Recombinant adenovirus construction and viral preparation
According to the cDNA sequence of PEDF in genebank, we designed a pair of PEDF primers that contain a Pme I restriction site (underlined in the following) in both primers (5'-AGCTTTGTTTAAAC ATGCAGGCCCTGGTGCTACTCCTC-3' and 5'-AGCTTTGTTTAAAC TTAGGGGCCCCTGGGGTCCAGAATC-3'). Using these primers, we amplified human PEDF cDNA with RT-PCR. PCR product was digested with Pme I and its sequence was confirmed. Using AdEasy system, we first clone PEDF cDNA into a shuttle vector pAdenoVator-CMV5 at Pme I and Bam H I site, in which PEDF expression is under the control of the constitutive cytomegalovirus (CMV) promoter. The recombinant shuttle plasmid was used to rescue the replication-defective adenovirus . Ad-luciferase and Ad-Null was prepared as the construction of Ad-PEDF, except luciferase gene or no objective gene was inserted.
The viral particles were amplified in human embryonic kidney (HEK293) cells (ATCC Rockville Maryland, USA), which were maintained in DMEM medium (Gibico BRL, Grand Island, New York, USA) with 10% fetal bovine serum (FBS) plus 100 μg/ml amikacin in a 37°C humidified chamber with 5% CO2 atmosphere. The harvested viral particles from the cultures were purified by double cesium chloride (CsCl) gradient ultracentrifugation followed by dialysis. Final aliquots of virus were measured by absorption (A260). The virus titer was quantified using a standard TCID50 assay.
Cell culture and adenovirus infection
Primary human umbilical vein endothelial cells (HUVECs) were collected and cultured as previously described . The CT26 mouse colon carcinoma and B16-F10 mouse melanoma cell lines were purchased from the American Type Culture Collection (ATCC, Rockville Maryland, USA) and cultured in RM1640 medium (Gibico BRL, Grand Island, New York, USA) supplemented with 10% FBS and 100 μg/ml amikacin. 2.5 × 105 CT26 or B16-F10 cells were plated into 6-well plates and grew to 70%~80% confluence. The cells were washed three times gently by serum-free medium and infected with Ad-PEDF or Ad-null (both at MOI50, 2.5 × 107 pfu per 5 × 105 cells) in 0.5 ml serum-free medium, with normal saline as the non-infection control. After incubation for 90 minutes at 37°C, 1.5 mL complete medium with 10% FBS was added to wells. Supernatants were collected after further culture for 72 hours and stored at -80°C for further analysis.
Western blotting analysis
Western blot analysis was performed as described previously . Briefly, the supernatant was concentrated by super filter (5 kDa, Minipore) and mixed with an equal volume of sodium dodecyl sulfate (SDS) sample buffer. The proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electronically transferred onto a polyvinylidene difluoride membrane (PVDF, Bio-Rad, Richmond, CA, USA). The blots were probed with a mouse anti-human PEDF monoclonal antibody (3:1000, mAb; R&D Systems, Boston, Massachusetts, USA) plus a peroxidase-conjugated secondary antibody, goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China). The protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, Illinois, USA).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay
The MTT Assay was used to determine the effect of PEDF derived from Ad-PEDF infected cells on human umbilical vein endothelial cells (HUVECs). Three types of supernatants from B16-F10 cells infected with Ad-PEDF, Ad-Null or NS, respectively, were prepared as described above. Each type of supernatant was further diluted into a series of 1/2 dilutions in six tubes (from 1:2 to 1:64). Each supernatant dilution was added into triplicate wells (50 μl/well) of HUVECs which were seeded on 96-well plates on the previous day (2 × 103 cells in 50 μl complete medium per well). The cells were incubated at 37°C in 5% CO2 for 72 hours. Then, each well received 10 μl MTT solution (5 mg/mL). After a 4-hour incubation, the media was removed and 150 μl dimethyl sulfoxide was added. After 20 min of incubation, the OD value was determined by a microplate reader (3550-UV, BIO-RAD, USA). The following formula was used to calculate the inhibition rate of HUVEC proliferation: [1 - (experimental group OD value - negative control OD value)/(positive control OD value - negative control OD value)] × 100%.
Six to 8 week-old female C57BL/6 and BALB/c mice were purchased from the West China Experimental Animal Center of Sichuan University (Sichuan, China). Mice were permitted 1 week to acclimate to their environment before manipulation. All surgical procedures were completed in accordance with the guidelines on the care and use of laboratory animals for research purposes by the West China Hospital Cancer Center's Animal Care and Use Committee. C57BL/6 mice were inoculated with 1 × 105 B16-F10 melanoma cells s.c. in the right flank. Primary tumors usually became palpable on day 7–8 and with an average diameter of 3 mm. On day 9, the tumor-bearing mice were randomly assigned into 3 groups and each group contained 8 mice. Each mouse in Ad-PEDF group received 5 × 108 IU Ad-PEDF virus in 0.1 ml via i.v. injection on day 9, 12, 15, and 18 with a total of 4 times. The mice in the control groups received 5 × 108 IU Ad-Null or normal saline (NS), serving as vector and injection control, respectively. The details of the treatment were described previously . Tumor dimensions were measured with calipers on day 9, 12, 15, 18, 21 and 24 with a total of 6 times. The tumor volumes were calculated according to the following formula: length × width2 × 0.52. Two mice from each group were bled to collect serum on day 22, which was used to examine the PEDF concentration in serum. Surviving mice in Ad-PEDF groups were monitored up to 42 days; all other mice become moribund by day 24 and were sacrificed. Subcutaneous tumors from sacrificed mice were removed and fixed in 4% formaldehyde solution for immunochemistry staining and histological analysis.
Detection of PEDF concentration in serum
Concentrations of PEDF in serum were determined using a commercial PEDF ELISA kit (ADL, Biotech. Dev. Co., USA) following the manufacturer's instructions. Briefly, 50 μl serum and 50 μl PEDF monoclonal antibody were added to every well of the pre-coated ELISA plate and the plate was incubated at 37°C for 1 hour. After wash, 80 μl of streptavidin-HRP was added and incubated at 37°C for 30 minutes. After wash, 50 μl substrate A and B was added, respectively, and incubated for 10 minutes at 37°C, followed by 50 μl stop solution. The absorbance was read immediately at 450 nm in a spectrophotometer . There were 2 serum samples in each group, and each sample were applied to 3 replicated wells.
Luciferase assay for virus distribution
Virus distribution was analyzed using the luciferase reporting system, as reported previously . C57BL/6 mice were inoculated with 1 × 105 B16-F10 melanoma cells s.c. in the right flank. On day 9, the tumor-bearing mice were randomly assigned into 2 groups and each group contained 3 mice. Experimental group received 5 × 1010 IU Ad-luciferase and control group received 5 × 1010 IU Ad-null virus in 0.1 ml via i.v. injection. Seven days later, the mice were sacrificed. Heart, liver, spleen, lung, kidney and tumor from each mouse were collected and individually stored in liquid nitrogen. Using a luciferase assay system kit (Promega, Madison WI, USA), we analyzed luciferase expression in each type of collected organ, according to the manufacturer's instructions. Briefly, the same organs from the same group were pooled and ground to a fine powder in a mortar containing liquid nitrogen. The fine powder was dissolved and further processed in CCLB solution in the assay kit. The resultant supernatants were collected and subjected to determination of relative light units (RLU, synergy 2, Biotek, Germany), along with a group of standard samples in the kit. The amount of luciferase in each sample was calculated on the basis of the standard curve.
Detection of apoptosis and microvessel density (MVD)
On day 24 after mouse inoculation with melanoma cells, subcutaneous tumors from Ad-PEDF, Ad-null and NS treated mice were collected, fixed, embedded in paraffin, and cut into 3–5 μm sections. The apoptotic cells within the tumor tissue were evaluated using the DeadEnd Colorimetric Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) System (Promega, Corporation, Madison, Wisconsin, USA) following the manufacturer's protocol. Ten high power fields on each slide and three slides from each animal were examined. Apoptosis index was calculated by dividing the number of apoptotic cells by the total number of cells in the field.
The method reported by Weidner et al was adopted to quantify MVD in tumor tissues . Briefly, 5 μm tumor sections were stained for the epithelial cell marker, CD31. The procedure of immunostaining for CD31 was previously described in detail . The following antibodies and reagents were used: goat anti-mouse CD31 mAb (1:200, Santa Cruz Biotechnology, Santa Cruz, California, USA), biotinylated polyclonal rabbit anti-goat (1:100, Santa Cruz Biotechnology, Santa Cruz, California, USA), ABC kit (Boster biological engineering company, Wuhan, China) and DAB visualization system (ZSJQ Biotechnology, Beijing, China). The resultant sections were first examined at low magnifications (×40 and ×100) to identify the vascular-rich area in the tumor. Within this area, the CD31-positive microvessels were counted in a single high-power (×200) field. Any CD31 stained single or cluster of cells was considered a single countable microvessel. Adjacent sections were stained with hematoxylin and eosin (H&E) and examined for tissue structure and histological morphology. Each group contains 2 mice, and 3 sections from each mouse.
Alginate-encapsulated tumor cell assay
The alginate-encapsulated tumor cell assay was used to measure tumor angiogenesis in vivo, as previously described [14, 18]. Briefly, B16-F10 or CT26 cells in 1.5% (m/v) sodium alginate solution (Sigma-Aldrich, St. Louis, Missouri, USA) was dropped into a swirling 0.25 M CaCl2 solution to prepare alginate beads (1 × 105 cells/bead). Four resultant beads were implanted s.c. on both dorsal sides of BALB/c female mice (2 beads/side). On the next day, mice were randomly assigned into 3 groups (n = 2), and each group was i.v. injected with 0.1 ml Ad-PEDF (5 × 108IU/mouse), Ad-Null (5 × 108 IU/mouse), or NS, respectively. After a week, this same treatment on each mouse was repeated. On day 11 after tumor cell implantation, all mice were injected i.v. with 100 μl FITC-dextran (Sigma-Aldrich, St. Louis, Missouri, US) solution (100 mg/ml), which is a plasma-borne tracer extravasating into tissue interstitial fluid from plasma within 20 minutes. Alginate beads were exposed surgically and photographed with a digital camera (model, Canon, Japan). Then, the beads were removed and vortexed in a tube containing 2 ml NS. After centrifugation, the supernatant was collected and subjected to a fluorescence spectrophotometer for the measurement of fluorescence intensity. The amount of FITC-dextran was calculated and used to estimate the amount of blood supply and angiogenesis status.
SPSS program (version 15.0, SPSS Inc., USA) was used for statistical analysis. Log-rank test was used to compare survival rate among groups. ANOVA was used to determine statistical significances in remaining comparisons in this study. The difference is considered as significant if p < 0.05.