The cell line SKNEP-1 (obtained from the American Type Culture Collection, Manassas, VA) was maintained in culture in 75-cm2 flasks with McCoy's 5A medium (Mediatech, Fisher Scientific, Springfield, NJ). Medium was supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies, Grand Island, NY). Cells were grown at 37°C in 5% CO2 until confluent, harvested, counted with trypan blue staining, washed, and resuspended in sterile PBS (Life Technologies) at a concentration of 107 per mL.
The Institutional Animal Care and Use Committee of Columbia University approved all experiments. Four- to 6-week-old female NCR nude mice (National Cancer Institute at Frederick, Frederick, MD) were housed in a barrier facility and acclimated to 12-hour light/dark cycles for at least 24 hours before experimental use.
Mice (n = 62) were anesthetized with intraperitoneal ketamine (50 mg/kg) and xylazine (5 mg/kg). The left flank was prepared in a sterile fashion, and an incision was made, exposing the left kidney. An inoculum of 106 SKNEP1 tumor cells in 0.1 ml of PBS was injected into the renal parenchyma using a 25-gauge needle. The flank musculature was closed with a single 4-0 Polysorb suture (U.S. Surgical, Norwalk, CT), and the skin incision was closed with staples.
Administration of SC236 and anti-VEGF antibody
One week after tumor implantation, animals were divided into four cohorts. Treated mice received either (1) SC236 (N = 17; Pfizer, Groton, CT) added to drinking water at a concentration of 30 μg/mL and changed thrice per week as previously described ; (2) the humanized monoclonal anti-VEGF antibody bevacizumab (N = 17; BV; Genentech, South San Francisco, CA) injected intraperitoneally at 10 mg/kg biweekly beginning at week 3; or (3) combined agents (N = 15). Control mice (N = 13) received standard drinking water and were injected with carrier vehicle on the same schedule. This concentration of SC236 is equivalent to ~6 mg/kg/d (anticipated SC236 plasma level 5 μg/ml). Plasma levels of SC-236 were previously confirmed in our model by high-performance liquid chromatography (HPLC) .
Harvesting of specimens and determination of metastases
At sacrifice, tumors and contralateral kidneys were removed, weighed, and then preserved in 4% paraformaldehyde for immunohistochemistry. Portions of tumor were flash frozen in liquid nitrogen and stored at -80°C. Both lungs were fixed in 10% formalin for histology. Slides of paraffin-embedded lung tissue were stained by routine H&E methods and examined by a surgical pathologist to determine the presence or absence of metastases, using three levels from each lung.
Endothelial cell immunofluorescent staining was done using a rat anti-mouse anti-platelet-endothelial cell adhesion molecule-1 (PECAM-1) monoclonal antibody (MAP0032, Angioproteomie, Boston, MA). Vascular mural cells (VMC) were visualized using a rabbit anti-human α-smooth muscle actin (αSMA) antibody (RB-9010, Lab Vision/Neomarkers, Fremont, CA). Macrophages were visualized using an anti-murine F4/80 antibody (Abcam, AB6640) and an anti-arginase antibody (Santa Cruz Biotech, SC-20150). All microscopy was done using a Nikon Eclipse E600 apparatus.
Quantification of proliferation
In vivo tumor proliferation (N = 3 for all groups) was examined by immunofluorescent staining for anti-phospho-histone H3 (Upstate, Inc., Lake Placid, NY), and quantified by computer-assisted image analysis (23-30 fields per group examined).
MMP-2 and -9 zymography
Tumor extracts (N = 3 for each group) were normalized for protein content, mixed with nonreducing sample buffer, and electrophoresed in 10% polyacrylamide gels copolymerized with 1 mg/mL of gelatin. After electrophoresis, gels were washed twice for 15 min in 2.5% Triton X-100, rinsed with water, and incubated overnight in activation buffer (50 mM/L Tris-HCl, pH 7.5, 5 mM/L CaCl2) at 37°C. Digested gelatin bands were visualized by staining with 0.1% Coomassie blue R-250 in 40% methanol/20% acetic acid. Pooled MMPs were used as a positive control (US Biological). Bands were optically scanned for quantification.
2 × 105 SKNEP cells/well were incubated on a 24-well plate (BD Labware, Franklin Lakes, NJ) for 24 hours. Media was then replaced with control media or media with 25 or 50 uM SC-236 (Pfizer, New York, NY) and incubated for 24 or 96 hours at 37°C in either normoxia (21% oxygen) or hypoxia (2.3% oxygen). After the incubation period, media was aspirated and replaced with serum-free media and MTT labeling reagent (Roche, Indianapolis, IN). After 24 hours, 600 uL isopropanol was added to each well and the resultant solution read in a Life Science UV spectrophotometer (Beckman, Fullerton, CA) at wavelengths of 550 and 690 nm. All samples were plated in triplicate and experiments were repeated 3 times. Assays were repeated with added bevacizumab (100 ng/ml).
SKNEP cells were serum-starved for 24 hours. 5 uM Cell Tracker Green (Invitrogen) was added to the cells and incubated for 45 min. Media was then aspirated and cells incubated in serum-free McCoy's solution for an additional 45 min. 8 × 105 cells/well were plated in 24-well, 8 um pore, Matrigel-coated Tumor Invasion System plates (BD Biosciences) with serum-free media containing 0 (control), 25, or 50 uM SC-236. The same media with 15% FBS was added to the lower well, and the plate incubated for 24 hours in either normoxia or hypoxia (as above). Invading cells were quantified using a Cytofluor series 4000 Fluorescence multi-well plate reader (Applied Biosystems, Foster City, CA) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. All samples were plated in triplicate and experiments were repeated 3 times.
105 tumor cells per well were plated on a 96-well Microtest Tissue Culture Plate (BD Labware). Either media or media with 25 or 50 uM SC-236 was added, and allowed to incubate in either normoxia or hypoxia for 20 hours. Apoptosis was analyzed using the Cell Death Detection Elisa kit (Roche, Indianapolis, IN). Briefly, media was aspirated, and cells lysed. After a 30 minute incubation period, the solution was centrifuged at 1250 rpm for 10 minutes and the supernatant added to a streptavidin-coated plate. A mixture of anti-histone-biotin and anti-DNA-peroxidase solution was added to each well and mixed for 2 hours. Substrate was then added, and color intensity read using a VersaMax tunable microplate reader (Molecular Devices, Sunnyvale, CA) at a wavelength of 405 nm. Camptothecin was used as a positive control for apoptosis.
Real-time PCR for lung metastasis signature genes
Total RNA was isolated from xenograft tumors (N = 4 each), using an Ambion ToTally RNA Extraction kit (Ambion, Austin, TX). Real-time PCR was performed using Taqman probes and an ABI 7300 (Applied Biosystems). Normalization of genes of interest was done utilizing the geNorm method described by Vandesompele et al . Six human housekeeping genes (ACTB, HMBS, UBC, GAPD, HPRT, PPIA) were used as reference controls.
Microarrays and gene set expression analysis
HG-U133A GeneChips (Affymetrix, Santa Clara, CA) were used to investigate gene expression in xenografts. cRNA probes were synthesized as recommended by Affymetrix. Briefly, total RNA was isolated in two steps using ToTALLY RNA Total RNA isolation kit (Ambion, Austin, TX) followed by RNeasy (Qiagen, Valencia, CA) purification. Double-stranded cDNA was generated from 5 μg of total RNA using a polydT oligonucleotide that contained a T7 RNA polymerase initiation site and the Superscript Choice System kit (Invitrogen, Carlsbad, CA). Biotinylated cRNA was generated by in vitro transcription using the Bio Array High Yield RNA Transcript Labeling System (Enzo, Farmingdale, NY). cRNA was purified using RNeasy and fragmented according to the Affymetrix protocol, and 15 μg of biotinylated cRNA hybridized to HGU133A microarrays (Affymetrix). Raw CEL files were processed using Bioconductor packages in an R environment http://www.bioconductor.org) . Briefly, quality controls were performed by inspecting Affymetrix® metrics using the simpleaffy package. Probe level signals were then background-corrected, normalized, and summarized using the GC-RMA function. Differential gene expression was computed using an Empirical Bayesian model implemented in the Limma package (Additional File 1 Table S1). To determine whether the addition of SC236 broadly changed inflammation-related pathways in BV-treated tumors, we used Gene Set Expression Analysis and GenePattern tools (GSEA, Broad Institute, Cambridge, MA). The MSigDB gene set database (5,542 total sets) was queried for gene sets containing inflammation-related pathways. From this, we constructed a 67 gene set matrix. GSEA was used to assess expression of gene sets in this matrix in BV- and BV+SC236-treated samples (N = 3, 4 respectively). To compute gene enrichment, we permuted by gene (as recommended for small sample sizes) 5000 times. We then computed odds ratios for the genesets identified as being significant by GSEA, defined as ratio of odds for the hits before and after the leading edge. In particular, for each geneset we computed the following:
oddsRatios =[(Hits/Misses) BeforeTheLeadingEdge]/[(Hits/Misses) AfterTheLeadingEdge]
We used the odds ratios to additionally filter the results, as nominal p-value is not informative in this setting, and the NES has a bias towards bigger genesets.
Enzyme-linked immunosorbent assay (ELISA)
ELISA for tumor-derived (human) IL4 was performed using the Human IL-4 Quantikine HS ELISA Kit HS400 (R&D Systems). All assays were performed in duplicate, and then repeated twice. Results of repeated assays were expressed as summary means ± standard error (square root of interassay variance) .
Tumor weights were compared by Kruskal-Wallis analysis. The presence or absence of lung metastases was evaluated by Fisher's exact test. Quantitative results of multiply-repeated MTT, invasion, and ELISA assays were assessed using Comprehensive Meta Analysis software (Biostat, Inc., Englewood, NJ). Differences in gene expression by real-time PCR were compared by Student's T-test.
Quantification of changes in vasculature
Digital images from immunofluorescence and fluorescein-labeled lectin studies were captured from a Nikon E600 fluorescence microscope with a Spot RT slider digital camera (Diagnostic Instruments, Sterling Heights, MI) and stored as TIFF files. Vessel radius was determined by immunofluorescence for αSMA and analyzed using software Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA) and Image Processing Toolkit (IPTK 5 IPTK 5, Reindeer Graphics, Raleigh, NC). Briefly, background fluorescence is subtracted (AutoLevelDark), Gaussian filter applied to reduce electronic and background noise (Gaussian Blur, radius = 1.0 pixel), and grayscale levels linearly expanded (Auto Levels). A common threshold level is applied that preserves correct vascular morphology (Threshold). The image is inverted (Invert) and dilated to lessen gaps (Classic Morphology > Dilate), small particles representing background removed (Reject Features), and vessel holes filled in (Fill Holes). The average of the inscribed radius is determined as a measure of vessel radius. 60 images from SK-NEP1-GFP (3 different tumors) and 49 images from SK-NEP1-Ang1* (3 different tumors) were analyzed. To assess MVD and vessel length, quantitative assessment of tumor vessel architecture was performed by computer-assisted digital image analysis as previously described . The fraction of fluorescein-labeled lectin-positive pixels per total field was quantified. Specific changes in vessel architecture are evaluated by quantifying branch points (nodes), end points, and total vessel length. Images are analyzed after application of a common threshold, image inversion, morphological erosion, and skeletonization, again using a combination of Photoshop and Image Processing Toolkit.