclear presentations of data in publications and failure to replicate the research. They proposed fundamental guidelines necessary for consideration in characterizing aptamers¹⁹. An aptamer binding assay is a critical step in screening and/or characterizing aptamers

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tubes with attached lid	Sigma-Aldrich	T6649
15 mL CellStar blue screw cap, conical bottom tube	Greiner Bio One	188271
5 mL serological pipettes	Greiner Bio One	606180
BD FACSCanto II Flow Becton Dickinson Cytometer	Becton Dickinson	N/A
BD FACSDiva V9.0	BD Biosciences	N/A
Bovine Serum Albumin (BSA), Lyophilized powder	Sigma-AldrichTM	A7906-50G
Bright-line Hemocytometer	Sigma-Aldrich	Z359629
Dulbecco’s Modified Eagle Medium (DMEM) High Glucose Media Powder	Life Technologies	12100046
Dulbecco’s Phosphate- Buffered Saline (DPBS)	Life Technologies	21300025
FlowJo, LLC 10.8.1	BD Biosciences	N/A
Foetal Bovine Serum (FBS)	Bovogen	SFBS-F
HEK293T	American Type Culture Collection	ACS-4500
Heracell 150i CO2 Incubator	Thermo Fisher Scientific	N/A
Heraeus Megafuge 16R Centrifuge	Thermo Fisher Scientific	N/A
Magnesium Chloride (MgCl2)	Sigma-Aldrich	M8266
MDA-MB-231	American Type Culture Collection	CRM-HTB-26
Microplate, PS, 96 well, F-bottom (Chimney well), Black	Greiner Bio One	655076
MiniAmp Thermal Cycler	Thermo Fisher Scientific	A37834
Phosphate-Buffered Saline (PBS) tablets	Life Technologies	18912014
Pyrogen- and RNase-free ultrapure water	Milli-Q
T75 Cell Culture flask	Cellstar	658170
TENN	Integrated DNA Technologies	N/A	5′-GC GCG TGCA CGC GC TA ACG GA TTCCTTT TCC GT-3
TEPP	Integrated DNA Technologies	N/A	5′-GC GCG GTAC CGC GC TA ACG GA GGTTGCG TCC GT-3′
Transfer RNA (tRNA)	Sigma-Aldrich	R8508-5X1ML
Trypan Blue Solution	Life Technologies	15250061
Trypsin-EDTA	Gibco	15400054