Antibodies and reagents
Selected antibodies were acquired as indicated: Rat IgG Isotype control (553993, BD Pharmingen), Rat IgG Fluorescent Isotype controls (sc-2831, sc-3788, sc-2895, sc 2872; Santa Cruz Biotechnology), Rat Anti-Mouse Flk-1-PE (555308, BD Pharmingen), Rat Anti-Mouse c-Kit-PE-Cy7 (558163, BD Pharmingen), Rat Anti-Mouse CD45-APC-Cy7 (557659, BD Pharmingen), Rat Anti-Mouse CD31-FITC (553372, BD Pharmingen), Mouse Anti-Human VEGFR2-PE (560872, BD Pharmingen), Mouse Anti-Human CD117-APC (561118, BD Pharmingen), Mouse Anti-Human CD45-APC-Cy7 (557833, BD Pharmingen), Mouse Anti-Human CD31-FITC (560984, BD Pharmingen), Rat Anti-Mouse CD31 (550274, BD Pharmingen), Goat Anti-Rat FITC (sc-2011, Santa Cruz Biotechnology), Goat Anti-Rat AlexaFluor594 (A-11007, Life Technologies), Anti-Rabbit Oct-4 (AB3209, EMD Millipore), and Goat Anti-Rabbit FITC (sc-2012, Santa Cruz Biotechnology). Additional reagents used: Hoechst’s 33258 Stain (94403, Sigma-Aldrich), acLDL-Dil (L3484, Life Technologies), acLDL-BODIPY (L3485, Life Technologies), acLDL (L35354, Life Technologies), Fibronectin (F1141, Sigma-Aldrich), and Alkaline Phosphatase Stain (A14353, Life Technologies).
HUVECs were grown in human endothelial growth media with supplementation (PM211500, Genlantis, San Diego, CA). The tumor cell lines: A549 lung carcinoma, U251 glioblastoma, and UMSCC-1 head and neck squamous cell carcinoma were grown in DMEM supplemented with 10% inactivated fetal bovine serum (Life Technology) and 1% penicillin-streptomycin. All cell lines were acquired from ATCC (Manassas, VA).
Female athymic nu/nu nude mice (aged 8–12 weeks) were acquired from NCI-Fredrick (Fredrick, MD). Mice were maintained in a germ-free environment and had access to food and water ad libitum. All animal procedures were approved by Stanford University’s Administrative Panel on Laboratory Animal Care (APLAC).
Tumors were implanted subcutaneously on the backs of nude mice approximately 2 cm above the base of the tail. A549, U251, and UMSCC-1 cell lines were implanted at a concentration of 3×106 cells in 100 μL PBS. Tumors reached a size of approximately 200 mm3 in 3–4 weeks with an efficiency of approximately 90%, 70%, and 90%, respectively.
EPC isolation was performed as reported previously with documented modifications [1, 55, 56]. Selected mouse tissues or tumor samples were surgically removed from euthanized nude mice. Tissues were kept in mouse endothelial media (M1168, CellBiologics, Chicago, IL) supplemented with the growth factor kit (VEGF, EGF, heparin, hydrocortisone, and L-Glumatine) and 10% fetal bovis serum (FBS) and then minced into small chunks with a #11 scalpel blade. Tissue fragments were then placed into a glass homogenizer and mechanically disrupted into cellular slurry. The slurry was transferred to 15 mL tubes and mixed with 1 mL of supplemented mouse endothelial media and combined with 2 mL of 0.1% Collagenase I (CLS-1, Worthington Biochem, Lakewood, NJ), 0.1% Collagenase IV (CLS-4, Worthington Biochem, Lakewood, NJ), and 0.001% Deoxyribonuclease I (DPRF, Worthington Biochem, Lakewood, NJ). Samples were incubated for 30 minutes at 37°C. Mouse endothelial medium was added to a total volume of 15 mL and solutions were poured through a 70 μM cell strainer into a new 15 mL tube. Tube were then spun at 4°C at 3000 rpm for 5 minutes. The supernatant was decanted and the resulting cell pellet was resuspended in 2 mL of RBC lysis buffer (1.5 M NH4CL, 100 mM NaHCO3, 10 mM Na2-EDTA; pH 7.4) and placed on ice. After 15 minutes, samples were spun down at 4°C at 3000 rpm for 5 minutes, supernatant removed, and resuspended in PBS and kept on ice. Live/Dead stain (L23105, Invitrogen) was added to the cells for 30 minutes with cells kept on ice and protected from light. Samples were then moved to eppendorf tubes and spun for 5 minutes, 4°C at 5000 rpm. Supernatant was removed and the cell pellet was washed twice in PBS, and resuspended in PBS and used for flow cytometry.
Flow cytometry was performed in the Stanford Flow Cytometry Core Facility using the BD Aria II (sorting) or BD LSR.II (analysis). Samples were isolated from mouse tissues or tumors as above or from trypsinized from tissue culture samples. Cells were blocked with CD16/CD32 Mouse Fc Block (553142, BD Pharmingen) for 30 minutes, then labeled with anti-Flk-1:PE, anti-c-Kit:PE-Cy7, anti-CD45: APC-Cy7, and as indicated, anti-CD31: FITC. Unstained samples and samples with antibody IgG controls were run in parallel. Additionally, color compensation for each fluorochrome was evaluated using IgG compensation beads (552843, BD Pharmingen) and ArC compensation beads (A10346, Invitrogen) with appropriate fluorescent marker controls. Once lasers were tuned and color compensation was set, at least 100,000 cells per sample were run, except for the adipose tissue samples which were run at 50,000 cells per sample. Gating was initially applied to isolate single cell populations of viable cells, then additional gating was used to select for the appropriate subpopulations (Flk-1+/c-Kit+/CD45-). Flow cytometric analysis was performed using FlowJo Software (TreeStar, Inc., Ashland, OR). For the flow cytometry sorting of live cells, the same staining process was used as above. Once subpopulations of single, viable cells were gated on Flk1+, c-Kit+, and CD45- and subsequently isolated, they were then collected in an eppendorf tube with 500 μL of mouse endothelial media (M1168, Cell Biologics, Chicago, IL) supplemented with the growth factor kit (VEGF, EGF, heparin, hydrocortisone, and L-Glumatine) and 10% FBS and managed as in vitro cultures as described below.
In vitro culturing of sorted EPCs
Sorted cell from nude mouse lung samples were placed in 5 μg/cm2 fibronectin treated T25 flasks in mouse endothelial medium (M1168, Cell Biologics, Chicago, IL) supplemented with the growth factor kit (VEGF, EGF, heparin, hydrocortisone, L-Glumatine, and 10% FBS) and incubated at physiologic concentrations of FiO2 (5%) at 37°C. Flasks were examined every 2–3 days and once attached cells began to generate colonies of 10–20 cells, flasks were moved to normoxic incubators (FiO2 21% at 37°C). As flasks became confluent, cells were passaged as usual. The EPC cell line and EPC_acLDL cell lines were used from passages 3–6 and the EPC_Late cell lines was used between passages 16–24.
Tumor and EPC cell lines were grown and treated in chamber slides (C1782, Sigma-Aldrich). The reagent acLDL-Dil or acLDL-BODIPY was mixed with cells at 2.5 μg/mL concentration and allowed to incubate for 4 hours at 37°C. Medium was then aspirated and cells were fixed in 4% paraformaldehyde for 10 min at room temperature. Paraformaldehyde was aspirated and the cells treated with a 0.2% NP40/PBS solution for 15 min. HUVEC cells and the human tumor cell lines used acLDL-BODIPY and the Anti-CD31-APC antibody while the murine derived EPC-related cell lines used acLDL-Dil and the Anti-CD31-FITC antibody for staining. Cells were then washed in PBS twice, and the selected anti-CD31 antibody at a dilution of 1:50 in 1% BSA was added and incubated overnight at 4°C. Cells were again washed twice in PBS before incubating in the dark with the appropriately labeled secondary antibody at a dilution of 1:100 in 1% BSA for 1 h. The secondary antibody solution was then aspirated and the cells washed twice in PBS. Cells were then incubated in the dark with Hoechst’s 33258 (1 μg/ml) in PBS for 30 min, washed twice, and coverslips mounted with an anti-fade solution (Dako Corp., Carpinteria, CA) and sealed with clear nail polish. Slides were examined on a Lecica DM6000B fluorescent microscope (Lecica, Buffalo Grove, IL). Images were captured by a CCD camera using the Image Pro Premier v9.0 (MediaCybernetics, Rockville, MD) software package.
Tissues were harvest from mice and placed in cassettes and covered in OCT media. Cassettes were held at -80°C until they were process on a cryotome and cut to 2 μm tissue sections. Slides were then kept at -80°C until processing. Slides were allowed to air dry for 10 min at room temperature and then were kept in -20°C methanol for 10 minutes. Methanol was removed and samples were allowed to air dry for 30 minutes and washed with PBS twice for 5 minutes each. The rat anti-CD31 antibody was used at a dilution of 1:20 in 2% BSA and incubated overnight at 4°C. Slides were again washed twice in PBS before incubating in the dark with a goat anti-rat AlexaFluor594-labeled secondary antibody at a dilution of 1:100 in 1% BSA for 1 h. The secondary antibody solution was then aspirated and the cells washed twice in PBS. Cells were then incubated in the dark with Hoechst’s (1 μg/ml) in PBS for 30 min, washed twice, and coverslips mounted with an anti-fade solution (Dako Corp., Carpinteria, CA) and slides were sealed with clear nail polish. Slides were examined and images captured as above.
Colony forming assay
Selected cell lines were cultured in methylcellulose-containing medium (M3434, StemCell Technologies, Vancouver, BC, Canada) with 50 ng/mL vascular endothelial (VE) growth factor (R&D Systems, Minneapolis, MN, USA), 50 ng/mL basic fibroblast growth factor (Wako, Osaka, Japan) and 10% fetal bovine serum (FBS) on 35-mm dishes. Cell densities were 1×103 for each cell lines. Dishes were plated in triplicate and kept in humidified incubator for 8–10 days until colonies started to develop. EPC colony forming units (CFUs) were defined as cluster-like collections of cells associated with attached spindle-shaped cells. The EPC-CFUs were identified by visual inspection with an inverted microscope under 20-40× magnification. Images were acquired on a Zeiss Observer. A1 (Zeiss Microscopy, Thornwood, NY) microscope using AxioVision 4.6.3 software (Zeiss Microscopy, Thornwood, NY).
As a modified protocol of Wu et al., matrigel (356237, BD Pharmingen) was applied in thin layers to wells of 24-well plates . Cells of selected cell lines were placed at indicated cellular concentrations: 1×104, 1×105, and 1×106 cells. Cells were incubated overnight and the next morning (18 hrs) evaluated under light microscopy at 20-40X magnification. Images were acquired on a Zeiss Observer. A1 (Zeiss Microscopy, Thornwood, NY) microscope using AxioVision 4.6.3 software (Zeiss Microscopy, Thornwood, NY).
Bone marrow transplant studies
Nude mice were exposed to two doses of 4.5 Gy given 4 hours apart and, as indicated, supplemental cells were given 2 hours after the final radiation dose . Normal bone marrow was collected from unirradiated 12–16 week old mice by isolating the femurs and tibia, cutting the end from the bone, and then flushing out the central cavity using 28 gauge needles with 2% FBS in Hank’s balanced salt solution. The cell solution was collected, spun down, and cell concentration calculated. Harvested bone marrow or the selected ex vivo cultured EPC cell lines were injected into the retro-orbital venous plexus of an anesthetized mouse at 3×106 cells in 100 uL PBS. Survival was assessed every two days and mice were sacrificed if weight had dropped more than 10% per animal protocol guidelines.
Gene array analysis
Sorted cell lines: EPC, EPC_acLDL, and EPC_late were grown out, collected, and RNA isolated (RNeasy mini kit, Qiagen). RNA concentration and quality was measured by NanoDrop analysis. RNA was stored in RNAase free water at -80°C until analysis. RNA was hybridized to MouseRef-8 v2.0 Beadchips (25 K, Illumina, San Diego, CA). Two separate experiments were performed independently and run in duplicate at the Stanford Gene Array Core Facility. Bead level intensity values were summarized without normalization and local background correction was applied by default using Beadstudio v3.1. Microarray gene expression data were processed and analyzed using Genespring VX (Agilent, Santa Clara, CA). Clustering was performed using non-centered Pearson correlation. Data was deposited at: http://www.ncbi.nlm.gov/projects/geo (GSE53681).
Tumor growth curves
U251 cells at a concentration of 3 × 106 were mixed with 3×105 EPC_acLDL cells (10% ratio). For co-injection studies, the cell mixture was implanted on the back of nude mice and tumor measurements were taken approximately three times a week. Tumor volume (mm3) was calculated as (height2*length)/2. For the systemic injection studies, Tumors (U251; 3×106) were implanted on the back of mice on day 0 and EPC_acLDL cells (3 × 105) were given by 100 μL retro-orbital injection on day 7 with controls receiving an injection of normal saline in an identical volume. Tumor measurements were then taken approximately three times weekly and tumor volumes calculated as above. Numeric data are presented as the mean +/- standard error.
Mean vessel density analysis
Images from the fluorescent immunohistochemical analysis were imported into ImageJ analysis software (http://www.rsbweb.nih.gov/ij/, NIH, Bethesda, MD). A threshold of CD31 pixel intensity was applied to all images and the total number of CD31 positive pixels was counted within the field of view. Total CD31 pixel counts from five random images from each tumor sample were analyzed and averages were calculated over a 0.1 mm2 area [59, 60]. Two tumors from two different experiments were used for analysis. Numeric data are presented as the mean +/- standard deviation.
Statistical comparisons of datasets were performed by a two-tailed Student’s T-test using Microsoft Excel (Redmond, Washington). Data was considered to be significantly different when P ≤ 0.05.