phung_tl20060814

							Cancer Cell, Volume 10

Supplemental Data

Pathological angiogenesis is induced by sustained Akt signaling and
inhibited by rapamycin
Thuy L. Phung, Keren Ziv, Donnette Dabydeen, Godfred Eyiah-Mensah, Marcela Riveros, Carole Perruzzi,
Jingfang Sun, Rita A. Monahan-Earley, Ichiro Shiojima, Janice A. Nagy, Michelle I. Lin, Kenneth Walsh,
Ann M. Dvorak, David M. Briscoe, Michal Neeman, William C. Sessa, Harold F. Dvorak,
and Laura E. Benjamin




Supplemental experimental procedures
Isolation of primary mouse endothelial cells
Four hearts were removed and collected in a tube containing cold HBSS with antibiotics. They were placed in a petri
dish, trimmed of extraneous tissues and washed once with HBSS. Hearts were finely minced with a scalpel and
mixed with 2% Worthington type 1 collagenase in Dulbecco’s PBS supplemented with Ca+2/Mg+2 that had been
prewarmed to 37°C for one hour. Hearts were digested for 1-1.5 hours at 37°C with agitation. Digested tissues were
gently pipeted up and down a few times to break up clumps and then filtered through a 100-micron cell strainer (BD
Discovery Labware). Digested filtrate was centrifuged and the pellet was washed twice in 0.1% BSA in PBS. The
filtrate was then incubated for 20 minutes at 4°C with 50 ul of magnetic beads (Dynal Biotech) that had been
conjugated with anti-mouse CD31 antibody (BD Biosciences). Cells with beads attached were collected using MPC
magnet (Dynal Biotech) and washed vigorously 6-8 times in 0.1% BSA/PBS. Washed cells were collected and
plated in 100-mm tissue culture plates that had been pre-coated with Type I collagen (Cohesion) and grown in
EBM-2 basal media (Cambrex) supplemented with 25 mg Endothelial Cell Mitogen (Biomedical Technologies,
Inc.), 10% fetal bovine serum and penicillin/streptomycin. After 2 days in culture, non-attached cells and excess
beads were removed and cells were fed. Cell growth was monitored and purity assessed by 1,1-dioctadecyl-3,3,3,3-
tetramethylindocarbocyanine perchlorate-acetylated low-density lipoprotein (DiI-acetylated LDL) (Biomedical
Technologies, Inc) and CD31 staining.

Histology and immunohistochemistry
LacZ staining of skin was performed following fixation in 4% paraformaldehyde, pH 7.4 for 2-4 hr at 4°C. Tissues
were then washed twice in 2 mM MgCl2 and 5 mM EGTA in PBS, pH 7.4, followed by two additional washes in 2
mM MgCl2, 0.01% Na deoxycholate and 0.02% NP-40 in PBS, pH 7.4. Tissues were transferred to freshly prepared
stain solution containing 1 mg/ml X-gal substrate (Specialty Media, Phillipsburg, NJ) dissolved in 2 mM MgCl2,
0.01% Na deoxycholate, 0.02% NP-40, 5 mM potassium ferricyanide, and 5 mM potassium ferrocyanide in PBS,
pH 7.4, and incubated in the dark at 37°C overnight.
Immuhistochemistry was performed on freshly harvested tissue, following immediate fixation in ice cold 4%
paraformaldehyde/PBS, pH 7.4 for 2-4 hours at room temperature, followed by dehydration in 30% sucrose in PBS
overnight at 4°C prior to embedding in OCT and stored at –80°C for subsequent use. 5-micron thick frozen sections
were cut in a standard cryostat and mounted on glass slides, washed in PBS, blocked in 5% goat serum, 0.1% triton
X-100 in PBS for 1 hour at room temperature. Tissues were then incubated with primary antibodies to
phosphorylated Akt (1:100 dilution, Cell Signaling Technologies), HA tag (1:100 dilution, Covance), Cy3-smooth
muscle actin (1:200 dilution, Sigma) and Bandeiraea simplicifolia (BS-1) lectin (1:200 dilution, Sigma) diluted in
5% goat serum in PBS, and incubated for 1-2 hours at room temperature, followed by 3 washes in PBS, 5 minutes
per wash. Secondary antibodies diluted to 1:200 in 5% goat serum in PBS were added to tissues and incubated at
room temperature for 1 hour, followed by 3 washes in PBS. Tissues were mounted onto coverslips with anti-fade
ImmunoGlo mounting medium (DakoCytomation). Images were captured using a Nikon TE200 inverted microscope
equipped with DIC/phase/fluorescence optics, connected to a Leica DC200 digital camera and analyzed using
DCViewer software.

MRI analysis
Double transgenic mice and control littermates were taken off tetracycline for 7 days to induce myrAkt1 expression
prior to MRI scan. The macromolecular contrast material biotin-BSA-Gadolinium-DTPA (biotin-BSA-Gd-DTPA)
was prepared as previously reported (Dafni et al., 2002a). Dynamic contrast enhanced MRI using biotin-BSA-Gd-
DTPA was done as reported (Ziv et al., 2004; Dafni et al., 2002b) in a 4.7 Tesla Biospec spectrometer (Brucker,
Karlsruhe) using a whole-body birdcage RF coil. A series of whole body precontrast 3D-gradient echo images with
pulse flip angles of 5, 15, 30, 50 and 70 degrees were acquired to determine the precontrast R1 (repetition time (TR)
10 ms, echo time (TE) 3.561 ms, 2 averages, spectral width 50000 Hz, field of view (FOV) 12x12x6cm, matrix
128x128x64 zero filled to 128x128x128 in plane resolution 937 µm, acquisition time 163 seconds per time point).
After acquisition of the precontrast 3D-gradient echo images, biotin-BSA-Gd-DTPA was administered as a bolus
through a tail vein catheter (10 mg/mouse in 0.2 ml). Consecutive postcontrast T1 weighted 3D gradient echo images
were acquired from 2 min to 30 min after administration of the contrast agent, (TR 10 ms, TE 3.561 ms, pulse flip
angle 15 degrees, 2 averages, spectral width 50,000 Hz, FOV 12x12x6 cm, matrix 128x128x64 zero filled to
128x128x128 in plane resolution 937 µm, acquisition time 163 sec per time point). Each study group (single
transgenic and double transgenic myrAkt1 expressing mice) had 6 animals.
MRI data was analyzed using Matlab (The MathWorks Inc.) as previously reported (Ziv et al., 2004). Briefly,
regions of interest (ROIs) analysis of brain, liver, kidney and hind limbs were used to generate concentration maps
of biotin-BSA-GdDTPA from 3D gradient echo data sets. Precontrast longitudinal relaxation rate (R1pre) was derived
by varying the pulse flip angle as reported (Dafni et al., 2002b). Postcontrast R1 values (R1post) were calculated from
pre- and post-contrast 3D-GE - signal intensities, and used for derivation of concentration maps based on the
relaxivity, R, of biotin-BSA-GdDTPA (178.9 mM-1s-1). Permeability surface area product (PS) was calculated as the
rate of contrast accumulation during the first 5 scans after contrast agent administration. PS was calculated for
selected regions of interest (ROIs) by linear regression using the equation PS= (Ct-C0)/Cblood*t, where Ct is the
averaged contrast concentration at the selected ROI as a function of time; t, C0 is the concentration extrapolated to
time zero; and Cblood is the blood volume concentration calculated from the vena cava. Blood volume fraction
(fBV) was calculated as the ratio of the extrapolated concentration of contrast agent in the tissue at time zero (C0)
and the concentration of contrast agent in the blood (measured from the vena cava at time zero, Cblood). Average
changes in PS and fBV for control and experiment groups were calculated from mean PS and mean fBV values of
each organ per mouse. The total blood volume was calculated from the dilution of contrast material in the circulation
as determined in the vena cava. At the end of the MRI follow-up, mice were sacrificed by anesthesia overdose, and
organs (brain, liver, kidneys, spleen, hind limb muscles, heart, lungs, skin, abdominal wall muscle and fat) were
excised for histological analysis.

Methods for analysis of vascular function and tumor growth
Lymphatic perfusion in mouse ear skin: Details of this method have been previously described (Nagy et al., 2002).
Briefly, mice were injected with colloidal carbon (India ink) through a 10-micron borosilicate glass micropipette
attached to a 500-microlitter Hamilton syringe. Images were captured with a SPOT Insight Digital Camera.
FITC-Dextran extravasation: Vascular leakage was visualized using FITC-conjugated dextran (M.Wt. 70 kDa,
Sigma) (Nagy et al., 2002; Pettersson et al., 2000) injected intravenously via the tail vein at a dose of 2 mg/20 gm
body weight. Forty-micron optical sections of fixed tissues were evaluated in a Bio-Rad MRC-1024 confocal
microscope equipped with an argon/krypton laser.
Miles assay for vascular permeability: Briefly, Nu/Nu mice were injected i.p. with AP-Cav or AP peptide for 45
minutes prior to performing Miles assay. For rapamycin studies, the mice were injected intraperitoneally with
rapamycin or control solvent for 24 hours prior to performing the assay. To measure acute permeability, Evans blue
(Boehringer-Mannheim) was injected intravenously via the tail vein at 50 mg/kg body weight and recombinant
human VEGF-A (National Cancer Institute) (50 ng in 50 ul volume) or sterile saline (50 ul) was injected
intradermally into the mouse ears and back skin. Thirty minutes later, the mice were sacrificed and the ears and back
skin removed, photographed and weighed. To measure chronic permeability, organs were harvested 8h after Evans
blue injection. For quantitative measurement of extravasated Evans blue, tissues were placed in 1 ml formamide and
incubated at 56°C for 48h. The amounts of extracted Evans blue was determined by measuring the absorbance at
620 nm and calculated against a standard curve of known Evans blue concentrations. Data is presented as
micrograms of dye normalized to grams of tissue weight.
Tumor growth and vascular permeability: C6 rat glioma tumor cells (0.5 x106 cells) that overexpress mouse VEGF-
A (Benjamin and Keshet, 1997) were inoculated subcutaneously on the back of Nu/Nu mice. The tumor was allowed
to grow to ~0.1 cm3 in size and then animals were injected i.p. with either rapamycin or vehicle solvent control.
Tumor size was measured every other day during the drug treatment. Tumor volume was calculated as a spheroid
using the formula V = 0.52 x (width)2 x length. Tumor vascular permeability was performed as described above and
the amount of extracted Evans blue was normalized to grams of tumor weight (Gratton et al., 2003).

Supplemental references
Benjamin, L.E. and E. Keshet, Conditional switching of vascular endothelial growth factor (VEGF) expression in
tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF
withdrawal. Proc Natl Acad Sci U S A, 1997. 94(16): p. 8761-6.
Dafni, H., et al., Overexpression of vascular endothelial growth factor 165 drives peritumor interstitial convection
and induces lymphatic drain: magnetic resonance imaging, confocal microscopy, and histological tracking of triple-
labeled albumin. Cancer Res, 2002a. 62(22): p. 6731-9.
Dafni, H., et al., MRI and fluorescence microscopy of the acute vascular response to VEGF165: vasodilation, hyper-
permeability and lymphatic uptake, followed by rapid inactivation of the growth factor. NMR Biomed, 2002b.
15(2): p. 120-31.
Gratton, J.P., et al., Selective inhibition of tumor microvascular permeability by cavtratin blocks tumor progression
in mice. Cancer Cell, 2003. 4(1): p. 31-9.
Nagy, J.A., et al., Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as
well as angiogenesis. J Exp Med, 2002. 196(11): p. 1497-506.
Pettersson, A., et al., Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular
permeability factor/vascular endothelial growth factor. Lab Invest, 2000. 80(1): p. 99-115.
Ziv, K., et al., Longitudinal MRI tracking of the angiogenic response to hind limb ischemic injury in the mouse.
Magn Reson Med, 2004. 51(2): p. 304-11.
Figure S1.
Low magnification staining of C6 tumor and adjacent skin with anti-pAkt antibody (red), anti-CD31 antibody
(green) and Hoescht dye to label cell nuclei (blue) shows the difference in pAkt staining in tumor compared to
‘normal’ stroma of the skin. The bright pAkt staining in skin is highlighting hair follicles and epidermis which often
auto-fluoresce, but not the regions of skin containing blood vessels, fibroblasts and other normal cells of that tissue.
Figure S2. Activation of Akt signaling pathways in double transgenic endothelial cells
A-C: Endothelial cells (A, bright field view) isolated from double transgenic mouse hearts were labeled with
endothelial cell markers DiI-acetylated LDL (B, yellow fluorescence) and CD31 (C, green fluorescence). The cell
nuclei were stained with Hoescht dye (blue).
D-E: myrAkt1 endothelial cells were treated ± tetracycline (2 ug/ml) for 48 hours and analyzed by western blot for
phosphorylated and total Akt, Erk-1/2, mTOR, GSK-3β, and eNOS.
Figure S3. Effects of eNOS inhibition on pathological vessel formation in myrAkt1 mice
A-B: Nu/Nu mice were pretreated with either control peptide AP (1.2 mg/kg) or eNOS inhibitor peptide AP-Cav
(2.5 mg/kg) for 45 minutes, followed by intravenous injection of Evans blue dye in the tail vein. Saline or VEGF-A
was immediately injected intradermally in the ears. Photographs of extravasated Evans blue in the ears of treated
animals are shown in (A). (B) Evans blue dye was extracted from the ears and the amount of extracted Evans blue
was measured. Data represented 4 mice per treatment group, and was calculated as micrograms of dye per gram
tissue weight (mean +/- SEM). p-value < 0.05 was considered statistically significant. NS, not statistically
significant.
C-I: Wild type (WT) and double transgenic (DT) littermates were taken off tetracycline, and starting on the same
day treated with AP (1.2 mg/kg/day), AP-Cav (2.5 mg/kg/day) or L-NAME (25 mg/kg/day) for 7 days. On day 8,
the mice were perfused with FITC-dextran for 60 minutes, and the flank skin was examined (C-F) (whole tissue
view, magnification x2.5), and harvested for confocal microscopy (G-I) (magnification x200). Arrows in H and I
indicate green fluorescent haze of extravasated FITC-dextran.