Reagents
The following primary antibodies were used: c-Myc (9E10, to detect Myc-tagged protein), DVL2 and DVL3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); DVL1 (R&D Systems, Abingdon, UK); active β-catenin (anti-ABC; Upstate, Billerica, VA, USA); α-tubulin (DM1A) (Neomarkers, Fremont, CA, USA); cyclin D1 (SP4) (Cell MARQUE, Rocklin, CA, USA) for immunohistochemistry (IHC); cyclin D1 (Chemicon, Billerica, MA, USA) for western blotting; bromodeoxyuridine (BrdU) (Roche, Basel Switzerland); p21Cip1 (OP64-100UG) (Oncogene Research Products, Cambridge, MA, USA); ERK and P-ERK (Thr202/Tyr204) (Cell Signaling Technology, Danvers, MA, USA); total β-catenin and CD31 (BD Pharmingen, Franklin Lakes, CA, USA); and active β1-integrin (Clone HUTS-4, MAB2079Z; Chemicon).
As secondary antibodies we used anti-rabbit and anti-mouse antibodies (GE Healthcare, Little Chalfont, UK; LI-COR Bioscience, Lincoln, NE, USA), anti-rat antibody (GE Healthcare) or anti-goat antibody (DAKO A/S, Glostrup, Denmark) coupled to horseradish peroxidase (HRP) or IRDye 800CW.
For IHC we used Biotin-SP-conjugated affinipure donkey anti-rabbit, anti-mouse, anti-rat IgG (Jackson ImmunoResearch, West Grove, PA, USA) and goat anti-rat ALEXA 568 (Molecular Probes, Eugene, OR, USA). Recombinant Wnt3a was purchased from R&D Systems. Y27632 was purchased from Sigma-Aldrich (St. Louis, MO, USA). The cDNA encoding Myc/His-tagged human sFRP1 in pCDNA was provided by Jeffrey Rubin (NCI, Bethesda, MD, USA) and was recloned into the pBabePuro retroviral vector. Conditioned media (CM) from Wnt1-producing cells, from sFRP1-producing cells, and purified sFRP1 were prepared as previously described [7].
T-cell factor reporter assay
MDA-MB-231/sFRP1-P1 cells and control-P1 cells were seeded on 12-well plates and were transfected with a mixture of Super TOPFlash plasmid and pRL-CMV (Promega, Madison, WI, USA) to assay TCF promoter activity using Fugene6 (Roche) according to the manufacturer's instructions. Luciferase activities were measured 48 hours later using the Dual-Luciferase Reporter Assay System (Promega) and Mithras LB940 (Berthold Technologies, Bad Wildbad, Germany) according to the manufacturer's instructions. The fold activation was normalized against renilla luciferase. Nine wells were used per condition and the average and standard error are shown in the graph.
Cell culture, transfections, retroviral infections, proliferation and anoikis assays
The human breast cancer cell line MDA-MB-231 (ATCC, Manassas, VA, USA) was cultivated in DMEM, 10% heat-inactivated FCS (Amimed, Allschwil, Switzerland) supplemented with penicillin and streptomycin (Sigma-Aldrich). All transfections were performed using FuGENE 6 Transfection Reagent (Roche) following the manufacturer's guidelines. MDA-MB-231 cells were stably transfected with pCDNA3.1(+) (Invitrogen, Carlsbad, CA, USA) encoding Myc/His-tagged human sFRP1 or empty pCDNA3.1(+) as control. After selection with 1 mg/ml G-418, three clones of MDA-MB-231/sFRP1 and three control clones were isolated. Equal cell numbers of these clones were pooled before some experiments (MDA-MB-231/sFRP1-P1 and MDA-MB-231/control-P1). A second pool of sFRP1-expressing MDA-MB-231 cells (MDA-MB-231/sFRP1-P2) and control cells (MDA-MB-231/control-P2), each representing > 100 clones, was generated by infecting the cells with pBabePuro encoding Myc/His-tagged human sFRP1 or empty pBabePuro followed by selection with 2 μg/ml Puromycin (Sigma-Aldrich).
Cell proliferation was measured either by counting cell numbers with a Vi-Cell XR cell viability analyzer (Beckman Coulter, Fullerton, CA, USA) on selected days after seeding 200,000 cells on six-well plates or using the YOPRO cell viability assay (Invitrogen) 3 days after seeding 1,000 cells on a 96-well plate, according to the manufacturer's instructions. Anoikis was measured by seeding cells in 1% FCS-containing medium on polyHema-coated plates to prevent adhesion. Cells were harvested 24 hours later, stained with propidium iodide. The cell cycle distribution was analyzed with a FACScalibur (Becton Dickinson, San Jose, CA, USA) using the Cellquest software. A representative cell cycle distribution of three MDA-MB-231/sFRP1 clones and three control clones is shown. Unless otherwise noted, P values were calculated using Student's t test.
Protein extraction and western blotting
Cells were lysed in 1% Nonidet P-40, 50 mM Tris pH 7.5, 120 mM NaCl, 5 mM ethylenediamine tetraacetic acid, 1 mM ethylene glycol tetra-acetic acid (EGTA), 2 mM sodium vanadate, 20 mM β-glycerophosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 0.5 mM phenylmethanesulphonylfluoride (PMSF), 50 mM NaF, 1 mM dithiothreitol for 5 minutes on ice before collecting lysates. Debris was removed by centrifugation at 4°C and the protein concentration was determined using the Bradford reagent (BioRad, Hercules, CA, USA).
For western blotting, protein loading buffer was added to 30 to 50 μg total protein and the samples were denatured for 10 minutes at 95°C prior to separation on SDS-polyacrylamide gels and blotting by semi-dry transfer for 90 minutes on PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked using 10% horse serum in Tris-buffered saline- Tween buffer for 1 hour (0.2 M NaCl, 25 mM Tris, pH 7.5, 0.5 ml/l Tween-20), except for p21Cip detection where PBS- Tween buffer was used instead of TBS-Tween buffer for blocking. Blots were incubated with primary antibodies at room temperature for 1 hour or at 4°C overnight, followed by 30-minute incubation with secondary antibodies. These antibodies were anti-rabbit-HRP, anti-mouse-HRP (1:5000) or anti-goat-HRP (1:5000) for detection of luminescence, which was carried out using ECL (GE Healthcare) according to the manufacturer's instructions and using X-OMAT LS films (Kodak, New York, NY, USA). Secondary antibodies IRDye 800CW goat anti-rabbit-IgG or anti-mouse-IgG (1:10,000; LI-COR Biosciences) were detected with the LI-COR Odyssey system according to the manufacturer's instructions (LI-COR Biosciences). Quantification of protein expression was carried out using Odyssey 2.1 (LI-COR Biosciences).
Wound healing assay
Cells were seeded on six-well plates and grown to confluency. Monolayers were scratched, and in the indicated experiments the media were changed to Wnt1 CM or control CM. When using recombinant Wnt3a and Y27632, 90 minutes before the scratch was made the media were changed to DMEM 10% FBS containing 100 μg/ml Wnt3a (R&D Systems) and/or 5 μM Y27632 (Sigma-Aldrich). Pictures of randomly-chosen nine wound edges per condition were taken at time 0 and at the indicated time points using Nikon DIAPHOT (Nikon, Tokyo, Japan). The recovered area was calculated using ImageQuant TL (GE Healthcare). In some experiments, purified sFRP1 was added to the CM [7].
Fluorescence-activated cell sorting analysis for active β1-integrin
MDA-MB-231/sFRP1-P1 and control-P1 cells (0.3 × 106) were incubated for 30 minutes on ice with an antibody recognizing active β1-integrin (final concentration 20 ng/μl) in HEPES/NaCl buffer. This was followed by incubation for 30 minutes on ice with fluorescein isothiocyanate-conjugated (FITC) donkey anti-mouse IgG secondary antibody (Jackson Laboratories) (diluted 1:250) in Flow PBS (1 × PBS, 2% horse serum, 0.1% sodium azide). Fluorescence was measured using a FACSCalibur machine (Becton Dickinson) and the percentage of gated cells stained with active β1-integrin was calculated.
In vivoexperiments with MDA-MB-231 cells
Female Balb/c nude mice 7 to 10 weeks old were obtained from Charles River Laboratories (L'Arbresle, France) and were maintained in accordance with the Swiss guidelines for animal safety. Mammary tumors were established in mice (5 to 8 mice per group) by injecting 0.5 to 1.0 × 106 control or sFRP1-expressing MDA-MB-231 cell lines in 100 to 150 μl PBS into the fourth right-side mammary fat pad. The tumor size was measured two or three times per week using a gage, and the volume was calculated considering the tumor as an oval according to the formula:
Statistical analyses were performed with two-way repeated-measures analysis of variance (repeated measures ANOVA (RM ANOVA)).
To assay tumor cell proliferation, 100 μg/g body weight of BrdU (Cell Proliferation Kit II; Roche) was intraperitoneally injected into tumor-bearing mice that were sacrificed 2 hours later. Tumors were excised and washed with PBS before fixation in 4% paraformaldehyde (PFA) at 4°C for 24 hours, and BrdU detection was performed as previously described [13]. To examine experimental metastasis, 1.0 × 106 MDA-MB-231/sFRP1-P2 cells or control-P2 cells were injected into the tail vein of female Balb/c nude mice (5 to 6 mice per group). Fifty-three days after the injection, the mice were sacrificed, lungs were dissected and the total number of surface lung metastases was determined. For western analyses, excised tumors were snap frozen and pulverized in liquid nitrogen and lysed in SDS buffer (100 mM Tris- HCl pH 7.6, 2% SDS, 10 mM dithiothreitol, 2 mM sodium vanadate, 0.5 mM ethylenediamine tetraacetic acid) by incubation at 95°C for 10 minutes.
Immunohistochemistry and functional vessel analysis on tumor sections
To detect functional vessels in tumors, 100 μl of 2 μg/μl solution of fluorescein-labeled Lycopersicon esculentum lectin (Vector Labs, Burlingame, CA, USA) was injected into tail veins of tumor-bearing mice [14], and the mice were sacrificed 5 minutes later. Tumors were excised, fixed in 4% paraformaldehyde in PBS for 48 hours at 4°C, followed by an overnight incubation in 30% sucrose in PBS at 4°C, and then embedded in tissue-Tec O.C.T. Compound 4583 (Sakura, Tokyo, Japan). Frozen sections (9 μm) were subjected to IHC analysis to detect tumor-associated vessels using rat anti-mouse CD31 (diluted 1:100; BD Pharmingen) and goat anti-rat ALEXA 568 (diluted 1:200; Molecular Probes). Staining was performed using Discovery XT (Ventana Medical Systems, Inc., Tucson, AZ, USA). Pictures were taken with a Z1 microscope (Carl Zeiss, Jena, Germany) and were analyzed with IMARIS (Bitplane, Zurich, Switzerland) to calculate the co-localized area. For detection of cyclin D1, frozen tumor sections (9 μm) were subjected to IHC using SP4 (diluted 1:100) and Biotin-SP-conjugated affinipure donkey anti-rabbit IgG (diluted 1:100). Staining was carried out using Discovery XT with sCC1 pretreatment. Pictures were taken with an Eclipse E600 (Nikon) and were analyzed with IMARIS (Bitplane) to calculate the signal intensity.
RNA isolation, real-time PCR and microarray analyses
Cultured cells were collected when plates were 70 to 80% confluent and total RNA was extracted using the RNeasy Mini kit (Qiagen, Venlo, The Netherlands). To extract total RNA from tumors, dissected tumors were put into RNAlater (Qiagen) overnight at 4°C, followed by RNA extraction using TRIzol reagent (Invitrogen) and washing using the RNAeasy Mini kit according to the manufacturer's instructions. RNA from mammary tumors (six MDA-MB-231/sFRP1-P1 tumors and five control tumors) and cultured cells (three MDA-MB-231/sFRP1 clones and three control clones) were individually amplified and labeled using the Ambion MesageAMP III RNA Amplification Kit (Applied Biosystems, Austin, TX, USA). Biotinylated, fragmented cRNA was hybridized to Affymetrix U133 plus 2.0 human GeneChips™ (Affymetrix, Santa Clara, CA, USA).
Expression values were estimated using the GC-RMA implementation found in Genedata's Refiner 4.5 software (Genedata AG, Basel, Switzerland). Quantile normalization and median scaling were performed in order to standardize array signal distributions to facilitate the comparison between in vitro cultured cells and in vivo tumor samples. Probesets showing statistically different expression profiles (one-way analysis of variance with P < 0.01; Benjamini and Hochberg Q values determined to minimize the false discovery rate) and specific pairwise fold changes were clustered by rank correlation with R > 0.8 for the first criterion and R > 0.885 for the second criterion using the Profile Distance Search function of Genedata's Analyst 4.5 tool (Genedata AG, Basel, Switzerland). All of the microarray data are stored in Gene Expression Omnibus [GEO:GSE13806].
For the quantitative real-time PCR, each sample cDNA was made from 2.5 μg RNA using Ready-To-Go™ You-Prime First-Strand Beads (GE Healthcare). Quantitative real-time PCR was performed with ABI Prism 7000 (Applied Biosystems, Austin, TX, USA) using ABsolute SYBR Green ROX Mix (THERMO Scientific, Waltham, MA, USA) following the manufacturer's guidelines. The primer sequences used for quantitative real-time PCR are as follows: human c-Myc forward, 5'-CCTACCCTCTCAACGACAG-3'; human c-Myc reverse, 5'-CTTGTTCCTCCTCAGAGTCG-3'; human β-actin forward, 5'-TGTCCACCTTCCAGCAGATGT-3'; and human β-actin reverse, 5'-CGCAACTAAGTCATAGTCCGCC-3'.