SCC61 cells (head and neck squamous cell carcinoma) were obtained from A. Weaver (Vanderbilt University Medical Center, Nashville, TN) and grown in DMEM containing 20% FBS and 4 µg/ml hydrocortisone (Sigma-Aldrich); NCI-H1792 and NCI-H23 lung cancer cells (a gift from J. Minna and A. Gazdar, University of Texas Southwestern, Dallas, TX) and BxPC3 pancreatic cancer cells (purchased from American Type Culture Collection) were grown in RPMI 1640 containing 10% FBS. PANC-1 and SU.86.86 pancreatic cancer cells (purchased from American Type Culture Collection) were grown in DMEM containing 10% FBS. FBS was obtained from HyClone. RPMI 1640 and DMEM (4.5 g/liter glucose) were purchased from Corning. All experiments were performed in complete medium in the absence of antibiotics.
Unless otherwise stated, cells were incubated for 16 h in a 1% O2 and 5% CO2 atmosphere in a humidified chamber in an incubation system (Xvivo; BioSpherix).
Reagents and antibodies
DAPT was purchased from Tocris Bioscience, TPA was purchased from Sigma-Aldrich, Echinomycin and compound E were obtained from Enzo Life Sciences, GM6001 was obtained from EMD, CRM-197 was obtained from BioAcademia, Gefitinib was purchased from Selleck Chemicals, and recombinant human HB-EGF was obtained from R&D Systems. Primary antibodies were HIF-1α (BD for immunoblotting and Novus Biologicals for immunohistochemistry), Notch1 (D1E11) and active Notch1 (D3B8; both obtained from Cell Signaling Technology), placental AP (H-330; Santa Cruz Biotechnology, Inc.), HB-EGF (AF-259; R&D Systems), ADAM12 (Proteintech), and γ-tubulin (Sigma-Aldrich). Goat IgG control was obtained from Santa Cruz Biotechnology, Inc. Alexa Fluor 568–conjugated phalloidin and Alexa Fluor 594– or Alexa Fluor 488–conjugated goat secondary antibodies for immunofluorescence were obtained from Invitrogen.
RNAi and treatment with inhibitors
siRNA oligos targeting HIF-1α (J-004018-08 and J004018-10), NOTCH1 (J-007771-10 and J-007771-12), NOTCH2 (pool, J-0171870-00-0005), ADAM12 (pool, L005118-00-0005), and JAG2 (pool, L-012235-00-0005) as well as nontargeting controls were obtained from Thermo Fisher Scientific. siRNA oligos were transfected at 20 nM (individual) or 80 nM (pools) using Lipofectamine 2000 (Invitrogen). 48 h later, cells were trypsinized and counted, and 4 × 104 cells were seeded on 18-mm glass in 12-well plates, transferred to hypoxia, and processed for invadopodia quantification or activity 16 h later. For RNA or protein extraction, 5–7 × 105 cells were plated in 60-mm dishes, transferred to hypoxia, and processed 16 h later. shRNA lentiviral constructs targeting NOTCH1 (TRCN0000350330 and TRCN0000003359) and ADAM12 (TRCN0000047033 and TRCN0000047034) were obtained from Sigma-Aldrich. Lentiviral particles were generated by the Sanford Burnham Medical Research Institute (SBMRI) viral vector shared resource. After lentiviral infection, cells were selected with Puromycin (ECM Biosciences) at a final concentration of 5 µg/ml (SCC61) or 2 µg/ml (H1792 and BxPC-3) for 7–10 d before performing the experiments. Treatment with inhibitors was performed at the time of plating.
The AP-tagged HB-EGF–expressing construct was described before (Tokumaru et al., 2000
). A fragment containing the AP-tagged HB-EGF–encoding DNA was subcloned into the lentiviral expression construct pCDH-CMV-MCS-EF1-neomycin (System Biosciences). Lentiviral particles were generated by the SBMRI viral vector shared resource. Cells were selected with G418 (ECM Biosciences) at a final effective concentration of 300 µg/ml for SCC61 cells and 400 µg/ml for BxPC3 cells for 7–10 d before performing experiments.
For HIF-1α immunoblotting, the nuclear fraction was extracted by lysing cells in a hypotonic NP-40–based lysis buffer for 30 min on ice. The lysate was spun 5 min at 2,500 rpm at 4°C, and the pellet containing nuclei was washed five times with the same lysis buffer. The pellet was then resuspended in a hypertonic lysis buffer, incubated 30 min on ice, and spun 15 min at 14,000 rpm at 4°C. Samples were separated in 8% polyacrylamide gels (Invitrogen). For NOTCH1 immunoblotting, cells were lysed in a Triton X-100–based lysis buffer. Cleared lysate was assayed for total protein content using the bicinchoninic acid protein assay (Thermo Fisher Scientific), and 80–100 µg of total protein per sample was separated in an 8% polyacrylamide gel (Invitrogen). Secondary antibodies were conjugated to Alexa Fluor 680 (Invitrogen) or IR800 (Rockland Immunochemicals), and membranes were scanned using an infrared imaging system (Odyssey; LI-COR Biosciences).
RNA extraction and qPCR
RNA was extracted with RNeasy kit (QIAGEN). cDNA synthesis was performed using a reverse transcriptase kit (SuperScript III First-Strand; Invitrogen). qPCR was performed by the SBMRI Gene Analysis Core facility using qPCR SuperMix power SYBR green (Applied Biosystems) and a real-time cycler (Mx3000P; Agilent Technologies). Actin or 18S mRNAs were used as normalizers.
Invadopodia detection and gelatin degradation assay
Cells were fixed in 4% PFA (Electron Microscopy Sciences) in PBS for 20 min at RT, incubated in 3% BSA and 0.1% Triton X-100 in PBS for 30 min at RT, and incubated with Alexa Fluor 568–conjugated phalloidin (1:100–1:500 in PBS containing 0.3% BSA and 0.1% Triton X-100) for 1 h at RT. For each independent experiment, the number of cells forming invadopodia was quantified in ≥15 microscope fields (40×) imaged randomly and representing a total of ~150 cells per experimental condition. For gelatin degradation assays, sterile coverslips were coated with a thin layer of Oregon green gelatin obtained from Invitrogen (0.2 mg/ml in PBS containing 2% sucrose), fixed in 0.5% glutaraldehyde for 15 min, washed in PBS, and incubated with 5 mg/ml sodium borohydride for 3 min at RT. After washing in PBS, coverslips were transferred to sterile multiwell plates and incubated in complete growth medium for 30 min at 37°C. Cells were plated on top and incubated in normoxia or hypoxia as indicated. After processing for F-actin staining, images were randomly taken at 40× for ≥15 fields per experiment. The percentage of degraded area normalized to the number of nuclei in that area was represented as “degradation.” The percentage of degraded area was quantified with ImageJ (National Institutes of Health).
Treatment with CM
SCC61 (2 × 105 cells per well) or BxPC3 (1.5 × 105 cells per well) were plated in 24-well plates in 0.5 ml of growing media and transferred to hypoxia or left in normoxia for 16 h. Medium was collected, spun, and immediately used to treat cells for 6 h in normoxia. Cells were processed for invadopodia staining, and invadopodia numbers were quantified.
Notch was activated by using immobilized recombinant human JAG2 according to a previous protocol with some modifications (Sahlgren et al., 2008
). Glass coverslips were coated overnight at RT with 100 µg/ml recombinant protein G (Invitrogen) in PBS. After three washes in PBS, coverslips were blocked with 10 mg/ml BSA in PBS for 2 h at RT, washed with PBS, and incubated with recombinant human JAG2 fragment crystallizable (Fc) chimera (R&D Systems) or purified human IgG Fc fragment (EMD Millipore) at 2 µg/ml in PBS containing 1 mg/ml BSA for 4–6 h at RT. Coverslips were washed, and cells (4 × 104
) were plated on top and incubated in normoxia in the presence of DAPT or DMSO. After 16 h, cells were processed for quantification of invadopodia.
AP staining was performed in the absence of detergents. Cells were fixed in 4% PFA. The AP antibody was used at 1:200 dilution overnight at 4°C. Cells were washed and incubated 1 h in PBS containing 0.3% BSA and 1:500 dilution of Alexa Fluor 488–conjugated anti–rabbit antibody.
Fluorescence microscopy images were obtained with a fluorescent microscope (Axioplan 2; Carl Zeiss) equipped with a charge-coupled device camera (AxioCam HRm; Carl Zeiss) and AxioVision software (Carl Zeiss). Images were acquired at RT at either 40 or 63× using Plan Neofluar 40×/0.75 NA or Plan Apochromat 63×/1.40 NA oil differential interference contrast objectives, respectively. Images in were inverted using Photoshop (Adobe).
Shedding experiments were performed essentially as previously described (Tokumaru et al., 2000
) with minor modifications. AP–HB-EGF–expressing cells were plated in 24-well plates (105
cells per well for SCC61; 1.5 × 105
cells per well for BxPC3) and transferred to hypoxia (1% O2
) 5 h after plating. After 16 h on hypoxia, cells were washed three times in PBS and incubated in 0.5 ml DMEM with 4.5 g/liter glucose without phenol red (Invitrogen) containing 60 nM TPA for 1 h in normoxia. CM was collected and spun for 5 min at 14,000 rpm. AP activity in the CM was assayed immediately using an AP activity kit (BioVision) in 80–100 µl of sample in quadruplicate in a 96-well assay plate. Incubation was performed at 37°C in the dark until color developed. Colorimetry was performed using an absorbance microplate reader (ELx800; BioTek Instruments, Inc.) with absorbance set at 405 nm. For normalization, each well containing cells was lysed in 100 µl Triton X-100–based lysis buffer, and protein content in the cleared lysate was calculated using the bicinchoninic acid assay. Colorimetric values for AP activity were normalized to the protein content (micrograms per milliliter) of the cells in the corresponding well. When indicated, cells were treated with inhibitors or vehicle at the time of plating, and fresh inhibitors or vehicle was added to the shedding media containing TPA. For siRNA experiments, cells were transfected 48 h before plating and assayed for AP activity 60 h after transfection.
All experiments were reproduced at least three times with similar results. In the figure legends, n is the number of experimental replicates averaged in a graph. Statistical significance was determined by calculating the p-value using the paired Student’s t test.
The Oncomine database (Compendia Bioscience) was used to compare gene expression profiles for HIF-1α and ADAM12 between normal and lung cancer samples. The rank of a gene is the median rank for that gene across each of the analyses, and the p-value for a gene is the p-value for the median-ranked analysis. P-values were calculated by Oncomine using Student’s t test.
Lung tumor samples embedded in paraffin were obtained from the SBMRI tumor bank. Adjacent tumor sections were stained with HIF-1α or ADAM12 antibodies (1:50) according to manufacturer’s instructions. Signal was developed with DAB or Alexa Fluor 488– or Alexa Fluor 546–conjugated antibodies. Slides were digitalized using slide scanner (ScanScope XT; Aperio), and images were exported as JPEGs.
Online supplemental material
Fig. S1 shows quantification of invadopodia in H23, H1792, PANC-1, and SU.86.86 cells. Fig. S2 shows quantification of invadopodia and GLUT1
mRNA levels in SCC61 after HIF-1α siRNA transfection, quantification of invadopodia after Echinomycin treatment on SCC61, and GSI treatment on PANC-1, SCC61, and BxPC3 cells. Fig. S3 shows quantification of invadopodia after Notch1 siRNA transfection on SCC61, H1792, and BxPC3 cells. Fig. S4 shows expression of Notch signaling pathway components in SCC61 and H1792 cells, effect of Notch2 knockdown on invadopodia formation in hypoxia, and invadopodia quantification after activation of Notch by recombinant JAG2 in BxPC3 and H1792 cells and, after JAG2 siRNA treatment, in H1792 cells. Fig. S5 shows quantification of invadopodia after ADAM12 siRNA in SCC61 and H1792 cells and, after ADAM12 shRNA, in H1792 cells. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201209151/DC1
. Additional data are available in the JCB DataViewer at http://dx.doi.org/10.1083/jcb.201209151.dv