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. Al....

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 p....

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<.......

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

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