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JoVE Journal Cancer Research

Cancer Research

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

Published: March 31, 2023 doi: 10.3791/64713
Automatic Translation
*1,3,4, *2,3,4, 1,3,4, 1,2,3,4
1Department of Chemistry, University of Illinois Urbana-Champaign, 2Department of Biochemistry, University of Illinois Urbana-Champaign, 3Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, 4Cancer Center of Illinois, University of Illinois Urbana-Champaign
* These authors contributed equally

Summary

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.

Abstract

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.

Introduction

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.

Figure 1
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. Please click here to view a larger version of this figure.

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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Protocol

1. Measure total ALDH1A1 activity in ovarian cancer cell homogenates via fluorimetry

  1. Thaw 1 × 106 cells in a T25 cell culture flask in 5 mL of the following cell culture media:
    1. IGROV-1 and PEO4: Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).
    2. BG-1 and Caov-3: Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS and 1% P/S.
    3. OVCAR-3: RPMI 1640 with 20% FBS, 1% P/S, and 0.01 mg/mL insulin.
  2. Maintain the cells in an incubator at 37 °C and 5% CO2 for two to three passages. Ensure the cells do not exceed 80%-90% confluency before passaging.
  3. Trypsinize the cells in 0.25% trypsin for 10 min, count them using an automated cell counter, and pellet 1 × 107 cells via centrifugation (180 × g) at 25 °C for 5 min.
  4. Remove the supernatant carefully via aspiration, wash the pellet by resuspending the cells in 1 mL of 1x PBS, and re-pellet the cells via centrifugation under the same conditions as described in step 4.
  5. Resuspend the cells in a solution of 1x protease inhibitor in 1x PBS. Use 1 mL of this solution per 2.5 × 106 cells.
  6. Sonicate the cell suspension on ice for 2 min (1 s pulse, 40% amplitude) with a cell homogenizer probe.
  7. Pellet insoluble/membrane fractions via centrifugation (3,200 × g) at 25 °C for 15 min. Remove and keep the supernatant, as this is the homogenate that will be used in subsequent steps. Separate the homogenates into three cuvettes to perform the experiment in triplicate.
  8. Add the probe to the homogenate to obtain a final probe concentration of 4 µM. Pipette up and down three times to mix the solution well.
  9. Measure the fluorescent signal immediately after addition and after desired incubation times on a fluorimeter. The incubation time can be optimized to see the maximum fold turn-on (1 h in this experiment). The incubation time must be constant across all cell lines compared.
    1. Set the excitation wavelength to 496 nm.
    2. Set the emission to 510-600 nm.
    3. Set the slit width to 0.5 mm.
    4. Pipette the solution into a 1 mL quartz cuvette, place into the fluorimeter, and hit scan.
  10. Divide the final fluorescence intensity at 516 nm (maximum fluorescent wavelength) by the intensity at the same wavelength from the initial reading to determine the fold activation of the isoform-selective dye.

2. Use of fluorescence microscopy to image cells with high ALDH1A1 activity

  1. Thaw 1 × 106 cells in a T25 cell culture flask in 5 mL of the appropriate cell culture media.
  2. Maintain the cells in an incubator at 37 °C and 5% CO2 for two to three passages. Ensure the cells do not exceed 80%-90% confluency before passaging.
  3. The day prior to confocal imaging, plate 4 × 105 cells in an 8-well chamber slide.
    1. Coat the bottom of each well with poly-L-lysine (0.1 mg/mL, 100 µL per well) for 10 min, subsequently aspirating.
    2. Wash each well 3x by adding cell culture grade water (100 µL per well) and aspirating.
    3. Trypsinize the cells in 0.25% trypsin for 10 min, count them using an automated cell counter, and plate the cells at 4 × 105 cells per well.
    4. Let the cells settle and attach overnight (12-16 h).
  4. Aspirate the growth media and add serum-free media (500 µL per well), supplemented with 2 µM of the probe or the control probe.
  5. Incubate with the probe at room temperature for 30 min and image the cells immediately.
  6. Turn on the confocal microscope and adjust for the sample.
    1. Load the sample carefully at the lowest magnification; find the cells to ensure proper positioning.
    2. Ensure the objective lens is at 10x magnification and locate the cells.
  7. For this experiment, the FITC channel (excitation: 488 nm laser; emission: 516-521 nm) and T-PMT (transmitted light) channel are required. Locate and focus on the cells using T-PMT to focus on the same z-plane for the entirety of the experiment to remove bias.
  8. Adjust the laser power and FITC gain to the appropriate setting, where the signal from the Ctrl-AlDeSense AM samples is minimally detectable, while still seeing signal in AlDeSense AM samples. Each parameter can be adjusted 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.
  9. Snap three images per well for a total of three wells per treatment condition (nine images in total). Focus on the proper plane using T-PMT to avoid bias rather than using the fluorescence channel or merged image.
  10. Process the images to determine the percentage of ALDH1A1+ cells.
    1. Using image processing software, split the czi file into different channels.
    2. Count the total number of cells and the total number of fluorescent cells.
    3. To determine the percentage of ALDH1A1+ cells, divide the number of fluorescent cells by the total number of cells in each image. It is imperative to count the same way for each image without manipulating the images, as, for instance, adjusting the brightness may add a confounding variable.

3. Application of flow cytometry to identify cells with high ALDH1A1 activity

  1. Thaw 1 × 106 cells in a T25 cell culture flask in 5 mL of the appropriate cell culture media.
  2. Maintain the cells in an incubator at 37 °C and 5% CO2 for two to three passages. Ensure the cells do not exceed 80%-90% confluency before passaging.
  3. Trypsinize, count, and pellet the cells in a 15 mL centrifuge tube via centrifugation (180 × g) at 25 °C for 5 min.
  4. Resuspend the cells in 1 mL of 2 µM probe/control probe solution in PBS. Rock the cells at room temperature for 60 min to ensure exposure to the dye is even.
  5. After the incubation period, pellet the cells via centrifugation (180 × g) at 25 °C for 5 min. Resuspend the cells in 0.5 mL of PBS. Run the cells through a cell strainer (35 µm nylon mesh) to remove cell clumps that may clog the flow cytometer. Immediately place the cells on ice.
  6. Turn the instrument on and run the start-up protocol.
  7. Check for sheath fluid and empty waste.
  8. Run the lines with 10% bleach and water for 5 min each.
  9. Run quality control beads to ensure proper function.
  10. In the settings tab, select FSC (forward scatter), SSC (side scatter), and FITC (fluorescein isothiocyanate) for the fluorescence filter.
  11. Draw the following graphs to gate for viability, singlets, and fluorescence. Optimize the laser power (specific to user's instrument) so that the cell populations are within the given parameters for further analysis.
    1. FSC-A versus SSC-A scatter plot: main cell population is near the center of the graph.
    2. FSC-A versus FSC-W scatter plot: narrow horizontal band indicative of singlets (rather than cell clumps).
    3. FITC-A versus FSC-A scatter plot: observe the distribution of cells sorted by the isoform-selective fluorogenic turn-on probe.
    4. FITC-A histogram: observe the shift in population based on FITC to determine the percentage of ALDH1A1+ cells.
  12. 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-A signal. Subsequently, run a sample with the control probe. A population shift should be observable to reveal the maximum dynamic range. 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.
  13. Run each sample for 10,000 counts (done in triplicate).
  14. Repeat step 3.12 for each cell line, as there will be variability in uptake and ALDH1A1 activity.
  15. After completion of sample collection, run the lines with 10% bleach and water for 5 min each, then initiate shutdown of the instrument.
  16. Process the data using the flow cytometry software and gate the desired cell population. Set the gates so that all events fall into either the ALDH1A1- or ALDH1A1+ gate. Using the rectangle gate selection, set the ALDH1A1- gate so that >99.5% of events in the control probe samples occur within this gate. The remaining cells will be considered ALDH1A1+. These same gates can then be applied to the probe sample to quantify the number of events considered ALDH1A1- and ALDH1A1+.

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Representative Results

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 ± 0.01); IGROV-1 (1.30 ± 0.03); Caov-3 (1.72 ± 0.06); PEO4 (2.51 ± 0.29); and OVCAR-3 (10.25 ± 1.46) (Figure 2).

Figure 2
Figure 2: The fold fluorescent turn-on of isoform-selective dye in homogenates of each ovarian cancer cell line measured by fluorimetry (mean ± standard deviation) (n = 3). Please click here to view a larger version of this figure.

Molecular imaging of ALDH1A1+ subpopulations in cultured ovarian cancer cells
Upon incubating the cells with a probe or control probe, the percentage of ALDH1A1+ cells was determined in each cell line. By tuning the laser power and gain to minimize signal in the Ctrl-AlDeSense AM treated sample, the fluorescence signal was optimized in the AlDeSense AM treated cells (Figure 3). By counting the number of ALDH1A1+ cells, the percentage of ALDH1A1+ cells was determined to be: BG-1 (3.2% ± 1.6%); PEO4 (18.0% ± 3.6%); OVCAR-3 (39.8% ± 3.9%); IGROV-1 (51.7% ± 5.4%); and Caov-3 (93.7% ± 3.4%) (Figure 4).

Figure 3
Figure 3: Representative confocal images. (A,C,E,G,I) Ctrl-AlDeSense AM stained cells and (B,D,F,H,J) AlDeSense AM stained cells. From left to right, the images exhibit brightfield, fluorescence, and merge. Scale bars are 50 µm. Please click here to view a larger version of this figure.

Figure 4
Figure 4: The mean percentage of ALDH1A1+ cells in each ovarian cancer cell line measured by confocal microscopy (mean ± standard deviation) (n = 9). The percentage of ALDH1A1+ cells are BG-1 (3.2% ± 1.6%); PEO4 (18.0% ± 3.6%); OVCAR-3 (39.8% ± 3.9%); IGROV-1 (51.7% ± 5.4%); and Caov-3 (93.7% ± 3.4%). Please click here to view a larger version of this figure.

Identifying population of ALDH1A1+ cells using flow cytometry
With the application of the isoform-selective fluorogenic probe, the population of ALDH1A1+ cells was successfully identified across different ovarian cancer cell lines. During the post-experimental data analysis, the ALDH1A1+ cell population within each cell line could be quantified by gating for the top 0.5% of the brightest cells within the Ctrl-AlDeSense AM treated population (Figure 5). Analysis of the panel of ovarian cancer cells has revealed the percentages of ALDH1A1+ cells are: BG-1 (4.9% ± 0.4%); PEO4 (5.66% ± 0.9%); OVCAR-3 (7.6% ± 0.1%); IGROV-1 (35.4% ± 2.8%); and Caov-3 (70.1% ± 2.4%) (Figure 6).

Figure 5
Figure 5: Representative scatter plots and histogram overlay. (A,D,G,J,M) Representative scatter plots of Ctrl-AlDeSense AM staining after a 1 h incubation with cells. (B,E,H,K,N) Representative scatter plots of AlDeSense AM staining after a 1 h incubation with cells. (C,F,L,J,O) Histogram overlay of Ctrl-AlDeSense AM and AlDeSense AM staining of these conditions. Cell lines being stained are (A-C) BG-1, (D-F) PEO4, (G-I) OVCAR-3, (J-L) IGROV-1, and (M-O) Caov-3. Please click here to view a larger version of this figure.

Figure 6
Figure 6: The mean percentage of ALDH1A1+ cells in each ovarian cancer cell line measured by flow cytometry (mean ± standard deviation) (n = 3). The percentage of ALDH1A1+ cells were BG-1 (4.9% ± 0.4%); PEO4 (5.66% ± 0.9%); OVCAR-3 (7.6% ± 0.1%); IGROV-1 (35.4% ± 2.8%); and Caov-3 (70.1% ± 2.4%). Please click here to view a larger version of this figure.

While several ALDH isoforms (i.e., ALDH1A1, ALDH1A2, ALDH1A3, and ALDH3A1) have been linked to CSCs, ALDH1A1 was selected as the target for this study because, in the context of ovarian cancer, expression levels are elevated21,27, and this isoform has been shown to exacerbate drug resistance28,29 and enhance tumorigenesis30,31. Results from confocal imaging and flow cytometry are in agreement in the order of the increasing population of ALDH1A1+ cells. Additionally, the cell homogenate experiments reveal the average activity within a cell line. In conjunction, conclusions about the amount of activity within ALDH1A1+ subpopulations of each cell line can be extrapolated. It can be concluded that BG-1 has the lowest ALDH1A1 activity and lowest ALDH1A1+ population. Of note, we selected the BG1 cell line to be used as a negative control in this study, owing to the negligible expression of ALDH1A1. Additionally, confocal imaging and flow cytometry revealed the largest percentage of ALDH1A1+ cells in Caov-3 cells, but the cell line's overall activity was only third highest. Alternatively, OVCAR-3 cells contained the third highest ALDH1A1+ population but exhibited the highest overall activity. Extrapolating from these results, the subpopulation of ALDH1A1+ OVCAR-3 cells have higher activity than the subpopulation of ALD1A1+ Caov-3 cells. Through further analysis of the ALDH1A1 population within cell lines or tissue samples, the role of ALDH1A1 can be further elucidated and the phenotypic changes as a result of elevated ALDH1A1 activity can be investigated.

Cell line Histological type Fluorimetry (fold turn on) Confocal (% positive) Flow (% positive)
BG-1 Poorly differentiated adenocarcinoma 1.12 3.2 4.9
PEO4 High grade serous cystadenocarcinoma 2.51 18.0 5.7
OVCAR-3 High grade serous adenocarcinoma 10.25 39.8 7.6
IGROV-1 Endometrioid adenocarcinoma 1.30 51.7 35.4
Caov-3 High grade serous adenocarcinoma 1.72 93.7 70.1

Table 1: Summary of results from cell homogenates, confocal microscopy, and flow cytometry.

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Discussion

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 fluorogenic probe used in this study represents the first rationally designed probe that reacts only with the ALDH1A1 isoform, even in the presence of competing enzymes that are present in large excess amounts11,12. Another distinguishing feature of this probe is its unique fluorogenic nature, which is characterized by low fluorescence emission prior to probe activation. This is in contrary to commercial kits such as the ALDEFLUOR assay, which features a fluorescent substrate (BODIPY aminoacetaldehyde [BAAA]) that is always in an 'on-state'32. BAAA identifies ALDH+ cells based on the premise that the activated carboxylate product (which is equally fluorescent) is retained to a greater extent in cells expressing ALDH at high levels. To account for non-specific accumulation of the probe in ALDH- cells, an inhibitor must be applied to block ALDHs that are present in a control sample. However, this is accompanied by several notable drawbacks. First, if the intent is to identify CSCs via ALDH1A1 activity, application of the inhibitor strategy fails because it also blocks other isoforms to varying extents. Second, the non-specific behavior of the inhibitor can impair the ability of a cell to detoxify reactive aldehydes on a global level. The first of these concerns is addressed in the design of AlDeSense, because it only reacts with a single isoform as mentioned above (Supplementary Figure 1). To ameliorate the second concern, Ctrl-AlDeSense replaces the role of the inhibitor. Specifically, the control reagent exhibits a nearly identical fluorescent signal as unactivated AlDeSense, is taken up to an equal extent by cells, and localizes predominantly to the cytoplasm. Any signal beyond the baseline established by the control must therefore represent ALDH1A1-mediated probe activation.

Supplementary Figure 1: The reactivity of AlDeSense and ALDEFLUOR. (A) Normalized fluorescence turn-on of AlDeSense after incubation with each ALDH isoform and (B) reactivity of ALDEFLUOR with each ALDH isoform. All measurements were done in triplicate; error bars are ± SD. Please click here to download this File.

The first application of the isoform-selective fluorogenic probe chosen to highlight is the measurement of ALDH1A1 activity in cell homogenates. Under circumstances where specialized instrumentation such as confocal microscopes or flow cytometers are not available, the use of common fluorimeters can rapidly report on total ALDH1A1 activity within a sample. Along these lines, the ease of sample preparation (homogenates can be accessed from samples ranging from cell cultures to excised tissue) broadens the applicability of this assay. There are a few key parameters that must be considered when optimizing the isoform-selective fluorogenic probe for use with homogenates. The first involves modulating the number of cells per lysate sample. For instance, if the dynamic range, defined as the extent of fluorescent signal change, is insufficiently low, the number of cells per sample can be increased. Likewise, the incubation time can also be adjusted such that more fluorogenic probe can be activated to yield a stronger fluorescent readout. However, it is noteworthy that a major limitation of this method is the inability to distinguish between subpopulations of cells exhibiting varying levels of ALDH1A1 activity. Since cells are combined and lysed regardless of their ALDH1A1 status, the amount of enzyme activity is averaged over all cells present in the sample. This results in a different outcome compared to confocal microscopy and flow cytometry analyses, where it is the percentage of ALDH+ cells that is being reported. While BG-1 cells display the lowest fold turn-on response, as well as the smallest population of ALDH1A1+ cells, the order of the remaining four cell lines becomes inconsistent. For instance, the homogenate assay identifies OVCAR-3 cells to have the highest overall ALDH1A1 activity, whereas it ranks only third based on confocal microscopy and flow cytometry. Our interpretation of this data is that an ALDH1A1+ cell must have varying levels of activity. Lastly, it is important to note this type of 'turn-on' experiment would not be possible using accumulation-based probes.

Additionally, the isoform-selective fluorogenic probe is a molecular imaging agent for the visualization of ALDH1A1+ cell populations. Of note, although confocal microscopy was employed in this demonstration, this fluorogenic probe is compatible with a large assortment of imaging setups, including automated cell counters, epi-fluorescence microscopes, and wide-field illumination instruments. One of the most important steps in this experiment is the use of Ctrl-AlDeSense to establish the appropriate thresholding parameters (e.g., pinhole size, laser power, FITC gain). In this regard, the settings should be adjusted such that the Ctrl-AlDeSense signal is just above the background. In the event where a user finds that it is necessary to increase the laser power to see this signal, extending the dye staining period or loading concentration is recommended over increasing the laser power (above 50%) due to increased phototoxicity or probe bleaching. A limitation of confocal imaging is variability when gating with Ctrl-AlDeSense. Despite this caveat, the observed cell line order based on increasing percentages of ALDH1A1+ cells were identical to the results obtained via flow cytometry. Unlike molecular imaging, where the maximum number of cells visualized is limited by the field of view, a typical flow cytometry experiment analyzes up to tens of thousands of cells. The considerations of staining time and loading concentration are similar to that discussed for molecular imaging. An additional element that must be considered is even dye staining in the cell suspension to ensure false positive ALDH1A1+ populations are not misidentified.

In closing, this article highlights the process to optimize AlDeSense for a variety of modalities, using ovarian cancer cells as an example. This represents an important first step toward answering why contradictory reports exists regarding ALDH1A1 expression in this cancer type21,22,23,24. Beyond what we have shown, we envision that this isoform-selective fluorogenic probe can be used in a high throughput screening campaign to identify ALDH1A1-selective inhibitors that may emerge as drug candidates. Additionally, this probe may continue to find use to identify CSCs in living animals, as we have demonstrated in our initial publication11. The green-light emissive characteristic of the isoform-selective fluorogenic probe primes it for multiplexing experiments where it can be used in tandem with red-emitting fluorescent proteins, small-molecule dyes, or analyte-responsive probes. Moreover, a red version of the isoform-selective fluorogenic probe exists, which can further expand this multiplexing capacity12. Finally, on the medical front, this probe can be used as a prognostic tool in the clinical setting for point-of-care treatment decision making. Quantifying ALDH1A1 activity and ALDH1A1+ cell populations can serve as a method to determine cancer aggressiveness, allowing for personalized treatment strategies for better quality-of-life. Moreover, using AlDeSense, a readout can be delivered and interpreted in a matter of hours.

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Disclosures

We disclose a pending patent (US20200199092A1) for the AlDeSense technology.

Acknowledgments

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.

Materials

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

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References

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AlDeSense Stratify Ovarian Cancer Cells Aldehyde Dehydrogenase 1A1 Activity Stem Cell Biomarker Flow Cytometry Live Cell Molecular Imaging Probe Activation High Signal To Background Ratio Selective ALDH1A1 Isoform Detection Michael Lee Sarah Gardner Rodrigo Tapia Hernandez Graduate Students Growth Media Eight Well Chamber Slide Serum Free Media Fluorogenic Probe Non-reactive Control Probe Confocal Microscope Imaging FITC Channel Transmitted Light Channels Z Plane Focus Laser Power FITC Gain
Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity
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Lee, M. C., Gardner, S. H., Tapia… More

Lee, M. C., Gardner, S. H., Tapia Hernandez, R., Chan, J. Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity. J. Vis. Exp. (193), e64713, doi:10.3791/64713 (2023).

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