Peptide synthesis and handling
The peptide pentastatin-1 (LRRFSTMPFMFCNINNVCNF) was synthesized using a solid-phase synthesis technique by a commercial provider (Abgent, San Diego, CA). The endogenous human and mouse sequences are identical for this peptide. The manufacturer provided HPLC and mass spectrometry analysis to guarantee >95% purity. The peptides were stored at -80°C in lyophilized form. Since pentastatin-1 is hydrophobic, it was solubilized using 10% dimethyl sulfoxide (DMSO) and water without any demonstrated effect on cell viability.
WST-1 cell viability experiments
In vitro viability assays were completed with pentastatin-1 on NCI-H82 small cell lung cancer and mouse 3T3 fibroblast cell lines. NCI-H82 human SCLC cells were obtained from the laboratory of Dr. D. Neil Watkins (JHMI, Oncology). The cells were propagated in RPMI 1640 cell medium (Invitrogen, Carlsbad, CA) supplemented with 10% v/v fetal bovine serum, 10 mM of HEPES, 2 mM of L-glutamine, 1% v/v of pen/strep, 1.5 g/l of sodium bicarbonate, and 1 mM of sodium pyruvate. The cells do not attach to the flask, but grow in small floating colonies and are passaged by centrifugation (1,000 RPM, 5 min) without trypsinization and subsequently resuspended in fresh media. 3T3 mouse fibroblast cells were acquired from ATCC (Manassas, VA) and grown under standard conditions in Dulbecco's Modified Eagle Medium (DMEM) (ATCC) with 10% FBS and 1% pen/strep.
The effects of the peptide on the NCI-H82 and 3T3 cell viability were measured using the colorimetric cell proliferation reagent WST-1 (Roche, Indianapolis, IN). Approximately 2 × 103 cells were seeded per well in a 96-well microplate, centrifuged at 1,000 RPM for 5 minutes, and exposed for 3 days to peptide concentrations of: 3.2, 6.3, 12.5, 25, 50, and 100 μg/mL. Cells were tested in triplicate for each concentration. Microplates containing NCI-H82s were centrifuged at 1,000 RPM for 5 minutes to minimize loss of non-adherent cells before application of WST-1. As an experimental control, equivalent to normal cell viability, the cells were cultured without any agent in complete medium, containing growth factors and serum without any exposure to peptide.
BrdU cell proliferation assay
Cell proliferation was measured by BrdU (5-bromo-2'-deoxyuridine) incorporation assay (Calbiochem/Merck, Whitehouse Station, NJ) on both NCI-H82 cells and 3T3 fibroblasts according to manufacturer's recommendations. Briefly, 10,000 cells/well were plated into 96 well plates in the presence of pentastatin-1 at 3.8, 15, and 60 μg/mL, and BrdU label (1:2000 dilution) for 24 hours. Plates were then washed, fixed with anti-BrdU antibody, and peroxidase goat anti-mouse IgG conjugate. Immunocomplex formation was measured using tetra-methylbenzidine solution, and the reaction terminated using 2.5 N sulfuric acid. The measured intensity is proportional to the amount of incorporated BrdU in the cells. Absorbance was measured at 450 nm using a Victor 3 V plate reader (PerkinElmer, Waltham, MA). Cells were tested at five wells per concentration.
Directed in vivo angiogenesis assay (DIVAA)
DIVAA (Trevigen, Gaithersburg, MD) is a quantitative in vivo method of assaying angiogenesis. Silicone cylinders of 20 μl volume and 100 μm of inner diameter (angioreactors) were closed on one side, and filled with an extract of extracellular matrix containing VEGF and fibroblast growth factor (FGF) with or without premixed peptides as a control. These angioreactors were then implanted subcutaneously in the abdominal region of C57BL/6 mice, two angioreactors per animal, ventrally one per each side of the peritoneal cavity. Capillary sprouts originating from the host vessels invaded the extracellular matrix and formed vessels in the angioreactor. 12 days after the implantation, the mice were euthanized and angioreactors were removed. The extracellular matrix, containing the developed vasculature, was removed from the cylinder and endothelial cells were stained using FITC-Lectin, quantified by fluorescence at 510 nm emission with 485 nm excitation using a fluorescence plate reader (Victor 3 V, Perkin Elmer, Waltham, MA). The intensity of the signal is proportional to the number of endothelial cells contained in each of the angioreactors. The results were normalized to the mean of the experimental controls. Statistical significance was tested by the Student's t-test at p < 0.05 *.
In vivo tumor xenograft model
Animal protocols were approved by the Animal Care and Use Committee at the Johns Hopkins Medical Institutions. Athymic nude mice were obtained from Harlan (Indianapolis, IN). The mice were 4-6 weeks of age and housed on a 12 hour light dark cycle with food and water provided ad lib. All mice were fed with a standard caloric diet for their age.
Mice were allowed to acclimate for one week prior to inoculation with NCI-H82 cells. NCI-H82 cells were grown under the same conditions for in vitro cell viability experiments. Prior to inoculation, the cells were prepared in a solution containing 1:1 mixture of PBS and Matrigel (BD Biosciences, San Jose, CA). 1 × 106 cells in 200 μl volume were injected subcutaneously in the right flank area of the mice. Tumors were inspected 5 to 6 days after inoculation. The tumor inoculation efficiency was approximately 75%.
Following growth incubation of 6-7 days, tumor size volume was estimated by measurement of tumor dimensions with digital calipers, and peptides were administered once per day for 12 days, intraperitoneally (i.p), at 5 mg/kg and 10 mg/kg. In most cases the initial tumor volume ranged from 150 mm3 to 200 mm3. Equivalent volumes of 10% DMSO in water were injected as an experimental control. Separate controls of scrambled peptide sequences were also made to test that the anti-angiogenic efficacy of pentastatin-1 was sequence-dependent. The injections were continued for 12 days, with a total of 8 animals per group used for the experiments per peptide per concentration. The location and side of the injection were alternated every day. The higher dose of 10 mg/kg was repeated twice with similar tumor suppression and without a statistically significant change.
The tumor dimensions were measured every three days. We measured two dimensions for each tumor, a long and short axis. In some cases the tumors developed as two lobes, in which we considered the whole tumor as a single lobe with two associated axes. In order to estimate the volume we considered the tumors as prolate spheroids with volume equal to (4/3)πab2, where a is the measurement for the long semi-axis and b is the measurement for the short semi-axis.
The average tumor size per condition and the standard error of the mean are reported over time. The statistical significance measured using the Student's t-test with significance defined at p < 0.05 * and p < 0.01 **. Statistical significance was tested among the peptide condition and the experimental control in addition to the scrambled peptide.
Immediately following the sacrifice of the mice, tumors were excised and stored in a zinc-based fixative (BD Biosciences, San Jose, CA) for 10-14 days, and sent to the JHMI Immunohistochemistry Core Facility for paraffin wax-embedding and processing. After deparaffinization and rehydration, 5 μm cross sections were treated overnight at room temperature with a monoclonal rat anti-mouse CD31 (PECAM-1) IgG antibody (1:100) (BD Pharmingen, San Jose, CA), which specifically recognizes an epitope on the surface of endothelial cells. Secondary antibody incubation was made using biotinylated rabbit anti-rat IgG antibody (1:200) for one hour, and lightly counterstained with hematoxylin.
Similar analysis was made for the apoptotic marker cleaved (activated) caspase-3. After deparaffinization and rehydration of slides, primary antibodies were applied at dilutions of 1:400 for cleaved caspase-3 (rabbit polyclonal antibody; Cell Signaling Tech., Boston, MA, Cat. #9661), diluted in antibody dilution buffer (ChemMat, San Dimas, CA, Cat.#ADB250) and incubated at 14 hours at 4°C. Primary antibodies were detected using the Power Vision Plus HRP-polymer detection system (Leica Microsystems, Bannockburn, IL, Cat.#PV6119) per manufacturer's instructions. DAB chromogen (Sigma Chemicals, St. Louis, MO, Cat.#D4293) was applied to develop the secondary detection reagent. Slides were counterstained with hematoxylin and mounted on cover slips.
All histological samples were digitized, then processed using Aperio Image Scope Software (Aperio Technologies Inc., Vista, CA), and quantified using FRiDA software (Johns Hopkins University, Baltimore, MD). For the CD31 antibody staining 100% represents the mean of the experimental controls. Apoptotic cell counts were made per frame at 20× magnification using FriDA. Each peptide concentration was compared to the control and scrambled peptide equivalent using the Student's t-test at p < 0.05 * and p < 0.01 **.