Name: application of aldesense to stratify ovarian cancer cells based on aldehyde dehydrogenase 1a1 activity
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Description: Watch this Scientific Journal Video about Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity at JoVE.com

This work was supported by The National Institutes of Health (R35GM133581 to JC) and the Cancer Center at Illinois Graduate Scholarship (awarded to SG). JC thanks the Camille and Henry Dreyfus Foundation for support. The authors thank Dr. Thomas E. Bearrood for his initial contribution to preparing stocks of AlDeSense and AlDeSense AM. We thank Mr. Oliver D. Pichardo Peguero and Mr. Joseph A. Forzano for their assistance with preparing various synthetic precursors. We thank Prof. Erik Nelson (Department of Molecular and Integrative Physiology, UIUC) for Caov-3, IGROV-1, and PEO4 cells. We thank Prof. Paul Hergenrother (Department of Chemistry, UIUC) for BG-1 cells. We thank the Core Facilities at the Carl R. Woese Institute for Genomic Biology for access to the Zeiss LSM 700 Confocal Microscope and corresponding software. We thank the Flow Cytometry Facility for access to BD LSR II CMtO Analyzer. We thank Dr. Sandra McMasters and the Cell Media Facility for assistance in preparing cell culture media.

1. Measure total ALDH1A1 activity in ovarian cancer cell homogenates via fluorimetry
<ol>
	<li>Thaw 1 &#215; 106 cells in a T25 cell culture flask in 5 mL of the following cell culture media:
	<ol>
		<li>IGROV-1 and PEO4: Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).</li>
		<li>BG-1 and Caov-3: Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 10% FBS and 1% P/S.</li>
		<li>OVCAR-3: RPMI 1640 with 20% ...

<ol>
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G., 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+novel+ALDH1A1+inhibitor+targets+cells+with+stem+cell+characteristics+in+ovarian+cancer.">A novel ALDH1A1 inhibitor targets cells with stem cell characteristics in ovarian cancer.</a> Cancers. 11 (4), 502 (2019).</li><li>Roy, M., Connor, J., Al-Niaimi, A., Rose, S. L., Mahajan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+dehydrogenase+1A1+(ALDH1A1)+expression+by+immunohistochemistry+is+associated+with+chemo-refractoriness+in+patients+with+high-grade+ovarian+serous+carcinoma.">Aldehyde dehydrogenase 1A1 (ALDH1A1) expression by immunohistochemistry is associated with chemo-refractoriness in patients with high-grade ovarian serous carcinoma.</a> Human Pathology. 73, 1-6 (2018).</li><li>Landen Jr, C. N., 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+aldehyde+dehydrogenase+cancer+stem+cells+in+ovarian+cancer.">Targeting aldehyde dehydrogenase cancer stem cells in ovarian cancer.</a> Molecular Cancer Therapeutics. 9 (12), 3186-3199 (2010).</li><li>Condello, 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=&#946;-catenin-regulated+ALDH1A1+is+a+target+in+ovarian+cancer+spheroids.">&#946;-catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids.</a> Oncogene. 34 (18), 2297-2308 (2015).</li><li>Storms, R. W., 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=Isolation+of+primitive+human+hematopoietic+progenitors+on+the+basis+of+aldehyde+dehydrogenase+activity.">Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity.</a> Proceedings of the National Academy of Sciences. 96 (16), 9118-9123 (1999).</li><li>Duellman, S. 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=A+bioluminescence+assay+for+aldehyde+dehydrogenase+activity.">A bioluminescence assay for aldehyde dehydrogenase activity.</a> Analytical Biochemistry. 434 (2), 226-232 (2013).</li><li>Minn, I., 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+red-shifted+fluorescent+substrate+for+aldehyde+dehydrogenase.">A red-shifted fluorescent substrate for aldehyde dehydrogenase.</a> Nature Communications. 5, 3662 (2014).</li><li>Doll&#233;, L., Boulter, L., Leclercq, I. A., van Grunsven, L. 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Gastrointestinal and Liver Physiology. 308 (7), 573-578 (2015).</li><li>Yagishita, A., 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+highly+selective+fluorescent+probe+enabling+flow-cytometric+isolation+of+ALDH3A1-positive+viable+cells.">Development of highly selective fluorescent probe enabling flow-cytometric isolation of ALDH3A1-positive viable cells.</a> Bioconjugate Chemistry. 28 (2), 302-306 (2017).</li><li>Maity, 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=Thiophene+bridged+aldehydes+(TBAs)+image+ALDH+activity+in+cells:+Via+modulation+of+intramolecular+charge+transfer.">Thiophene bridged aldehydes (TBAs) image ALDH activity in cells: Via modulation of intramolecular charge transfer.</a> Chemical Science. 8 (10), 7143-7151 (2017).</li><li>Koenders, S. T. A., 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+retinal-based+probe+for+the+profiling+of+retinaldehyde+dehydrogenases+in+cancer+cells.">Development of a retinal-based probe for the profiling of retinaldehyde dehydrogenases in cancer cells.</a> ACS Central Science. 5 (12), 1965-1974 (2019).</li><li>Oe, 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=Deep-red/near-infrared+turn-on+fluorescence+probes+for+aldehyde+dehydrogenase+1A1+in+cancer+stem+cells.">Deep-red/near-infrared turn-on fluorescence probes for aldehyde dehydrogenase 1A1 in cancer stem cells.</a> ACS Sensors. 6 (9), 3320-3329 (2021).</li><li>Yagishita, A., Ueno, T., Tsuchihara, K., Urano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+BODIPY-based+blue+fluorescent+probes+for+aldehyde+dehydrogenase+1-expressing+cells.">Amino BODIPY-based blue fluorescent probes for aldehyde dehydrogenase 1-expressing cells.</a> Bioconjugate Chemistry. 32 (2), 234-238 (2021).</li><li>Okamoto, A., 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=Identification+of+breast+cancer+stem+cells+using+a+newly+developed+long-acting+fluorescence+probe,+C5S-A,+targeting+ALDH1A1.">Identification of breast cancer stem cells using a newly developed long-acting fluorescence probe, C5S-A, targeting ALDH1A1.</a> Anticancer Research. 42 (3), 1199-1205 (2022).</li></ol>

Pan-selectivity is a major limitation of many ALDH probes; however, several isoform-selective examples have recently been reported32,33,34,35,36,37,38,39,40,41. The isoform-selective fluo...

Cancer stem cells (CSCs) are a subpopulation of tumor cells that exhibit stem cell-like properties1. Similar to their non-cancerous counterparts, CSCs possess the extraordinary ability to self-renew and proliferate. Together with other built-in mechanisms, such as the upregulation of ATP-binding cassette transporters, CSCs are often spared from initial surgical debulking efforts, as well as subsequent adjuvant therapy2. Owing to their critical role in treatment resistance3, relapse4, and metastasis5, CSCs have become a priority in cancer research. Although there are a variety of cell surface antigens (e.g., CD133) that can be used to identify CSCs6, leveraging the enzymatic activity of aldehyde dehydrogenases (ALDHs) found in the cytoplasm has emerged as an attractive alternative7. ALDHs are a superfamily of 19 enzymes responsible for catalyzing the oxidation of reactive endogenous and xenobiotic aldehydes to the corresponding carboxylic acid products8.
In general, aldehyde detoxification is crucial in protecting cells from undesirable crosslinking events and oxidative stress that may damage stem cell integrity9. Moreover, the 1A1 isoform controls retinoic acid metabolism, which in turn influences stemness via retinaldehyde signaling10. AlDeSense11,12, a small-molecule activity-based sensing (ABS) probe to selectively detect ALDH1A1 activity, was recently developed. ABS designs achieve analyte detection through a chemical change rather than a binding event, allowing for high selectivity and decreased off-target responses13,14,15,16. The design principle of the isoform-selective fluorogenic probe relies on a donor-photoinduced electron transfer (d-PeT) quenching mechanism17, originating from the aldehyde functional group, which serves to suppress the fluorescent signature of the probe18. Upon ALDH1A1-mediated conversion to the carboxylic acid, radiative relaxation is unlocked to yield a highly fluorescent product. Because d-PeT quenching is never 100% efficient, the residual fluorescence that may lead to possible false positive results was considered when establishing this assay through the development of Ctrl-AlDeSense, a non-responsive reagent with matching photophysical characteristics (e.g., quantum yield) and an identical cytoplasmic staining pattern in cells. When used in tandem, this unique pairing can reliably distinguish cells with high ALDH1A1 activity from those that exhibit low levels via fluorimetry, molecular imaging, and flow cytometry. Several key advantages are associated with the use of isoform-selective activatable dyes over traditional immunohistochemical methods. For instance, CSCs are hypothesized to be buried deep within a tumor, and thus are more accessible to a small molecule relative to large antibodies19. Additionally, the turned-over fluorescent product does not covalently modify any cellular component, meaning it can be readily removed via wash cycles to leave a CSC in an unmodified state. Lastly, the turn-on response only identifies viable cells and functions, much like the MTT assay, owing to its reliance on the NAD+ cofactor.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64713/64713fig01.jpg" /> 
Figure 1: Schematic demonstrating fluorescent turn-on of AlDeSense. The isoform-selective dye is activated by ALDH1A1 and can be used to identify elevated ALDH1A1 activity in ovarian cancer cells via fluorimetry, molecular imaging, and flow cytometry. <a href="https://www.jove.com/files/ftp_upload/64713/64713fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In past work, the isoform-selective fluorogenic probe assay successfully stratified ALDH high (ALDH+) cells from ALDH low (ALDH-) cells in K562 human chronic leukemia cells, MDA-MB-231 human breast cancer cells, and B16F0 murine melanoma cells. This is important because, for many cancer types, high ALDH1A1 protein expression signifies a worse clinical prognosis20. This presumes that elevated levels of ALDH1A1 are indicative of CSCs which can evade treatment, develop resistance, and disseminate throughout the body. However, in the case of ovarian cancer, there are studies reporting the opposite finding (high ALDH1A1 expression is linked to improved patient survival)21,22,23,24. While this may appear contradictory at first glance, expression does not necessarily correlate to enzyme activity, which may be influenced by changes in the tumor microenvironment (e.g., pH flux, oxygen gradients), availability of the NAD+ cofactor or aldehyde substrates, levels of carboxylic acids (product inhibition), and post-translational modifications that can alter enzyme activity25. Additionally, ovarian cancer is divided into five main histological types (high-grade serous, low-grade serous, endometrioid, clear cell, and mucinous), which we hypothesize will feature variable levels of ALDH1A1 activity26. With the goal of investigating ALDH1A1 activity in ovarian tumors, an isoform-selective fluorogenic probe assay was employed to identify ALDH1A1+ populations in a panel of five ovarian cancer cell lines belonging to the different histological types mentioned above. The cell lines tested in this study include BG-1, Caov-3, IGROV-1, OVCAR-3, and PEO4 cells, covering clear cell and serous histotypes. Herein, the versatility and generalizability of the probe was highlighted to identify CSCs for the researchers that seek to perform similar studies in other immortalized cancer cell lines as well as patient samples. The use of AlDeSense will shed light on the biochemical pathways involved in CSC maintenance in complex tissue microenvironments and potentially serve as a clinical tool for determining prognosis and measuring cancer aggressiveness.

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	<tbody>
		<tr>
			<td>&#160;0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate</td>
			<td>Corning</td>
			<td>&#160;&#160;25-050-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffer Saline</td>
			<td>Corning</td>
			<td>21-040-CMX12</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuSpin Micro 17R</td>
			<td>Fisher Scientific</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>AlDeSense</td>
			<td></td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>BG-1</td>
			<td></td>
			<td></td>
			<td>A gift provided by the Prof. Paul Hergenrother Lab, University of Illinois Urbana-Champaign</td>
		</tr>
		<tr>
			<td>BioLite 25cm2 Flask</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>130189</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet 1300 series A2</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Caov-3</td>
			<td></td>
			<td></td>
			<td>A gift provided by the Prof. Erik Nelson Lab, University of Illinois Urbana-Champaign</td>
		</tr>
		<tr>
			<td>Cell homogenizer</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5180R</td>
			<td>Eppendorf</td>
			<td>22627040</td>
			<td></td>
		</tr>
		<tr>
			<td>Contrl-AlDeSense</td>
			<td></td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>DMEM, 10% FBS, 1% P/S</td>
			<td></td>
			<td></td>
			<td>Prepared by UIUC cell media facility</td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 5mL</td>
			<td>Corning</td>
			<td>352003</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS Express 6</td>
			<td></td>
			<td></td>
			<td>Provided by UIUC CMtO</td>
		</tr>
		<tr>
			<td>FL microscope</td>
			<td>EVOS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorometer</td>
			<td>Photon Technology International</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Series II Water-Jacketed CO2&#160;Incubator</td>
			<td>Fisher Scientific</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr>
			<td>IGROV-1</td>
			<td></td>
			<td></td>
			<td>A gift provided by the Prof. Erik Nelson Lab, University of Illinois Urbana-Champaign</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Innova 42R Incubated Shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 700</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSR II</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Lab-Tek Chambered #1.0 Borosicilate Coverglass System</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>155383</td>
			<td></td>
		</tr>
		<tr>
			<td>OVCAR-3</td>
			<td>ATCC</td>
			<td>HTB-161</td>
			<td></td>
		</tr>
		<tr>
			<td>PEO4</td>
			<td></td>
			<td></td>
			<td>A gift provided by the Prof. Erik Nelson Lab, University of Illinois Urbana-Champaign</td>
		</tr>
		<tr>
			<td>Pierce Protease Inhibitor Tablets</td>
			<td>Thermo Scientific</td>
			<td>A32963</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Cultrex</td>
			<td>3438-100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI, 10% FBS, 1% P/S</td>
			<td></td>
			<td></td>
			<td>Prepared by UIUC cell media facility</td>
		</tr>
		<tr>
			<td>RPMI, 20% FBS, 1% P/S, 0.01 mg/mL Insulin</td>
			<td></td>
			<td></td>
			<td>Prepared by UIUC cell media facility</td>
		</tr>
	</tbody>
</table>

application of aldesense to stratify ovarian cancer cells based on aldehyde dehydrogenase 1a1 activity

Relapse after cancer treatment is often attributed to the persistence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are characterized by their remarkable tumor-initiating and self-renewal capacity. Depending on the origin of the tumor (e.g., ovaries), the CSC surface biomarker profile can vary dramatically, making the identification of such cells via immunohistochemical staining a challenging endeavor. On the contrary, aldehyde dehydrogenase 1A1 (ALDH1A1) has emerged as an excellent marker to identify CSCs, owing to its conserved expression profile in nearly all progenitor cells including CSCs. The ALDH1A1 isoform belongs to a superfamily of 19 enzymes that are responsible for the oxidation of various endogenous and xenobiotic aldehydes to the corresponding carboxylic acid products. Chan et al. recently developed AlDeSense, an isoform-selective "turn-on" probe for the detection of ALDH1A1 activity, as well as a non-reactive matching control reagent (Ctrl-AlDeSense) to account for off-target staining. This isoform-selective tool has already been demonstrated to be a versatile chemical tool through the detection of ALDH1A1 activity in K562 myelogenous leukemia cells, mammospheres, and melanoma-derived CSC xenografts. In this article, the utility of the probe was showcased through additional fluorimetry, confocal microscopy, and flow cytometry experiments where the relative ALDH1A1 activity was determined in a panel of five ovarian cancer cell lines.

Total ALDH1A1 activity of ovarian cancer cell homogenates 
The average fold turn-ons for each cell line obtained from this assay are: BG-1 (1.12 &#177; 0.01); IGROV-1 (1.30 &#177; 0.03); Caov-3 (1.72 &#177; 0.06); PEO4 (2.51 &#177; 0.29); and OVCAR-3 (10.25 &#177; 1.46) (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64713/64713fig02.jpg" /> 
Figure 2<...

Watch this Scientific Journal Video about Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity at JoVE.com

Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity

Methods to measure ALDH1A1 activity in live cells are critical in cancer research due to its status as a biomarker of stemness. In this study, we employed an isoform-selective fluorogenic probe to determine the relative levels of ALDH1A1 activity in a panel of five ovarian cancer cell lines.

Relapse after cancer treatment is often attributed to the persistence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are ...

Our protocol allows users to measure ALDH1A1 activity, a stem cell biomarker using diverse modalities ranging from flow cytometry to live cell molecular imaging. The major advantages of this approach are that probe activation involves the turnaround response characterized by a high signal to background ratio, as well as selective ALDH1A1 isoform detection. Demonstrating this procedure will be Michael Lee, Sarah Gardner, and Rodrigo Tapia Hernandez graduate students from my laboratory.To begin, aspirate the growth media from the cultured cells of interest in eight well chamber slide. Add 500 microliters per well of serum free media supplemented with a two micromolar isoform selective fluorogenic probe or a non-reactive control probe. Incubate the slide at room temperature for 30 minutes.After incubation, proceed to confocal microscope imaging immediately. Locate the cells at 10 x magnification. The FITC channel and transmitted light channels are required for this experiment.Locate and focus the cells using the transmitted light channel to focus on the same Z plane for the entirety of the experiment to remove bias. Adjust the laser power and FITC gain to the appropriate setting where the signal from the control AlDeSense AM samples is minimally detectable, while still seeing signal in AlDeSense AM samples. Adjust each parameter by sliding the corresponding bar.The settings may have to be adjusted a few times to identify the correct parameters. Once optimized, complete the rest of the experiment within that cell line using identical parameters. Snap three images per well for a total of three wells per treatment condition and save the images.Ensure to focus using the transmitted light channel only while switching between planes and wells to avoid bias. Next, using the image processing software split the CZI file into different channels and count the total number of cells and fluorescent cells. To determine the percentage of aldehyde dehydrogenase 1A1 positive cells, divide the number of fluorescent cells by the total number of cells in each image.Count the same way for each image to avoid confounding variables. Take out a T25 cell culture flask containing the cells maintained in the incubator. Add trypsin for cell detachment, and count the cells using an automated cell counter.Then pellet the cells in a 15 milliliter centrifuge tube by centrifugation at 180G for five minutes at 25 degrees Celsius. Re-suspend the cells in one milliliter of a two micromolar probe or control probe solution in PBS. Rock the cells at room temperature for 60 minutes to ensure even exposure to the dye.After the incubation period, pellet the cells by centrifugation at 180 G for five minutes at 25 degrees Celsius. Then re-suspend the cells in 0.5 milliliters of PBS. Run the cells through a 35 micrometer nylon mesh cell strainer onto a tube placed on ice to remove cell clumps that may clog the flow cytometer.Turn the flow cytometer instrument on and run the startup protocol. Check for sheath fluid and empty waist. Run the lines with 10%bleach and water for five minutes each.Then run quality control beads to ensure proper function. In the settings tab, select forward scatter side scatter and FITC for the fluorescence filter. Draw a forward scatter area versus a side scatter area plot for the main cell population near the center of the graph.Next, draw a forward scatter area, versus a forward scatter width plot for the narrow horizontal bands indicating singlets. Then draw an FITC area versus a forward scatter area plot to observe the distribution of cells sorted by the isoform selective fluorogenic turn on probe. Next, draw an FITC area histogram to observe the shift in population based on FITC and determine the percentage of aldehyde dehydrogenase 1A1 positive cells.To optimize the FITC laser power, run a sample with the probe so that the right tail of the histogram curve is near the maximum FITC area signal. The laser power optimization step may have to be repeated multiple times, but the laser power should not be altered across samples, once a setting has been designated for an experiment. Subsequently, add a sample to the sift, and run it with the control probe.A population shift should be observable to reveal the maximum dynamic range. Run each sample for 10, 000 counts done in triplicate. After completion of sample collection, run the lines with 10%bleach and water for five minutes each, then shut down the instrument.Process the data using flow cytometry software and gait the desired cell population. Using the rectangle gate selection, set the aldehyde dehydrogenase 1A1 negative gait, so that more than 99.5%of events in the control probe samples occur within this gate. The remaining cells will be considered aldehyde dehydrogenase 1A1 positive.Apply the same gates to the probe sample. To quantify the number of events considered aldehyde dehydrogenase 1A1 negative and 1A1 positive. The average fold turn-ons for each cell line were determined to quantify the total aldehyde dehydrogenase 1A1 activity.The percentage of aldehyde dehydrogenase 1A1 positive cells was determined in each cell line by tuning the laser power and gain. To minimize the signal in the control AlDeSense AM treated sample the fluorescence signal was optimized in the AlDeSense AM treated cells. By counting the number of aldehyde dehydrogenase A1A positive cells, the percentage of aldehyde dehydrogenase A1A positive cells was determined.With the application of the isoform selective fluorogenic probe, the aldehyde dehydrogenase 1A1 positive cell population was quantified within each cell line by gating for the top 0.5%of the brightest cells within the control AlDeSense AM treated population. Analysis of the panel of ovarian cancer cells has revealed the percentage of aldehyde dehydrogenase 1A1 positive cells. From the obtained results of the tested cancer cell lines, it can be concluded that BG-1 has the lowest aldehyde dehydrogenase 1A1 activity and lowest aldehyde dehydrogenase 1A1 positive population.Additionally, confocal imaging and flow cytometry revealed the largest percentage of aldehyde dehydrogenase 1A1 positive cells in Caov-3 cells. But the cell line's overall activity was only the third highest. Alternatively, OVCAR-3 cells contained the third highest aldehyde dehydrogenase 1A1 positive population, but exhibited the highest overall activity.AlDeSense is a versatile and generalizable probe to identify CSCs in immortalized cell lines and in patient samples. We hope to see it as a prognostic tool in the clinical setting.

application-aldesense-to-stratify-ovarian-cancer-cells-based-on

Research

JoVE Journal

Cancer Research

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

A HMCA-based imaging plate is presented for invasion assay performance. This plate facilitates the formation of three-dimensional (3D) tumor spheroids and the measurement of cancer cell invasion into the extracellular matrix (ECM). The invasion assay quantification is achieved by semi-automatic analysis.

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, transcription-replication conflicts, or amplified oncogenes, inevitably resulting in the generation of single-stranded DNA (ssDNA). Quantifying ssDNA, therefore, presents an opportunity to assess the level of replication stress in different cell types and under various DNA-damaging conditions or treatments. Emerging evidence also suggests that ssDNA can be a predictor of responses to chemotherapeutic drugs that target DNA repair. Here, we describe a detailed immunofluorescence-based methodology to quantify ssDNA. This methodology involves labeling the genome with a thymidine analog, followed by the antibody-based detection of the analog at the chromatin under non-denaturing conditions. Stretches of ssDNA can be visualized as foci under a fluorescence microscope. The number and intensity of the foci directly co-relate with the level of ssDNA present in the nucleus. We also describe an automated pipeline to quantify the ssDNA signal. The method is rapid and reproducible. Furthermore, the simplicity of this methodology makes it amenable to high-throughput applications such as drug and genetic screens.

Here, we describe an immunofluorescence-based method to quantify the levels of single-stranded DNA in cells. This efficient and reproducible method can be utilized to examine replication stress, a common feature in several ovarian cancers. Additionally, this assay is compatible with an automated analysis pipeline, which further increases its efficiency.

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

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Human Omentum Specimen Processing and Co-Culture with Ovarian Cancer Cells

Standardizing Ovarian Cancer Organoid Biobanking

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

Author Spotlight: Advancing Personalized Medicine in Ovarian Cancer 

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...