SWCNT characterization and preparation
SWCNT were obtained from Carbon Nanotechnology (CNI, Houston, Texas) and were purified by acid treatment to remove metal contaminates. Elemental carbon analysis was performed by NIOSH Manual of Analytical Methods (NMAM 5040), whereas trace metal analysis was performed by nitric acid dissolution and inductive coupled plasma-atomic emission spectrometry (ICP-AES, NMAM 7300). The specific surface area was measured at -196°C by the nitrogen absorption-desorption technique (Brunauer Emmet Teller method, BET) using a SA3100 Surface Area and Pore Size Analyzer (Beckman Coulter, Fullerton, CA). The diameter and length distribution of the SWCNT were measured by field emission scanning electron microscopy. SWCNT were dispersed by acetone/sonication method [16, 24, 32] or by using Survanta® as previously described . For acetone/sonication method, SWCNT were treated with acetone and placed in an ultrasonic bath for 24 h. The dispersed SWCNT were then filtered from the solution using a 20-μm nylon mesh screen followed by a 0.2-μm polytetrafluoroethylene filter, which had been weighed prior to use. After filter collection, the dispersed SWCNT were washed thoroughly with distilled water, weighted, and suspended in phosphate-buffered saline with 2–3 minute sonication (Sonic Vibra Cell Sonicator, Sonic & Material Inc, Newtown, CT, USA). For Survanta® dispersion, 1 mg of SWCNT were dispersed in 1 mL of phosphate-buffer saline (PBS) containing 150 μg/mL of Survanta® (Abbott Laboratories, Abbott Park, IL) using light sonication and were diluted in culture medium to obtain the desired concentration.
Chemicals and reagents
Crocidolite asbestos (CAS# 12001-28-4) was obtained from the National Institute of Environmental Health Sciences (Research Triangle Park, NC). Hoechst 33342, cis-diamminedichloroplatinum II (cisplatin), etoposide, antimycin A, and antibody against -actin were obtained from Sigma-Aldrich (St. Louis, MO). Doxorubicin was obtained from EMD Biosciences (La Jolla, CA). Human pluripotent stem cell array was obtained from R&D Systems (Minneapolis, MN). Antibodies against Nanog, E-cadherin, and peroxidase-labeled secondary antibody were obtained from Cell Signaling Technology (Boston, MA). Antibodies against SOX2 and SOX17 were obtained from Millipore (Billerica, MA). Antibody against p53 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorochrome-conjugated antibodies against human CD24, CD44, and CD133 were obtained from Miltenyi Biotec (Auburn, CA).
Primary human small airway epithelial cells (SAECs) immortalized with hTERT were kindly provided by Dr. Hei (Columbia University, NY) . SAECs were cultured in SABM medium supplemented with Clonetics SAGM SingleQuots (Lonza, Walkersville, MD) which contain 0.4% v/v bovine pituitary extract, 0.1% insulin, 0.1% hydrocortisone, 0.1% retinoic acid, 1% bovine serum albumin, 0.1% transferrin, 0.1% triiodothyronine, 0.1% epinephrine, 0.1% human epidermal growth factor and 0.1% gentamicin. Human bronchial epithelial BEAS-2B cells were obtained from American Type Culture Collection (ATCC; Manassas, VA). They were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. Non-small lung cancer cell (NSCLC)-H460 cells were obtained from American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 medium supplemented with 5% FBS, 2 mM L-glutamine, and 100 units/mL penicillin/streptomycin. All cells were maintained in a humidified atmosphere of 5% CO2 at 37°C.
Plasmid and transfection
p53 and control GFP plasmids were obtained from Invitrogen (Carlsbad, CA). Cells were transfected with p53 or GFP plasmid by nucleofection using Nucleofector® (Amexa Biosystems, Cologne, Germany), according to the manufacturer’s instructions. Briefly, cells were suspended in 100 μL of nucleofection solution with 2 μg of plasmid and nucleofected using the device program T020. The cells were then resuspended in 500 μL of complete medium and seeded in 60-mm cell culture dishes. Cells were allowed to recover for 48 hours before each experiment. The efficiency of transfection was determined by using a green fluorescent protein reporter plasmid and was found to be ~80%.
Chronic SWCNT exposure and derivation of SWCNT-transformed cells
Lung epithelial BEAS-2B and SAEC cells were continuously exposed to low-dose SWCNT (surface area dose of 0.02 μg/cm2 or concentration dose of 0.1 μg/mL) in culture for 6 months. The cells were passaged weekly at preconfluent densities using a solution containing 0.05% trypsin and 0.5 mM EDTA (Invitrogen, Carlsbad, CA). SWCNT-exposed BEAS-2B cells were designated as BSW cells, whereas SWCNT-exposed SAEC cells were designated as SASW cells. Parallel cultures grown in SWCNT-free medium with the same background level of dispersant provided passage-matched controls and were designated as BC and SAC cells for the cells originated from BEAS-2B and SAEC cells, respectively. After 6 months of exposure, the cells were cultured in normal complete medium, and their cancer and CSC phenotypes were assessed as described below. Human non-small cell lung cancer H460 cells were used as a positive control in the studies.
Soft agar colony formation assay
Soft agar assay was performed as previously described with minor modifications . Passage-control BC and SAC cells, and chronic SWCNT-exposed BASW and SASW cells (3 × 104 cells) were mixed with culture medium containing 0.5% agar to a final agar concentration of 0.33%. Cell suspensions were immediately plated onto dishes coated with 0.5% agar in culture medium. Colonies were examined under a light microscope after 2 weeks of culture.
Tumor sphere assay
Tumor sphere assay was performed under non-adherent and serum-free conditions as previously described as stem cell-selective conditions [31, 36]. Briefly, cells were resuspended in 0.8% methylcellulose (MC)-based serum-free medium (Stem Cell Technologies, Vancouver, Canada) supplemented with 20 ng/mL epidermal growth factor (BD Biosciences, San Jose, CA), basic fibroblast growth factor and 4 mg/mL insulin (Sigma) and plated at 5 × 103 cells (BC, BSW, and H460) or 1 × 104 cells (SAC and SASW) in ultralow adherent 24-well plates (Corning, Corning, NY). Cells were cultured for two or three weeks. In order to assess the self-renewing property of cells, spheres were collected by gentle centrifugation, dissociated into single cell suspensions, filtered and cultured under conditions described above (second spheres).
Side population analysis and isolation
Cells were detached by trypsinization and 1 × 106 cells were labeled with 5 μg/mL of Hoechst 33342 in DMEM-F12 medium containing 2% FBS in the presence or absence of 25 μM ABCG2 inhibitor fumitremorgin C (FTC; EMD Biosciences, San Diego, CA) at 37°C for 90 minutes. The cells were then centrifuged and resuspended in ice-cold Hank’s buffer salt solution (HBSS). SP analysis and sorting were performed using BD FACSAria fluorescence-activating (flow cytometry)-based cell sorter (BD Biosciences). The Hoechst dye was excited with a UV laser and its fluorescence was measured with both a 450/20 filter (Hoechst Blue) and 675 LP filter (Hoechst Red). SP fraction was calculated based on the disappearance of SP cells in the presence of FTC using the formula: SP percentage in the absence of FTC - SP percentage in the presence of FTC.
Apoptosis was determined by DNA condensation/fragmentation assay using Hoechst 33342 dye. Cells were incubated with 10 μg/mL of Hoechst 33342 for 30 minutes and visualized under a fluorescence microscope (Leica Microsystems, Bannockburn, IL). Cells having intensely condensed and/or fragmented nuclei were considered apoptotic. Approximately 1000 nuclei from 10 random fields were analyzed for each sample. The apoptotic index was calculated as the percentage of cells with apoptotic nuclei over total number of cells.
Cell migration and invasion assays
In vitro cell migration and invasion were determined using a 24-well Transwell® unit with polycarbonate (PVDF) filters (8-μm pore size). The membrane was coated with Matrigel® (BD Biosciences, NJ) for the invasion assay, while control inserts were used for the migration assay. Briefly, cells at the density of 3 × 104 cells per well (invasion) or 1.5 × 104 cells per well (migration) were seeded into the upper chamber of the Transwell® unit in serum-free medium. The lower chamber of the unit was filled with a normal growth medium containing 5% FBS. Chambers were incubated at 37°C in a 5% CO2 atmosphere for 48 hours. The non-migrating or non-invading cells were removed from the inside of the insert with a cotton swab. Cells that migrated or invaded to the underside of the membrane were fixed and stained with 10 μg/mL Hoechst 33342 for 30 minutes. Inserts were visualized and scored under a fluorescence microscope (Leica DM, IL).
Xenograft mouse model
Animal care and experimental procedures described in this study were performed in accordance with the Guidelines for Animal Experiments at West Virginia University with the approval of the Institutional Animal Care and Use Committee (IACUC #12-0502). Immunodeficient NOD/SCID gamma mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG; Jackson Laboratory, Bar Harbor, ME), were maintained under pathogen-free conditions within the institutional animal facility. Food and tap water were given ad libitum. Mice were subcutaneously injected with 5 × 103 - 1 × 105 sorted SP and NSP cells derived from the transformed BSW, positive control H460 cells, or passage-control BC cells suspended in 100 μL of ExtraCel® hydrogel (Advanced BioMatrix, San Diego, CA). Mice were inspected daily for any signs of distress such as weight loss, hunching, failure to groom, and red discharge from the eyes. Tumor growth was monitored daily and tumor size was measured at 21, 28 and 35 days post-injection by using an external caliper (VWR International, Batavia, IL). Tumor volume was calculated using the formula: tumor volume [mm3] = 1/2 (length [mm]) × (width [mm]2 ). At the end of experiments, mice were euthanized and tumors were dissected and weighted.
Tumor samples from each tumor were formalin-fixed and paraffin-embedded. Tumor specimens were cut into 5-μm sections and stained with hematoxylin and eosin (H&E) to define the morphology and cellular structure within the tumor region. All tissue sectioning and staining were performed at the West Virginia University Pathology Laboratory for Translational Medicine. The presence of multinucleated cells and condensation of heterochromatin (hematoxylin staining) were considered as cancer-specific patterns.
Human stem cell array
The Proteome Profiler™ array of human pluripotent stem cell array was commercially obtained from R&D Systems (Minneapolis, MN) and was used according to the manufacturer’s instruction. Briefly, a total of 150 μg of protein lysates were incubated overnight with nitrocellulose membranes dotted with duplicate spots for 15 stem cell markers and control antibodies. Bound proteins were detected with horseradish peroxidase (HRP)-conjugated antibodies using a chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ) and quantified using analyst/PC densitometry software.
Western blot analysis
After specific treatments, cells were incubated in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM Na3VO4, 50 mM NaF, 100 mM phenylmethylsulfonyl fluoride, and a commercial protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN) at 4°C for 20 minutes. The lysate was collected and determined for protein content using the Bradford method (Bio-Rad Laboratories, Hercules, CA). Proteins (40 μg) were resolved under denaturing conditions by 7.5-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Bio-Rad). The transferred membranes were blocked for 1 hour in 5% nonfat dry milk in TBST (25 mM Tris–HCl, pH 7.4, 125 mM NaCl, 0.05% Tween 20) and incubated with the appropriate primary antibodies at 4°C overnight. Membranes were washed twice with TBST for 10 minutes and incubated with HRP-coupled isotype-specific secondary antibodies for 1 hour at room temperature. The immune complexes were detected by an enhanced chemiluminescence detection system and quantified using analyst/PC densitometry software.
Stem cell surface marker analysis
Cells were detached by trypsinization and 2 × 105 cells in 100 μL of FACS buffer were labeled with 10 μL of fluorochrome-conjugated antibodies against CD24, CD44, and CD133 (Miltenyi Biotec) in a dark refrigerator for 15 minutes. The cells were then washed, fixed in 2% paraformaldehyde, and resuspended in FACS buffer for analysis by flow cytometry.
The data represent means ± SD from three or more independent experiments as indicated. Statistical analysis was performed by Student’s t test at a significance level of p < 0.05.