Antibodies and reagents
Primary antibodies: Anti-rat-E-cadherin, anti-rabbit-β-actin, anti-mouse-vimentin, anti-rabbit-fibronectin all from Abcam, Cambrigde, MA; anti-rabbit-β-catenin (Sigma-Aldrich, St. Louis, MO), CD68-Alexa-488 and F4/80-Alexa-647 (AbD Serotec, Düsseldorf, Germany) and anti-mouse-active-β-catenin (Millipore, Billerica, MA). Secondary antibodies: Goat-anti-rat-IgG-TRITC (Sigma-Aldrich, St. Louis, MO); chicken-anti-rabbit-Alexa-594 (Molecular Probes, Carlsbad, CA); biotin-SP-donkey-anti-rabbit-IgG, biotin-SP-donkey-anti-rat-IgG, and biotin-SP-donkey-anti-mouse-IgG (Jackson ImmunoResearch Laboratories, INC, Suffolk, UK). Streptavidin-HRP (Biolegends, San Diego, CA) was used to detect biotin labeled secondary antibodies. Recombinant TGF-β1 and TGF-β neutralizing antibody was purchased from R&D Biosystems (Minneapolis, MN) and recombinant EGF was kindly provided by Dr. A. Mueller, IMCR, University of Zürich, Switzerland. LEAF (low endotoxin azide free) purified mouse IgG1, κ isotype control antibody was obtained from BioLegends (San Diego, CA). Recombinant IL-4 and IL-13 were from Biosource (Camarillo, CA).
Cell lines and conditioned media
F9-teratocarcinoma cells (ATCC CRL-1720) were grown on 0.01% gelatin. NMuMG-cells (CRL-1636) were kindly provided by Prof. G. Christofori, Center for Biomedicine, University of Basel, Switzerland. Both cell lines were cultured at 37°C, 5% CO2
in DMEM/10% FBS/0.8% penicillin-streptomycin. RAW264.7 macrophages (Sigma-Aldrich, St. Louis, MO) were cultured at 37°C, 5% CO2
in RPMI1640/10% FBS/0.8% penicillin-streptomycin/1% Na-pyruvate (GIBCO, Basel, Switzerland). Conditioned medium was generated by culturing cells at 80% confluence in DMEM/10% FBS/0.8% penicillin-streptomycin for 24 h followed by sterile filtration. RAW264.7 macrophages were M2 polarized by culturing in DMEM/10% FBS plus recombinant IL-4 and IL-13 for 48 h (each 10 ng/ml) as previously described [43
F9 tumors and macrophage depletion
F9-tumors were generated in female SV129S1 mice (Charles River, Sulzfeld, Germany) and liposomes were prepared as previously described [11
]. Mice were kept in standard housing and normal diet at the animal facility of the University of Zürich. Animal studies were approved by the Veterinary Department of the Canton Zürich and performed under license 183/2006 issued to R.A. Schwendener. The control group (n = 6) received empty liposomes (100 μl/20 g body weight, i.p.), the test group (n = 6) clodrolip (1.5 mg clodronate/20 g body weight, i.p.) starting 6 h post tumor inoculation and followed by the same dosage every 3rd day for 20 days. Tumors subjected to immunohistochemistry and protein analysis were stored in Hanks salt buffer (GIBCO, Basel, Switzerland) at -80°C. Tumors subjected to q-PCR were stored in RNAlater as described by the provider (Qiagen, Valencia, CA). Data are shown from two independent experiments and non-responders as assayed by q-PCR of Csfr-1
were excluded from the study unless otherwise noted.
H&E staining, immunohistochemistry and quantification of frozen F9 tissue sections
Frozen sections (8 μm) were acetone fixed. The sections were either stained with haematoxylin and eosin (DAKO, Glostrup, Denmark) following the providers protocol or blocked with 1% BSA/TBS prior to immunostaining. For immunohistochemistry, the sections were incubated with primary and secondary antibodies overnight at 4°C. Nuclei were stained with DAPI (1 μg/ml). The sections were mounted with Vectashield (Vector Labs, Burlingame, CA) and visualized with an Olympus fluorescence microscope (1X81) using the CellR software (Olympus, Hamburg, Germany). The pictures were merged in Adobe Photoshop CS4. Macrophage density was quantitatively estimated by counting the absolute number of CD68+ and F4/80+ cells and the total number of DAPI positive cells in defined areas throughout tumor sections using ImageJ software (NIH, Bethesda, MD). Quantification and correlation of macrophage density and tumor cell expression of EMT-associated markers was similarly done in a quantitative manner, using ImageJ to count the absolute number of CD68+ and EMT-marker positive cells in defined areas throughout the tumor sections. The quantifications were done on 6-10 individual sections from various control and clodrolip treated tumors sampled from two independent experiments.
In vitro induction of EMT and immunofluorescence analysis of F9-and NMuMG-cells
F9-and NMuMG-cells were cultured on sterile glass coverslips in F9-CM, N-CM, M-CM +/- LEAF purified IgG1 control antibody (1 μg/ml) or TGF-β neutralizing antibody (1 μg/ml), DMEM/10% FBS +/- rEGF (50-100 ng/ml) or +/- rTGF-β1 (2 ng/ml). The medium was renewed every 24 h. The cells were harvested at the time points annotated, fixed with 3% formaldehyde, stained and visualized as described for frozen sections.
In vitro invasion assay
The cells were starved in serum free medium for 6 h and seeded (100.000 cells/well) in Boyden chambers (Corning, NY, 8 μm pore size) coated with 50 μl 1% Matrigel (BD Biosciences, Rockville, MD). F9-CM, N-CM and M-CM +/-LEAF purified IgG1 control antibody (1 μg/ml) or +/- TGF-β neutralizing antibody (1 μg/ml) were used as chemoattractants. The assay was incubated for 48 h at 37°C, 5% CO2. The relative number of invading cells was estimated by resazurin live cell detection using the provider's protocol (Invitrogen, Carlsbad, CA). Fold invasion was calculated relative to control conditions.
TOPFLASH reporter assay
The TOPFLASH reporter assay was established as previously described [44
]. The fold values were calculated as TOPFLASH/FOPFLASH, where TOPFLASH is the plasmid expressing luciferase downstream of three wild type β-catenin/Tcf binding sites, and FOPFLASH is the plasmid with mutated binding sites. Renilla pRL SV40 was included as transfection control. Luciferase was detected using the Dual Glo Luciferase detection kit (Promega, Madison, WI). The cells were transfected two days prior to luciferase readout using a standard in-house transfection protocol. Fold changes were calculated relative to controls.
F9-and NMuMG-cells were lysed 1% NP-40, 100 mM orthovanadate, 100 mM 3-indoleacetic acid (IAA), 100 mM phenylmethylsulfonylfluoride (PMSF) at annotated time points and snap frozen in liquid nitrogen. Frozen tumors were cut into small pieces and soaked in lysis buffer and homogenized using an Ultra Turrax T8 homogenizer (IKA-Werke, Staufen, Germany). Protein concentration was determined by Bradford analysis (Bio-Rad, Reinach BL, Switzerland). The blots were quantified using ImageQuant 5.2 software (Amersham Biosciences, Piscataway, NJ). Protein expression was normalized to β-actin levels.
Quantitative real time PCR
Total RNA was isolated from homogenized F9-tumors using the RNAeasy kit (Qiagen, Valencia, CA). cDNA was synthesized using the Omniscript reverse transcriptase kit (Qiagen, Valencia, CA). Q-PCRs were carried out using the LightCycler 480 instrument (Roche Diagnostics, Rotkreuz, Switzerland). PCR program: 95°C, 5 min, 45 cycles of 10 s 95°C, 25 s annealing and 15 s 72°C. Primers were obtained from Microsynth, Switzerland, (for primer sequences and annealing temperatures, see Additional file 1
: Table S1). The quality of the PCR-products was assayed on 1.5% agarose gels. Expression of all target genes was normalized to β-actin and GAPDH. All samples were run in duplicates; n = 5-6/per group. Fold change was calculated as clodrolip treated versus control tumors using the Pfaffl equation [45
NSCLC tissue microarrays and patient cohort
The selection of NSCLC patient tissue samples and manufacture of the tissue microarrays (TMAs) were done as previously described [26
]. In brief, formalin-fixed and paraffin-embedded tumor tissues of 532 NSCLC patients were reviewed by two pathologists and two representative tissue cores (0.6 mm) were assembled into 3 TMAs. Patients having obtained neo-adjuvant chemotherapy were excluded. Sarcomatoid carcinomas were excluded from this study and EMT was strictly defined by expression of EMT-associated protein markers and not by morphology (n final = 491). The study was approved by the institutional review board of the University Hospital Zürich under reference number StV-29-2009.
NSCLC immunohistochemistry and interpretation
Immunohistochemistry on 4 μm sections from the TMA blocks was performed using automated immunohistochemistry platforms from either Ventana (Ventana Medical Systems, Tucson, AZ) or Bond (Vision Biosystems, Melbourne, Australia). Following primary monoclonal antibodies were used: anti-CD68 (DAKO-Cytomation, clone PG-M1, 1:50 dilution, Glostrup, Denmark), anti-E-cadherin (Cell Marque, clone EP700Y, 1:200), anti-β-catenin (BD Transduction laboratories, clone 14, 1:50, Lexington, KY), anti-vimentin (DAKO-Cytomation, 1:250), Pab anti-periostin (BioVendor, 1:500, Modrice, Czech Republic) and anti-TGF-β1 (Santa Cruz, 1:100 dilution). Detection was done using the UltraVIEW-DAB (Ventana Medical Systems) or the Refine-DAB (Bond) detection kits, including respective secondary antibodies. Distinct intra-epithelial CD68+ macrophage density was quantitatively scored by AKB and by two pathologists (VT and AS) on a multi-headed microscope (Zeiss Axioscope 2 MOT) using a four-tiered system: 0 (negative), 1+ (few to some CD68+ macrophages), 2+ (moderate number of CD68+ macrophages), and 3+ (multiple CD68+ macrophages). Membranous β-catenin (AKB and VT) and membranous E-cadherin (VT and AS) were evaluated for staining intensity according to a four-tiered system: 0 (negative, no detectable staining), 1+ (weak, faint discontinuous membrane staining), 2+ (moderate and continuous membrane staining), 3+ (strong and continuous membrane staining). Cytoplasmic β-catenin (AKB and VT), cytoplasmic vimentin (AKB and AS), cytoplasmic periostin (VT and AS) were scored due to staining intensity: 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong) and TGF-β1 (AKB and VT) was scored for intraepithelial staining intensity: 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong).
Statistical methods and correlation interpretation
The statistical analyses of all in vitro assays were performed using the GraphPad Prism 5 software (GraphPad Software, La Jolla, CA). All data are reported as mean ± SEM. The significance levels were evaluated by two tailed, unpaired t-tests; *P < 0.05, **P < 0.01. The statistical and correlation analyses of the NSCLC tissue samples were done in SPSS 16.0 for windows (IBM, Somers, NY) using the Spearmann correlation coefficient as a readout for degree of correlation with *P < 0.05.