The murine hemangioendothelioma cell line (EOMA) CRL-2586™ was obtained from the American Type Culture Collection (ATCC, Manassas, VA). EOMA cells were maintained in Dubelco's Modified Eagle's Medium (DMEM) (Gibco, Carlsbad, CA), supplemented with 10% Fetal Bovine Serum (FBS) and 0.05 mg/ml gentamicin sulfate (Gibco).
Retroviral Vectors and Transduction
The pBABE-HA-HoxA5 retroviral vector was constructed and transduced as described in Rhoads et al (12
). EOMA cells were transduced with control plasmid (pBabe) and pBabe HA-HoxA5, using 2 ml of virus-containing media in the presence of 8 μg/ml of polybrene (Sigma-Aldrich, St. Louis, MO) for 16 hours at 37°C. Twenty-four hours later, cells were selected in 1 μg/ml puromycin (Sigma-Aldrich) for 7 days, and the pooled cell population was used for subsequent experiments. Cells were used at passages 4 through 6.
RNA Isolation and Quantitative Real-time Polymerase Chain Reaction (PCR)
One μg of total RNA from transfected cells, extracted using the RNeasy isolation kit (Qiagen, Valencia, CA), was reverse-transcribed using MMuLVRT (Invitrogen, Carlsbad, CA) in a total volume of 25 μl. Quantitative real-time PCR was carried out in triplicate with a 10−20 fold dilution of first-strand cDNA using human HoxA5 and murine TSP-2 taqman probes, and primers purchased as Assays on Demand (Applied Biosystems, Foster City, CA). As a reference, we used β-glucuronidase or GUS. An ABI PRISM SDS 7000 (Applied Biosystems) was used according to the manufacturer's instructions with the following cycling protocol: one cycle at 50°C, 2 minutes; one cycle at 95°C, 10 minutes; 40 cycles at 95°C, 15 seconds; 50°C, 1 minute. Data were analyzed with an ABI Prism SDS 7000 companion software.
EOMA Cell Transplantation in the CD-1 Mouse Brain
EOMA cells were grown to 80% confluence in DMEM-10% FBS. Afterwards, they were trypsinized in 0.25% trypsin for 5 minutes at 37°C. Cells were then diluted with culture medium and centrifuged at 1200 rpm for 5 minutes at 4°C. The pellet was washed once with PBS and spun down again at 1200 rpm for 5 minutes at 4°C. Finally, EOMA and EOMA-HoxA5 cells were diluted with PBS to reach appropriate concentration for injection.
Eight-week old CD-1 male mice were anesthetized with 50mg/kg body weight ketamine and 10mg/kg body weight xylazine (Sigma Aldrich) intraperitoneally. Following induction of anesthesia, mice were placed in a stereotactic frame with a mouse hold (David Kopf Instruments, Tujunga, CA), and a burr hole was drilled to the pericranium 3 mm lateral to the sagittal suture and 1 mm posterior to the coronal suture. A 10 μl syringe (Hamilton Company, Reno, NV) was inserted into the burr hole 3 mm under the cortex. 5×105 EOMA or EOMA-HoxA5 cells in a volume of 3 μl were injected stereotactically into the lateral caudoputamen at a rate of 0.2 μl/minutes. The needle was then withdrawn over the course of 5 min, the hole sealed with bone wax, and the wounds closed with suture. Animals were put back into their cages for recovery. After 1, 2 and 3 weeks, EOMA-control (n=6 for each time point) and EOMA-HoxA5 (n=6 for each time point) injected mice were sacrificed and brains were harvested for further analysis.
Hematoxylin and Eosin (H&E) and Immunofluorescence Staining
The mice were euthanized 1, 2, and 3 weeks after surgery respectively. They were perfused-fixated with 4% paraformaldehyde/phosphate-buffered saline solution. The brains were removed, dehydrated with graded alcohols, fixed in xylene, and finally embedded in paraffin blocks. The brain coronal sections were made at the thickness of 5μm for use. For H&E staining, sections were deparaffinized first in 3 changes 100% xylene for 5 minutes each, then hydrated through graded alcohol (100%, 95% and 70%, respectively; 5 min each), and rinsed in phosphate-buffered saline ready to use. We then followed standard H&E staining protocol.
Brain paraffin or frozen sections were incubated in 5% normal goat serum for 30 minutes before they were incubated in primary antibody diluted in 5% normal goat serum overnight at 4°C. Primary antibodies include rat anti-mouse CD31 (1:100 dilution, BD Pharmigen, San Jose, CA) and Licopersicum sculentum lectin (1:200, Vector Lab, Burlingame, CA) for endothelial cells, rabbit anti-mouse alpha-actin for smooth muscle cells (1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), anti-HIF-1α (1:100 dilution, Novus Biologicals) . The sections were incubated for one hour with either horseradish peroxidase conjugated anti-rat or -rabbit IgG (1:1000, Alexa 594 for red and Alexa 488 for green) (Invitrogen, Carlsbad, CA). Finally, slides were mounted and evaluated using a fluorescence microscope (Nikon Microphoto-SA, Melville, NY) with a filter cube (excitation filter, 450−490nm) for fluorescent isothiocyanate labeling and a filter cube (excitation filter, 515−560nm) for Texas-red. Photomicrographs for double-labeling illustrations were obtained by changing the filter cube without altering the position of the section or focus.
For quantification of the Hif1α positive cells, we chose three different sections for each mouse brain sample; the level of needle track, 0.5 mm before and 0.5 mm after the needle track, and the stained sections were taken from the corresponding levels from each brain. Three 40X images were randomly taken in the tumor area of each section. Samples were taken from at least 3 mice per group at each time point. For Hif1α positive cell quantification, the number of positive cells (red color) visible in each 40X field from each of 3 areas (a total of 3 mice per group at each time point) was counted blindly. Data are reported as the mean.
Tumor Volume Measurement
Two groups of mice (EOMA and HOXA5-treated EOMA) were sacrificed by decapitation 1 to 3 weeks following transplantation. The brains were removed and frozen immediately on dry ice for 5 minutes. Cryostat sections (20 μm thickness) distal from the frontal pole were cut and mounted on slides. The sections were dried and then stained following standard H&E staining protocol. With the use of NIH Image 1.63 software, a line was drawn around the imaged tumor on each section in order to measure the area. Total tumor volume was calculated by multiplying the tumor areas by the thickness of the sections.
Western Blot Analysis
Brain frozen tissue was resuspended in RIPA buffer, then sonicated and centrifuged at 1,200 rpm for 10 min at 4°C. Protein concentration was determined using the BCA protein assay reagent (Thermo Scientific, Rockford, IL). 40 μgs of protein from EOMA and EOMA HoxA5 tissues were separated in a 7% SDS-PAGE gel and transferred to PVDF membranes. After blocking for non-specific binding, the membrane was probed with a mouse anti-TSP-2 antibody (1:500) (BD Biosciences, San Jose, CA), followed by goat anti-mouse-HRP at 1:10000. Excess antibody was removed by extensive washing, and blots were developed by using ECL system (Amersham Biosciences, Piscataway, NJ). The membrane was then stripped and treated with a polyclonal β-actin antibody (1:1000 dilution, Abcam Inc., Cambridge, MA), followed by donkey anti-rabbit–HRP at 1:8000 dilution and detected by ECL system.
All the experiments were performed at least 3 times. For Real-time PCR and Western blot analysis, we used a t-test for comparison. For tumor volume and average number of HIF-1 positive cells per field, we used a log transform to normalize the distribution. Data were analyzed using ANOVA (Statview 5.0) to examine tumor volume and average number of HIF-1 positive cells. There were two grouping variables: timing (one, two or three weeks after injection), and group (control or treated). Post hoc testing was performed using Fisher's PLSD. The data are presented as mean±standard deviation (SD); untransformed raw data are shown in the figures. A P value less than 0.05 was considered significant for the comparisons.