The authors acknowledge the Institute for Mental and Physical Health and Clinical Translation (IMPACT) SEED funding, the &#34;Alfred Deakin Postdoctoral Research Fellowship&#34; program at Deakin University, and the &#34;Australian Government Research Training Program Scholarship&#34;.&#160;

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
<ol>
	<li>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 &#176;C. 
	NOTE: Each aptamer requires a unique folding condition. This includes the SELEX buffer and folding temperature conditions. Care should be taken to fully replicate the methods from the original paper describing the aptamer10. In this experiment, all buffers are prepared in Dulbecco&#39;s phosphate-buffered saline (DPBS). The buffer volume required in each experiment depends on the number of cell lines, number of replicates, and number of aptamer concentrations that are tested.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2"></td>
			<td colspan="2">Ingredients</td>
			<td rowspan="2">Volume required</td>
		</tr>
		<tr>
			<td>Item</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>SELEX buffer</td>
			<td>MgCl2</td>
			<td>5 mM</td>
			<td>50 &#181;L per sample + 10% pipetting error</td>
		</tr>
		<tr>
			<td rowspan="4">Blocking Buffer</td>
			<td>MgCl2</td>
			<td>5 mM</td>
			<td rowspan="4">500 &#181;L per cell line</td>
		</tr>
		<tr>
			<td>BSA a</td>
			<td>1 mg/mL</td>
		</tr>
		<tr>
			<td>tRNA b</td>
			<td>0.1 mg/mL</td>
		</tr>
		<tr>
			<td>FBS c</td>
			<td>10% (v/v)</td>
		</tr>
		<tr>
			<td>Wash Buffer</td>
			<td>MgCl2</td>
			<td>5 mM</td>
			<td>1 mL for the first wash + 100 &#181;L per test sample + 10% pipetting error</td>
		</tr>
		<tr>
			<td rowspan="4">Binding Buffer</td>
			<td>MgCl2</td>
			<td>5 mM</td>
			<td rowspan="4">50 &#181;L per sample + 10% pipetting error</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>2 mg/mL</td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>0.2 mg/mL</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>20% (v/v)</td>
		</tr>
	</tbody>
</table>
Table 1: Buffers required for the binding assay. aBovine Serum Albumin, bTransfer Ribonucleic Acid, cFetal Bovine Serum.
2. Preparation of aptamers
NOTE: The aptamers used in the assay are tagged with a fluorescence reporter molecule, and therefore care should be taken to protect them from light.
<ol>
	<li>Prior to the experiment, prepare a 100 &#181;M stock (stock A) of test and control aptamers using pyrogen- and RNase-free ultrapure water (Figure 1). 
	NOTE: For long-term preservation, stock A should be kept in a freezer at -20 &#176;C.</li>
	<li>Prepare stock B as the working concentration of aptamers by diluting stock A using SELEX buffer (Table 1). To follow this protocol, dilute stock A to a 1,000 nM stock to prepare stock B (Figure 1).</li>
	<li>To make the aptamer ready for the formation of the 3-dimensional (3D) structure, in a 250 &#181;L tube, dilute stock B with SELEX buffer to prepare the required volume and concentration of the aptamer for folding. 
	NOTE: The folded aptamers will be exposed to an equal volume of cells. Therefore, the concentration of the aptamer that is set for folding should be 2x more concentrated than the desired final concentration. Use equation (1) to calculate the required volumes and concentrations. Remember to consider an extra 10% volume for the pipetting error. 
	Concentrationstock A &#215; Volumestock A = Concentrationstock B &#215; Volumestock B&#160; (1)</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64304/64304fig01.jpg" /> 
Figure 1: A diagram showing the steps in the preparation of aptamers. 1Stock 1 is stored at -20 &#176;C for long-term preservation. 2Working concentrations are prepared in SELEX buffer and are not stored. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64304/64304fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Maintenance of cancer cells
NOTE: Prior to commencement of the study, make sure that the cells are at their early passage numbers, show their typical morphological features, and are mycoplasma free. To test the selectivity and specificity of the aptamer, cell lines that are high, moderate, and low/negative expressors of the protein of interest are ideally required.
<ol>
	<li>Seed the cells in a T75 culture flask, using appropriate culture conditions. Grow them in a 5% CO2 humidified incubator, at 37 &#176;C. 
	NOTE: In this study, Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (complete medium) was used.</li>
	<li>When the cells reach ~80% confluency, passage them to a new flask containing fresh complete medium. 
	NOTE: Depending on the protein of interest and the cell line, 80% confluency could provide a suitable cell population for the binding assay. For the cell lines in this experiment, MDA-MB-231 and HEK 293T, 80% confluency is suitable. At this stage, proceed to section 4, the binding assay. Always check the expression of the protein of interest, using mAbs specific for that protein.</li>
</ol>
4. Binding assay
NOTE: Figure 2 summarizes the steps required in the binding assay in adherent cells.
<ol>
	<li>In a class II biosafety cabinet, collect the cells of each flask in tubes as follows:
	<ol>
		<li>Collect and discard the media in the flask, add 2 mL of PBS, spread it over the cells, and then, collect and discard the PBS. Repeat this step twice more to remove all traces of media that may inactivate trypsin. Add 1 mL of 0.25% of trypsin/EDTA to each flask and incubate for 5-10 min at 37 oC. Visualize the detachment of cells under a microscope.</li>
		<li>Add 1 mL of complete medium to the cells, and pipette the cells up and down to make a single-cell suspension. Pipette the cells into an appropriate tube and centrifuge at 200 &#215; g for 5 min. 
		NOTE: For non-adherent cells, collect the cells in a tube, centrifuge (200 &#215; g, 5 min), and proceed to step 4.1.3.</li>
		<li>Discard the supernatant and resuspend the cells in 1 mL of fresh medium. Count the cells using trypan blue staining, by diluting a certain volume of cell suspension with trypan blue. Distribute ~15 &#181;L of the mixture between a hemocytometer and a cover glass. Count the cells as previously described18, using equation (2): 
		<img alt="Equation 1" src="/files/ftp_upload/64304/64304eq01.jpg" style="margin: auto;" /> (2) 
		NOTE: Use the minimum possible volume of cell suspension and take a note of the dilution factor. For example, mixing equal volumes of cell suspension and 0.04% trypan blue gives a dilution factor of 2. Ensure high viability (live cells/total cells &#215; 100) of ~90% for most adherent cell lines before proceeding. Dead cells non-specifically take up aptamers and alter the results19. It is possible to use other cell counting techniques, such as using a cell counter.</li>
		<li>Collect the required number of cells, making sure to have 10 &#215; 104 cells per test sample. Consider an extra 10% volume for pipetting error. 
		NOTE: It is important to always keep the same cell count between experiments and replicates.</li>
		<li>Incubate the cells at 37 &#176;C for 2 h to allow for the stabilization of the protein of interest on the cell membrane following enzymatic detachment. 
		NOTE: This incubation period might differ according to the protein of interest.</li>
	</ol>
	</li>
	<li>During this 2 h incubation:
	<ol>
		<li>Set the temperature of the centrifuge to 4 &#176;C. Leave tRNA and stocks of aptamer at room temperature or on ice to thaw. To protect the fluorescence reporter molecule, protect the aptamer tubes from light. 
		NOTE: The role of tRNA is to block the nucleic acid binding sites.</li>
		<li>Prepare the SELEX buffer, BB, WB, and BiB (see section 1), keeping them all on ice or at 4 &#176;C. Set the thermocycler machine on an empty cycle. Place a 96-well black plate and flow cytometry tubes on ice. 
		NOTE: Setting the thermocycler on an empty cycle prepares the cooling and heating system and helps generate more reproducible results.</li>
	</ol>
	</li>
	<li>Following the 2 h incubation, centrifuge the cells at 500 &#215; g for 5 min. Discard the supernatant and resuspend the cells in 500 &#181;L of BB. Incubate the cells at 4 &#176;C for 30 min with intermittent mixing.</li>
	<li>During this 30 min incubation, perform aptamer folding as follows:
	<ol>
		<li>Make up the 2x concentrations of aptamers (see section 2), and then mix and incubate the aptamers in the thermocycler machine, according to the required folding conditions. For this EpCAM aptamer, use the following folding conditions of 95 &#176;C, 5 min, followed by 22 &#176;C, 10 min, and 37 &#176;C, 15 min. 
		NOTE: Always include a negative control (i.e., SELEX buffer without aptamers).</li>
	</ol>
	</li>
	<li>Following the 30 min incubation, centrifuge the cells (500 &#215; g, 5 min, 4 &#176;C), remove the supernatant, add 1 mL of WB, and centrifuge the cells again (500 &#215; g, 5 min, 4 &#176;C). Remove the supernatant and resuspend the cells in a suitable volume of the BiB.</li>
	<li>Pipette 50 &#181;L of the resuspended cells into each well of an ice-cold, 96-well black plate. Keep the cells on ice to inhibit internalization of the protein of interest.</li>
	<li>Pipette 50 &#181;L of the aptamers onto a 50 &#181;L volume of cells, mix, and incubate in darkness at 4 &#176;C for 30 min. Centrifuge the plate at 500 &#215; g, 5 min, 4 &#176;C, and carefully remove the supernatant.</li>
	<li>Carefully resuspend the pellet in WB and centrifuge at 500 &#215; g for 5 min. Repeat the wash step (4.7) 2x and resuspend in 100 &#181;L of WB for flow cytometric analysis. 
	NOTE: See Figure 3 for a diagram of interactions between aptamers and cells.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64304/64304fig02.jpg" /> 
Figure 2: A diagram depicting the steps in performing an aptamer-protein-binding assay. Abbreviations: SELEX = Systematic Evolution of Ligands by EXponential Enrichment; BB = Blocking Buffer; WB = Wash Buffer; BiB = Binding Buffer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64304/64304fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64304/64304fig03.jpg" /> 
Figure 3: A diagram showing the different types of cells and aptamers required to perform the aptamer binding assay. Abbreviation: EpCAM = epithelial cellular adhesion molecule. This figure was created using Biorender.com.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64304/64304fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Flow cytometry and data analysis
NOTE: Before turning on the flow cytometer, make sure that there are no &#34;bubbles&#34; in the membrane filter units for the shut-down solution, cleaning solution, and sheath fluid (0.9% NaCl). &#34;Bleed out&#34; bubbles if there are bubbles in the capsules. Make sure that the waste container is empty, and containers of sheath fluid, water, and 1% bleach in ultrapure water are full.
<ol>
	<li>Turn on the flow cytometer and then the computer. 
	NOTE: The details of running the flow cytometer explained here are specific to the machine and software demonstrated in the video (see Table of Materials). Other software would require appropriate training to use.</li>
	<li>Open the flow cytometry analysis software, log in to the program, and under the Cytometer tab, run Fluidics Start up.</li>
	<li>To create a new experiment, under the experiment tab, click New Folder and name the folder/experiment appropriately.</li>
	<li>Click on new folder to highlight, then under the experiment tab again, click New Experiment and name the experiment appropriately.</li>
	<li>To add the first sample/specimen, under the experiment tab, click New Specimen and name this specimen appropriately (name of cell line/control sample/ experiment sample).</li>
	<li>To add a tube sample, highlight the specimen (group) and under the experiment tab, click New Tube. Add the appropriate number of tubes and name.</li>
	<li>To prepare the required graphs, under the worksheet tab, open a new worksheet. Once the new worksheet window pops up, open the following using the worksheet screen (hover the mouse across the logo/pictures to find the names):
	<ol>
		<li>Prepare a dot blot graph of forward scatter (FSC) versus side scatter (SCC) to select the population of interest. Define the first gate by identifying and selecting the population of interest (P1) in a forward and side scatter density plot. Exclude the debris, which constitutes the population with the lowest forward scatter signal. 
		NOTE: The FSC parameter detects cells or events based on their size and the SCC discriminates them based on their granularity20.</li>
		<li>Prepare a dot blot graph of FSC-area (FSC-A) versus FSC-height (FSC-H) to select the single-cell population. Define the second gate by excluding doublet cell populations, as doublet cells considerably affect the results and conclusions. Exclude doublets by using FSC-H versus FSC-A density plots, where cells of the same size show a similar area and height. Hence, the singlets get clustered diagonally and separated from doublets. 
		NOTE: FSC is roughly proportional to the cell size. The voltage pulses are defined as FSC-H, the intensity of the signal, FSC-width that reflects cell size and the duration of the signal, and FSC-A, which is H &#215; W. Doublets have a double width and area value; therefore, gating for singlets is based on detecting disproportions between H, W, and A caused by doublets.</li>
		<li>Prepare a histogram of the number of events against the fluorophore of interest.</li>
	</ol>
	</li>
	<li>Before starting flow cytometry, ensure that the acquisition dashboard for controlling the sample acquisition, inspector, and cytometer to adjust voltage parameters, as well as the worksheet with all the graphs are open. 
	NOTE: At least 100 &#181;L of a 10 &#215; 104 cell suspension in a flow cytometry tube is needed to perform the analysis. Especially in case of lower viabilities, propidium iodide staining can be performed to select the viable cell population21,22.</li>
	<li>To run the first sample, on the left-hand side of the screen make sure the arrow pointing to the tube is green. If this arrow is not green, click on the arrow to make it green.</li>
	<li>Using a pipette, transfer each sample from the 96-well black plate to a flow cytometry tube. Run the untreated, unstained control sample on a low speed.</li>
	<li>On the acquisition dashboard, choose an appropriate number of events to record (30,000), change the flow rate to low, and click Acquire Data.</li>
	<li>Adjust the voltage for the FSC and SCC parameters. Ensure the cell population is centralized within the dot plot and that no cells are touching either axis of the graph to avoid losing the cells of interest.</li>
	<li>Increase the acquisition speed to medium or high to analyze the samples faster but do not exceed more than 200 events/s. Then, click Record Data.</li>
	<li>Perform the gating for P1 (Figure 4A) and the single-cell population (Figure 4B). Construct the histogram of events against the used fluorochrome and select P1 based on the data (allophycocyanin (APC) in this case) (Figure 4C).</li>
	<li>After adjusting the voltage, gating and recording the data, take out the sample and click Next Tube.</li>
	<li>Insert the next sample, and repeat recording data for all control and test samples (Figure 3).</li>
	<li>Once all the data are collected, wash the flow cytometer by running three tubes of 50% bleach, FACS rinse, and ultrapure water, each for 5 min at a high flow rate.</li>
	<li>Then, from the Cytometer dropdown menu, click Fluidics Shut Down.</li>
	<li>Prior to closing the software and turning off the machine and the computer, export the results as .fcs files to a USB drive to transfer and analyze them, as follows:
	<ol>
		<li>In the analysis software, press the NEW button to create a new document and window to handle the analysis. Drag the sample files into the new window.</li>
		<li>Double-click to open the unstained sample. Choose the P1 population, double-click on the P1 population to create a FSC-H versus FSC-A graph, and gate the single-cell population.</li>
		<li>Double-click on the gated single cells to create a histogram of events against the used fluorochrome.</li>
		<li>In the original window, select P1 and single cells and drag them to All Samples so that all samples now contain the same gating.</li>
		<li>Click on the Layout Editor button to open the Layouts window. Drag two samples (control and test) over one another to create an overlay histogram.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Cancer. World Health Organization Available from: <a target="_blank" href="https://www.who.int/news-room/fact-sheets/detail/cancer#:~:text=Cancer%20is%20a%20leading%20cause.and%20rectum%20and%20prostate%20cancers">https://www.who.int/news-room/fact-sheets/detail/cancer#:~:text=Cancer%20is%20a%20leading%20cause.and%20rectum%20and%20prostate%20cancers</a> (2022)</li><li>Liu, J. K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+of+monoclonal+antibody+development+-+Progress,+remaining+challenges+and+future+innovations.">The history of monoclonal antibody development - Progress, remaining challenges and future innovations.</a> Annals of Medicine and Surgery. 3 (4), 113-116 (2014).</li><li>Nakhjavani, M., Shigdar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+of+PD-1/PD-L1+axis+modulation+for+the+treatment+of+triple-negative+breast+cancer.">Future of PD-1/PD-L1 axis modulation for the treatment of triple-negative breast cancer.</a> Pharmacological Research. 175, 106019 (2022).</li><li>Bukari, B., Samarasinghe, R. M., Noibanchong, J., Shigdar, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+delivery+of+therapeutics+into+the+brain:+the+potential+of+aptamers+for+targeted+delivery.">Non-invasive delivery of therapeutics into the brain: the potential of aptamers for targeted delivery.</a> Biomedicines. 8 (5), 120 (2020).</li><li>Wu, X., Chen, J., Wu, M., Zhao, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamers:+active+targeting+ligands+for+cancer+diagnosis+and+therapy.">Aptamers: active targeting ligands for cancer diagnosis and therapy.</a> Theranostics. 5 (4), 322 (2015).</li><li>Feng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aptamer+functionalized+nanocomposite+used+for+prostate+cancer+diagnosis+and+therapy.">The aptamer functionalized nanocomposite used for prostate cancer diagnosis and therapy.</a> Journal of Nanomaterials. 2022, (2022).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+aptamer-based+biomarker+discovery.">Advances in aptamer-based biomarker discovery.</a> Frontiers in Cell and Developmental Biology. 9, 571 (2021).</li><li>Ashrafuzzaman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamers+as+both+drugs+and+drug-carriers.">Aptamers as both drugs and drug-carriers.</a> BioMed Research International. 2014, (2014).</li><li>Nakhjavani, M., Samarasinghe, R. M., Shigdar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triple-negative+breast+cancer+brain+metastasis:+an+update+on+druggable+targets,+current+clinical+trials,+and+future+treatment+options.">Triple-negative breast cancer brain metastasis: an update on druggable targets, current clinical trials, and future treatment options.</a> Drug Discovery Today. , (2022).</li><li>Macdonald, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+bifunctional+aptamer+targeting+the+transferrin+receptor+and+epithelial+cell+adhesion+molecule+(EpCAM)+for+the+treatment+of+brain+cancer+metastases.">Development of a bifunctional aptamer targeting the transferrin receptor and epithelial cell adhesion molecule (EpCAM) for the treatment of brain cancer metastases.</a> ACS Chemical Neuroscience. 8 (4), 777-784 (2017).</li><li>Macdonald, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bifunctional+aptamer-doxorubicin+conjugate+crosses+the+blood-brain+barrier+and+selectively+delivers+its+payload+to+EpCAM-positive+tumor+cells.">Bifunctional aptamer-doxorubicin conjugate crosses the blood-brain barrier and selectively delivers its payload to EpCAM-positive tumor cells.</a> Nucleic Acid Therapeutics. 30 (2), 117-128 (2020).</li><li>Shigdar, S., Agnello, L., Fedele, M., Camorani, S., Cerchia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+cancer+cells+by+cell-SELEX:+use+of+aptamers+for+discovery+of+actionable+biomarkers+and+therapeutic+applications+thereof.">Profiling cancer cells by cell-SELEX: use of aptamers for discovery of actionable biomarkers and therapeutic applications thereof.</a> Pharmaceutics. 14 (1), 28 (2021).</li><li>Rahimizadeh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+cell-specific+aptamers:+recent+advances+and+insight+into+the+selection+procedures.">Development of cell-specific aptamers: recent advances and insight into the selection procedures.</a> Molecules. 22 (12), 2070 (2017).</li><li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+cell-SELEX+technology+and+its+application+in+cancer+diagnosis+and+therapy.">Development of cell-SELEX technology and its application in cancer diagnosis and therapy.</a> International Journal of Molecular Sciences. 17 (12), 2079 (2016).</li><li>Shigdar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+sensitive+chemical+antibodies+for+diagnosis:+detection+of+low+levels+of+EpCAM+in+breast+cancer.">The use of sensitive chemical antibodies for diagnosis: detection of low levels of EpCAM in breast cancer.</a> PLoS One. 8 (2), 57613 (2013).</li><li>Ni, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+EpCAM+(CD326)+in+prostate+cancer+metastasis+and+progression.">Role of the EpCAM (CD326) in prostate cancer metastasis and progression.</a> Cancer and Metastasis Reviews. 31 (3), 779-791 (2012).</li><li>Ni, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+adhesion+molecule+(EpCAM)+is+associated+with+prostate+cancer+metastasis+and+chemo/radioresistance+via+the+PI3K/Akt/mTOR+signaling+pathway.">Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway.</a> The International Journal of Biochemistry &amp; Cell Biology. 45 (12), 2736-2748 (2013).</li><li>McKeague, M., Kruse, P. F., Patterson, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tissue Culture. , 395-397 (1973).</li><li>McKeague, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minimum+aptamer+publication+standards+(MAPS+guidelines)+for+de+novo+aptamer+selection.">The minimum aptamer publication standards (MAPS guidelines) for de novo aptamer selection.</a> Aptamers. 6, 10-18 (2022).</li><li>Schoofs, G., Van Hout, A., D'huys, T., Schols, D., Van Loy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+cytometry-based+assay+to+identify+compounds+that+disrupt+binding+of+fluorescently-labeled+CXC+Chemokine+ligand+12+to+CXC+Chemokine+receptor+4.">A flow cytometry-based assay to identify compounds that disrupt binding of fluorescently-labeled CXC Chemokine ligand 12 to CXC Chemokine receptor 4.</a> Journal of Visualized Experiments. (133), e57271 (2018).</li><li>McKinnon, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+an+overview.">Flow cytometry: an overview.</a> Current Protocols in Immunology. 120 (1), 1-11 (2018).</li><li>Kamiloglu, S., Sari, G., Ozdal, T., Capanoglu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+cell+viability+assays.">Guidelines for cell viability assays.</a> Food Frontiers. 1 (3), 332-349 (2020).</li><li>Ruscito, A., DeRosa, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+binding+aptamers:+Selection+strategies,+characterization,+and+applications.">Small-molecule binding aptamers: Selection strategies, characterization, and applications.</a> Frontiers in Chemistry. 4, 14 (2016).</li><li>McKeague, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+analytical+comparison+of+strategies+used+for+small+molecule+aptamer+evaluation.">Comprehensive analytical comparison of strategies used for small molecule aptamer evaluation.</a> Analytical Chemistry. 87 (17), 8608-8612 (2015).</li><li>Henri, J., Bayat, N., Macdonald, J., Shigdar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+using+nucleic+acid+aptamers+in+cell+based+assays.">A guide to using nucleic acid aptamers in cell based assays.</a> Aptamers. 23, (2019).</li><li>Mao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+and+regularity+of+quenching+the+effect+of+bases+on+fluorophores:+the+base-quenched+probe+method.">The mechanism and regularity of quenching the effect of bases on fluorophores: the base-quenched probe method.</a> Analyst. 143 (14), 3292-3301 (2018).</li><li>McKeague, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+in+vitro+aptamer+selection+parameters.">Analysis of in vitro aptamer selection parameters.</a> Journal of Molecular Evolution. 81 (5), 150-161 (2015).</li><li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+negative+surface+charges+of+cancer+cells+by+multifunctional+nanoprobes.">Targeting negative surface charges of cancer cells by multifunctional nanoprobes.</a> Theranostics. 6 (11), 1887 (2016).</li><li>Shigdar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+aptamers+targeting+cancer+stem+cell+marker+CD133.">RNA aptamers targeting cancer stem cell marker CD133.</a> Cancer Letters. 330 (1), 84-95 (2013).</li><li>Amraee, M., Oloomi, M., Yavari, A., Bouzari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+aptamer+identification+and+characterization+for+E.+coli+O157+detection+using+cell+based+SELEX+method.">DNA aptamer identification and characterization for E. coli O157 detection using cell based SELEX method.</a> Analytical Biochemistry. 536, 36-44 (2017).</li><li>Yu, X. -. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+and+characterization+of+a+novel+DNA+aptamer,+Apt-07S+specific+to+hepatocellular+carcinoma+cells.">Selection and characterization of a novel DNA aptamer, Apt-07S specific to hepatocellular carcinoma cells.</a> Drug Design, Development and Therapy. 14, 1535 (2020).</li></ol>

The authors have no conflicts of interest to disclose.

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 aptamers23, which is widely used by researchers in the field. Since no single guideline exists to display the step-by-step protocol, a flow cytometry method, which is commonly used to study the aptamer-protein binding, is demonstrated in the accompanying video protocol.
There are several methods that measure the interaction of the aptamer and its target. Flow cytometry is one of these methods. Other examples include fluorescence polarization, surface plasmon resonance, capillary electrophoresis, and isothermal titration calorimetry24. Choosing the correct method depends on the application of the aptamer. However, it is important to know that each method has its limitations, and that the application of several assays is more beneficial for the characterization of small molecule aptamers24. The method described here has several advantages. It is one of the most reliable, precise, and accurate methods, and is also rapid and cost-effective. The flow cytometric analysis of aptamer binding can be applied in various steps of aptamer development, including candidate screening, truncation and optimization, characterization, and validation.
With fluorescence labeling, there are choices to either label or use the intrinsic fluorescence of the target or label the aptamer24. In the authors&#39; experience, using labeled aptamers is reliable and easy to set up. Aptamers can be labeled with a fluorophore at any end (3&#39; or 5&#39;)25; however, the end with less guanine is more favorable, as guanine can quench the fluorescence and its detection26. A disadvantage of this method is that the aptamer should be tagged to a fluorophore. Furthermore, although this method reflects the binding of the aptamer to the specific protein, it does not display the location of the interaction site. Hence, further studies, such as fluorescence microscopy, might be required to confirm the aptamer-protein interaction location25.
There are some critical notes to be considered while performing this assay. It is important to consider that each aptamer has specific requirements for handling and folding. This includes the choice of reconstitution and dilution buffers and the folding conditions. Reconstitution of lyophilized aptamers occurs in purified sterile pyrogen- and RNase-free ultrapure water. This is to minimize the concentration of ions around the nucleotides. Ion concentration highly affects the formation of 2D structures of aptamers and their affinity and stability. Hence, for further reconstitution of the aptamer stock, care should be taken in the correct preparation of other buffers and metal cationic solutions, such as MgCl225,27. The optimum concentration of metal cationic solution shields the negative charge of the aptamer yet, does not inhibit the interaction of the aptamer with its target.
Furthermore, because the aptamer has a potential for a non-specific, off-target binding, the application of blocking buffer plays a critical role in the binding assay. In addition to the negatively charged BSA, which is commonly used for antibodies, salmon sperm DNA or tRNA is also required here. This mixture blocks the positively charged proteins and nucleic acid binding sites. This blocking stage is specifically important for the selectivity and specificity of aptamers towards cancer cells, due to their negative charge compared to neutral and positively charged normal cells28. Furthermore, the 3D folding of aptamers is dependent on other factors such as temperature. The importance of conditions affecting the 3D formation of the aptamer, including the choice of tube and the duration of PCR phases, has been reviewed25. Moreover, the incubation time of the aptamer with the target should be optimized for every specific aptamer. The incubation temperature is also highly important. Maintenance of a temperature of 4 &#176;C is especially critical for cell surface proteins that can easily be internalized, such as EpCAM25.
The other important factor in this assay is the application of proper controls. Cancer cells that either express or do not express the target protein should be used to evaluate the selectivity of the aptamer. In each experiment, in addition to the aptamer of interest, a negative control aptamer (a random sequence or a scrambled sequence) is required to show the specificity of the aptamer. Ideally, this control should have a similar length of nucleotides and undergo similar folding as the aptamer. In this experiment, a negative control (TENN), an aptamer with a similar structure to TEPP shown to have a low binding affinity with EpCAM, was used10.
The protocol presented here is a qualitative assay and can be further used for the quantitative assessment of binding affinity and the determination of the dissociation constant (Kd)29,30,31. However, it is important to show the reproducibility of the results using technical and biological replicates. To achieve this, it is highly important to consider major determinants to properly perform this assay. This could include, but is not limited to, using a low passage number of mycoplasma-free cells that are properly grown in their optimal conditions, using a constant number of cells to be exposed with the aptamer in each replicate, the application of proper temperature and folding conditions, maintaining similar experimental conditions for both the aptamer and the control, maintaining minimal exposure of the aptamers to environmental light, using optimized aptamer-cell exposure time and temperature, maintaining a temperature of 4 &#176;C for the cells, which includes precooling the centrifuge, the 96-well plate, and the flow cytometry tubes, and accurately preparing the buffers, with an ionic concentration as close to the claimed concentrations as possible.

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 was to improve the efficacy of chemotherapeutics and minimize their adverse reactions. However, the administration of mAbs also became a challenge. This was not only because of the mAb-induced immunological reactions but also due to the animal-dependent and expensive production costs and difficult storage conditions3. Introduction of aptamers in the 1990s4 raised new hopes in cancer treatment, as the application of aptamers could address the challenges associated with mAbs.
Aptamers are short nucleic acid sequences that are specifically produced for a certain target. Systematic evolution of ligands by exponential enrichment (SELEX) is a common method in aptamer production. In SELEX, the protein of interest is incubated with a library of random nucleotide sequences, and through a series of iterative cycles, the aptamer specific for that protein is purified. Aptamers have similar target selectivity and specificity to mAbs, and therefore drug development in this field shows promising future applications. Aptamers specific for cancer biomarkers could be applied as single drugs and cancer diagnostic tools5,6,7. Due to their nano-sized structure, these aptamers could also act as drug carriers to deliver cytotoxic agents specifically to the tumor8. This would increase the efficacy of targeted drug delivery and decrease chemotherapy-associated, off-target adverse reactions. Moreover, these nanomedicines have a high tissue penetration, which makes them a desirable candidate for deep-tumor drug delivery and treatment. Aptamers can also be designed to target the transporters expressed on the blood-brain barrier (BBB) to improve drug delivery to brain tumors9. A good example of such an aptamer are bifunctional aptamers, targeting the transferrin receptor (TfR)10 to enhance drug delivery across the BBB, and delivering a cytotoxic drug payload to tumor cells11.
Despite all the advantages of aptamers, drug development in this field has not yet yielded a marketed, successful anticancer drug. One reason for this could be the lack of standard and reproducible methods that could be followed globally by researchers in the field. In this paper, a step-by-step protocol of an aptamer binding to a native protein expressed on the cell surface is demonstrated. This protocol is a prerequisite step in the preclinical assessment of anticancer aptamers. The assay is performed to show the selectivity and specificity of the purified aptamer collected from SELEX or a published aptamer sequence for confirmation of selectivity and specificity. This flow cytometric-based assay is a rapid, reliable, sensitive assay that accurately shows the selectivity and specificity of the aptamer, where the aptamer is being tested against proteins on the cell surface12,13,14. This method is demonstrated using the binding of an aptamer specific for EpCAM shown in this paper15. EpCAM, as a transmembrane glycoprotein, plays roles in tumor cell signaling, progression, migration, and metastasis16,17. To show the selectivity and specificity of this aptamer, EpCAM-positive and -negative cancer cells were used. The previously developed EpCAM specific aptamer, TEPP (5&#8242;-GC GCG GTAC CGC GC TA ACG GA GGTTGCG TCC GT-3&#8242;), and a negative control aptamer, TENN (5&#8242;-GC GCG TGCA CGC GC TA ACG GA TTCCTTT TCC GT-3), were used as EpCAM-binding and -non-binding aptamers, respectively10. The 3&#39; end of both TEPP and TENN were labeled with a TYE665 fluorophore.
TEPP is a bifunctional aptamer that targets EpCAM from one end and TfR on the other. This has made TEPP a suitable candidate for drug delivery to EpCAM+ brain tumors. Using its TfR-specific end, TEPP traverses the blood-brain barrier, and using the EpCAM-specific end, finds the tumor and delivers its cargo (e.g., cytotoxic drugs) to the tumor. TENN has a similar length and 2D structure as TEPP, but it does not have affinity for the EpCAM or TfR, and hence is a suitable negative control aptamer. Using TEPP and TENN, testing the binding of an aptamer to the target protein using flow cytometry is shown in this paper. This protocol applies to the development of cell-specific aptamers. It is also applicable to further complementary and confirmation analyses of the aptamer sequences available in the literature. The protocol can also be used by those new to the aptamer field who are looking at using a previously published aptamer for their research and development (R&#38;D) purposes. In this paper, two aptamer sequences available in the literature are studied.

<table><tbody><tr><td>1.5 mL microcentrifuge tubes with attached lid</td><td>Sigma-Aldrich</td><td>T6649</td><td></td></tr><tr><td>15 mL CellStar blue screw cap, conical bottom tube</td><td>Greiner Bio One</td><td>188271</td><td></td></tr><tr><td>5 mL serological pipettes</td><td>Greiner Bio One</td><td>606180</td><td></td></tr><tr><td>BD FACSCanto II Flow Becton Dickinson Cytometer</td><td>Becton Dickinson</td><td>N/A</td><td></td></tr><tr><td>BD FACSDiva V9.0</td><td>BD Biosciences</td><td>N/A</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA), Lyophilized powder</td><td>Sigma-AldrichTM</td><td>A7906-50G</td><td></td></tr><tr><td>Bright-line Hemocytometer</td><td>Sigma-Aldrich</td><td>Z359629</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM) High Glucose Media Powder</td><td>Life Technologies</td><td>12100046</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate- Buffered Saline (DPBS)</td><td>Life Technologies</td><td>21300025</td><td></td></tr><tr><td>FlowJo, LLC 10.8.1</td><td>BD Biosciences</td><td>N/A</td><td></td></tr><tr><td>Foetal Bovine Serum (FBS)</td><td>Bovogen</td><td>SFBS-F</td><td></td></tr><tr><td>HEK293T</td><td>American Type Culture Collection</td><td>ACS-4500</td><td></td></tr><tr><td>Heracell 150i CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Fisher Scientific</td><td>N/A</td><td></td></tr><tr><td>Heraeus Megafuge 16R Centrifuge</td><td>Thermo Fisher Scientific</td><td>N/A</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>MDA-MB-231</td><td>American Type Culture Collection</td><td>CRM-HTB-26</td><td></td></tr><tr><td>Microplate, PS, 96 well, F-bottom (Chimney well), Black</td><td>Greiner Bio One</td><td>655076</td><td></td></tr><tr><td>MiniAmp Thermal Cycler</td><td>Thermo Fisher Scientific</td><td>A37834</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS) tablets</td><td>Life Technologies</td><td>18912014</td><td></td></tr><tr><td>Pyrogen- and RNase-free ultrapure water</td><td>Milli-Q</td><td></td><td></td></tr><tr><td>T75 Cell Culture flask</td><td>Cellstar</td><td>658170</td><td></td></tr><tr><td>TENN</td><td>Integrated DNA Technologies</td><td>N/A</td><td>5&amp;prime;-GC GCG TGCA CGC GC TA ACG GA TTCCTTT TCC GT-3</td></tr><tr><td>TEPP</td><td>Integrated DNA Technologies</td><td>N/A</td><td>5&amp;prime;-GC GCG GTAC CGC GC TA ACG GA GGTTGCG TCC GT-3&amp;prime;</td></tr><tr><td>Transfer RNA (tRNA)</td><td>Sigma-Aldrich</td><td>R8508-5X1ML</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>Life Technologies</td><td>15250061</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>15400054</td><td></td></tr></tbody></table>

a flow cytometry-based cell surface protein binding assay for assessing selectivity and specificity of an anticancer aptamer

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.

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 shows that the protein of interest only responds to a single drug candidate. Here, by using an EpCAM non-binding aptamer, TENN, it was shown that only TEPP attached to EpCAM. In EpCAM-positive cells (MDA-MB-231), overlaying the histograms representing cells treated with TEPP and TENN shows that the TEPP-treated cells are shifted to the right compared to the TENN-treated cells. This shows the binding of the aptamer, TEPP, to the protein of interest, EpCAM (Figure 4D). In negative control HEK 293T cells, overlaying histograms of TEPP and TENN does not reflect any shift (Figure 4E). This means that in EpCAM-expressing cells, TEPP as the EpCAM aptamer attached to its receptor, and furthermore, no binding was observed in EpCAM-negative cells. These results confirm the selectivity and specificity of the developed aptamer.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64304/64304fig04.jpg" /> 
Figure 4: Gating and histograms showing the binding of the cells to the aptamer. (A) Selecting the population of cells, (B) the single cells, and (C) the histogram of the cells attached to the aptamer (200 nM). The binding of EpCAM aptamer (TEPP) versus a non-EpCAM-binding aptamer (TENN) in (D) EpCAM+ MDA-MB-231 cells and (E) EpCAM- HEK 293T cells is compared. Abbreviations: EpCAM = epithelial cellular adhesion molecule; FSC-A = forward scatter-peak area; SSC-A = side scatter-peak area; FSC-H = forward scatter-peak height; APC = allophycocyanin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64304/64304fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer at JoVE.com

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

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.&#160;

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

a-flow-cytometry-based-cell-surface-protein-binding-assay-for

Nanyang Technological University

Research

JoVE Journal

Cancer Research

3.5K Views.  Deakin University. Dit protocol zal onderzoekers die nieuw zijn in het werken met aptamers helpen om ze in hun projecten te kunnen gebruiken en ook om de individuele kleine stappen te begrijpen die nodig zijn om het protocol te voltooien. Aptameren zijn zeer veelzijdig en dit protocol laat zien hoe gemakkelijk en eenvoudig ze zijn in een immunofenotyperingstest. Aptameren kunnen worden gebruikt voor zowel diagnostische als therapeutische toepassingen.Dit protocol is de eerste stap die wordt ondernomen om...

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

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

Biology

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Medicine

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Immunology and Infection

In Vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses

Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, causing delays in treatment and further spread of the virus. Developing new sensors that can inform on the infectability of clinical or environmental samples will meet this unmet challenge. However, very few methods can obtain sensing molecules that can recognize an intact infectious virus and differentiate it from the same virus that has been rendered non-infectious by disinfection methods. Here, we describe a protocol to select aptamers that can distinguish infectious viruses vs non-infectious viruses using systematic evolution of ligands by exponential enrichment (SELEX). We take advantage of two features of SELEX. First, SELEX can be tailor-made to remove competing targets, such as non-infectious viruses or other similar viruses, using counter selection. Additionally, the whole virus can be used as the target for SELEX, instead of, for example, a viral surface protein. Whole virus SELEX allows for the selection of aptamers that bind specifically to the native state of the virus, without the need to disrupt of the virus. This method thus allows recognition agents to be obtained based on functional differences in the surface of pathogens, which do not need to be known in advance.

We provide a protocol that can be generally applied to select aptamers that bind to infectious viruses only and not to viruses that have been rendered non-infectious by a disinfection method or to any other similar viruses. This opens the possibility of determining infectivity status in portable and rapid tests.

Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, ...

Bioengineering

Thyroid Cancer Cells as Effectors in an ADCC Bioassay for Cancer Immunotherapy

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in cancer immunotherapy. Evaluating ADCC activity in cancer cells is essential for the development and optimization of antibody-based treatments. Here, we propose a methodological approach of utilizing an ADCC bioassay kit for quantitative assessment of ADCC reaction using thyroid cancer cells as effector cells. The protocol involves the co-culture of effector cells with target cancer cells in different ratios in the presence of a therapeutic antibody. The ADCC bioassay kit used in this experiment includes the genetically engineered effector cells expressing a luciferase reporter gene under the control of Nuclear Factor of Activated T-cell (NFAT) response elements. Upon the binding of the surface antigen on the target cells with the antibodies and effector cells, the effector cells release luciferase, enabling quantification of cytotoxicity through measurement of luminescence signal. In contrast to conventional ADCC assays, this method proved the binding of target antigen with antibodies and effector cells, which can produce reliable results in a short period.

Author Spotlight: Optimizing Antibody-Based Cancer Treatments via Antibody-Dependent, Cell-Mediated Cytotoxicity Assay

Here, we present a protocol for an antibody-dependent, cell-mediated cytotoxicity (ADCC) assay using an ADCC bioassay kit. This method offers a valuable tool for elucidating the ADCC mechanism and evaluating the therapeutic potential of antibodies in cancer immunotherapy.

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in assessing their efficacy and is a prerequisite for clinical application. However, traditional cytotoxicity assays are conducted as bulk assays and do not provide detailed information about the functional heterogeneity of the CAR T cell population. In this study, we describe a double-emulsion droplet-based method that allows for large-scale co-encapsulation of single effector CAR T cells with single target cells while enabling dual quantification of both cytotoxic effector molecules from T cells and cell death of the target cell. The protocol outlines a method for the generation and purification of CD19-specific CAR T cells, followed by their co-encapsulation in droplets with the CD19+ cell line JeKo-1, along with reagents to visualize cytotoxic effector molecule secretion (Granzyme B) and cell death (using propidium iodide, PI). We demonstrate how to generate droplets containing single CAR T and target cells using a commercially available microfluidics device for generating double emulsion droplets. Additionally, we provide examples of how to assay the functional diversity of CAR T cells in droplets using standard flow cytometry equipment. Finally, we briefly describe the temporal kinetics and heterogeneity of CD19-specific CAR T-cell killing. While this method focuses on cell death following a CAR T cell attack, it is also adaptable for examining other types of T cells, cytotoxic immune cells, and effector cell functions, such as cytokine secretion.

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video