Patients and tissue collection
Tissue was obtained from the Department of Pathology at Rush University Medical Center, Chicago, IL. All procedures followed an Institutional Review Board (IRB) approved protocol. Ovarian tissue was obtained from women with normal ovaries at hysterectomy (mean age 47.4 ± 3.4 years; n = 11), patients with benign serous ovarian tumors (mean age 56.1 ± 13.6 years; n = 9) and primary ovarian cancer patients with malignant serous ovarian tumors (mean age 58 ± 11.1 years; n = 8). The tumor histology and tumor grade were determined by diagnostic evaluation by a pathologist. Malignant serous tumors comprised Grade 3 (n = 6) and Grade 1 (n = 2) with Stage II (n = 3) and Stage III (n = 5) pathology. The criterion for inclusion in the study was women ≥ 40 years old (range 43-76 years; mean age 54.2 ± 11.6 years) and for the patients with benign or malignant ovarian tumors the inclusion criteria included primary serous ovarian tumors. The criteria for exclusion were previous history of any cancer and prior chemotherapy or radiation treatment.
Assessment of mRNA expression
Total RNA was isolated using TRIZOL reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendation. RNA was measured at an optical density (OD) of 260 nm and the purity was evaluated using an OD 260/280 nm absorbance ratio ≥1.7. Before the first strand synthesis, 1 μg of total RNA was treated with DNase to remove trace genomic DNA. cDNA was synthesized using 500 ng of DNase treated RNA with a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) according to manufacturer's recommendation. Primer pairs were designed using Oligoperfect Designer software (Invitrogen) for ALDH1A1 [GenBank: NM_000689; in-between exon 6 and exon 7]. The Primer sequences were: ALDH1A1 Forward (5'- TTGGAATTTCCCGTTGGTTA-3') and Reverse (5'- CTGTAGGCCCATAACCAGGA-3'); Actin Forward (5'-CTGTGGCATCCACGAAACTA-3') and Reverse (5'- ACATCTGCTGGAAGGTGGAC -3'). The PCR amplifications were carried out in a 25 μl reaction volume containing 25 ng of cDNA using Platinum Taq DNA Polymerase (Invitrogen) according to manufacturer's recommendation. The mixture was denatured at 94°C (3 minutes) followed by 35 cycles at 94°C (30 seconds) and 54°C (30 seconds) to anneal and 72°C (1 minute) for extension followed by a final extension at 72°C (10 minutes) in a programmable Peltier Thermo Cycler (PTC-200, MJ Research Inc. Ramsey, MN). The PCR products were separated by electrophoresis in a 3% (W/V) agarose gel (Invitrogen) and visualized using ethidium bromide stain (Fischer Scientific, Pittsburg, PA). Amplicon from one positive sample each from normal ovary and ovarian serous carcinoma was purified using a QIAquick PCR purification kit (QIAGEN, Valencia, CA) and sequenced at DNA sequencing facility (University of Illinois at Chicago) using an ABI 3100 Genetic analyzer (Applied Biosystems). The amplicon sequences were blasted against the NCBI RefSeq human mRNA database and confirmed with a perfect match for ALDH1A1 gene [GenBank: NM_000689.3]. Quantitative Reverse Transcriptase-PCR (qRT-PCR) was carried out using SYBR green master mix in an ABI 7500 RT-PCR system and analyzed using the ΔCt method with human Actin as an internal control according to the manufacturer's recommendation (Applied Biosystems). The ΔΔCt was determined by subtracting ΔCt of each sample from the average ΔCt of normal ovary. The differences in ALDH1 mRNA expression levels were calculated as the fold change using the formula 2-ΔΔCt as previously described .
Immunohistochemical (IHC) detection of protein expression and localization
Tissues were fixed in formaldehyde, embedded in paraffin and sectioned (6 μm thick). Sections were mounted on microscope slides (Fischer Scientific, Pittsburg, PA), dried (16 hours; 37°C), deparaffinized in xylene, rehydrated in graded alcohols and rinsed with tap water. Sections were examined for histopathology following routine staining with hematoxylin and eosin (H&E; Sigma-Aldrich, St. Louis, MO). ALDH1, CD44, CD117 and CD133 expression was visualized using mouse anti-human ALDH1 mAb (clone 44, BD Transduction Lab San Jose, CA), mouse anti-human CD44 mAB (clone IM7; BioLegend, San Diego, CA), rabbit anti-human CD117 polyclonal antibody (C-19; c-Kit; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-human CD133 mAb (clone EMK08; eBioscience, San Diego, CA) respectively. Staining was carried out according to the manufacturer's protocol (Vector Laboratories, Burlingame, CA). In brief, antigens were unmasked by treating with antigen Unmasking solution (Vector Laboratories) and boiling in a microwave. Endogenous peroxidase was inactivated using substrate (0.3% H2O2 in methanol; 20 minutes; 22°C). Sections were washed with phosphate buffer and non-specific binding sites were blocked with normal horse serum (30 minutes). The sections were then incubated with mouse anti-human ALDH1 antibody (1:200) diluted in phosphate buffer containing 1% Bovine Serum Albumin (BSA; Sigma-Aldrich, St. Louis, MO) in a humid chamber (2 hours, 22°C). The bound anti-human ALDH1 antibody was detected using ABC Universal kit and the antigen-antibody reaction was visualized with 3, 3-diaminobenzidine peroxide substrate (DAB; brown color). As a control for secondary antibody binding directly to sections, the ALDH1 antibody was omitted. Sections were briefly rinsed in water, counterstained with hematoxylin (Fischer Scientific) and rinsed in running water (15 minutes). Double label immunostaining was carried out according to the manufacturer's multiple labeling protocol (Vector Laboratories). In brief, the ALDH1 stained sections were further treated with normal horse serum (30 minutes) to block non-specific binding sites. Sections were then incubated with anti-human CD44 or CD177 or CD133 antibody (1:100, diluted in 1% BSA in phosphate buffer) and processed as described for anti-ALDH1 alone, except that the color was developed with DAB and Nickel peroxide substrate (gray/black color). Finally, the sections were dehydrated in graded alcohols and xylene, and covered using Permount (Fischer Scientific). Sections were examined by light microscopy (Olympus BX-41, Center Valley, PA) and images captured and evaluated with MicroSuite Five software (Olympus).
ALDH1 protein expression and localization was assessed using a unbiased cell counting stereology method with a microscope (Olympus BX60, Center Valley, PA) interfaced with a digital camera (CX9000; MBF Bioscience Williston, VT), motorized stage and image analysis software (StereoInvestigator 8.1, MBF Bioscience, Williston, VT). Cell estimation was performed using optical fractionator procedure . Three sections/sample (triplicates) were evaluated. Briefly sections were outlined and scanned at low magnification (×12.5). The thickness of each section was measured at higher magnification (×600) in three separate areas, and the average thickness of each section was calculated. Cells were counted under higher magnification (×600) using an oil immersion objective. Cell counts were estimated within a dissector height of 7 μm, using an 800 × 800 μm2 grid size and a 60 × 60 μm2 counting frame size. The coefficient of error was calculated based on the Gundersen equation . ALDH1 staining was quantified using average number of ALDH1 positive cells divided by the average number of Hematoxylin counterstained cells in each group and expressed as % mean ± standard deviation (SD).
Western blot and densitometry analysis
Total protein was extracted from tissue and separated by one-dimensional Western blot using 10% gradient Tris-HCl gels (Bio-Rad, Hercules, CA; 10 μg total protein/lane) using standard procedures as described previously . Proteins were transferred to a nitrocellulose membrane (0.45 μm; Bio-Rad). Recombinant ALDH1A1 (rALDH1; 1 μg/lane) produced in collaboration with Dr. Jim Dias (University of Albany, Albany, NY) was used as a positive control. Mouse anti-human ALDH1 (1:2000; clone 44, BD Transduction Lab San Jose, CA) and peroxidase-conjugated donkey anti-mouse IgG (1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA) antibody was used to detect ALDH1. Human β-actin was used as a loading control and was detected with mouse anti-actin (1:2000; Sigma, St. Louis, MO). Antibodies were diluted in Blocker solution (Sigma) containing 0.05% Tween 20 (Bio-Rad). The membranes were washed after each step using Tris-buffered saline (10 mM Tris and 0.15 M NaCl, pH7.5) containing 0.05% Tween 20. The protein bands were detected using SuperSignal West Dura substrate (Thermo Scientific, Rockford, IL). MagicMark XP Western standards (Invitrogen, Carlsbad, CA) were used to estimate molecular weight. Digital images were obtained with a Chemidoc XRS Imaging System (BioRad) and analyzed by Quantity One software (Bio-Rad) according to manufacturer's recommendation. The relative density of each ALDH1 band was expressed as a ratio of the density of ALDH1 band and the corresponding β-actin band.
Assessment of ALDH1 expression and enzyme activity by flow cytometry
The tissue was dissociated mechanically and enzymatically using a solid human tissue dissociation protocol (Stemcell Technologies, Vancouver, BC) with minor modifications. In brief, tissue was minced, washed in cold Dulbecco's Phosphate Buffered Saline (DPBS; Invitrogen, Carlsbad, CA) and suspended in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Invitrogen) supplemented with 5% Fetal Bovine Serum (FBS; Invitrogen), collagenase type I (Worthington, Lakewood, NJ) and DNase 1 (Stemcell Technologies) followed by incubation with gentle agitation (2 hours; 37°C). The cell pellet and tissue fragments were separated by centrifugation (5 minutes; 100 × g) followed by a wash with DPBS. A single-cell suspension was obtained after filtering through 40 μm sterile nylon mesh (BD Falcon, San Jose, CA). The flow through was collected in a fresh tube, centrifuged (5 minutes; 100 × g), washed and suspended in DPBS. To remove and lyse red blood cells the cells were treated with ammonium chloride solution (BioLegend, San Diego, CA; 10 minutes; 4°C). Cells were then suspended in DPBS with 2% BSA and the cell count was determined using a Coulter Counter (Beckman, Brea, CA).
Aldehyde dehydrogenase enzyme activity in viable cells was determined using a fluorogenic dye based ALDEFLOUR assay (Stemcell Technologies) according to the manufacturer's instructions. In brief, cells were suspended (0.5 × 106 cells/mL) in ALDEFLUOR assay buffer containing ALDH substrate (Bodipy-Aminoacetaldehyde) and incubated (45 minutes; 37°C). As a reference control, the cells were suspended in buffer containing ALDEFLUOR substrate in the presence of diethylaminobenzaldehyde (DEAB), a specific ALDH1 enzyme inhibitor. Propidium iodide (2 μg/mL; Sigma, St. Louis, MO) was used to exclude dead cells. The cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, Rockville, MD) and the data was analyzed using FlowJo 7.6.1 software (Tree Star, Ashland, OR).
The outcome variables were expressed as mean ± SD. SPSS (Student version 7.5, SPSS Inc., Chicago, IL) was used for statistics. The independent samples t-test was used to test the statistical difference between groups. Correlation was analyzed by calculating a Pearson correlation coefficient (r). P values < 0.05 were considered statistically significant.