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

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. AlDeSense^11,12, 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 responses^13,14,15,16. 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: 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.

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+.

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).

Discussion

Pan-selectivity is a major limitation of many ALDH probes; however, several isoform-selective examples have recently been reported^{32,33,34,35,36,37,38,39,40,41}. The isoform-selective fluo...

Materials

List of materials used in this article

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