Cell lines and culture conditions
Highly metastatic human ovarian cancer cell lines HeyA8 and SKOV3ip1, as described previously, were used in the present study.32
Cells were cultured in RPMI1640 medium supplemented with 15% fetal bovine serum and 0.5% gentamicin, maintained on plastic and incubated at 37°C in a mixture of 5% CO2
and 95% air. The tumor cells were free of pathogenic murine viruses and Mycoplasma (assayed by M.A. Bioproducts, Walkersville, MD). The cells were maintained for less than 10 weeks after recovery from frozen stock.
Female athymic nude mice (NCr-nu) were purchased from the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD). The mice were kept under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in agreement with current regulations and standards of the NIH, the United States Department of Health and Human Services, and the United States Department of Agriculture. According to institutional guidelines, mice used in these experiments were 8–12 weeks old.
Drugs and reagents
Bevacizumab and docetaxel were purchased from the M.D. Anderson Cancer Center pharmacy (Houston, TX). AX102 was generously provided by Archemix Corp. (Cambridge, MA). AX102 is a 34-nucleotide DNA aptamer that specifically binds to and inhibits the activity of PDGF-B.
Orthotopic implantation of tumor cells and tumor collection procedures
Sub-confluent cultures of HeyA8 and SKOV3ip1 cells were lifted with either 0.25% trypsin-EDTA (Life Technologies, for SKOV3 ip1) or 0.1% EDTA (for HeyA8), neutralized with RPMI 1640 medium containing 10% fetal bovine serum, centrifuged at 1,000 rpm for 5 minutes, washed in serum-free medium and resuspended in HBSS (Invitrogen). Single-cell suspensions with >95% viability, as determined by trypan blue exclusion, were used for the in vivo injections. Cells were then injected intraperitoneally (i.p) into mice at a concentration of 2.5 × 105 cells/0.2 ml for HeyA8 cells and 1 × 106 cells/0.2 ml for SKOV3ip1 cells. Mice were sacrificed at 28–42 days after tumor cell implantation, and body weight, tumor weight and location, number of nodules, and the amount of ascites were recorded. Tumor tissues were collected for further studies. For immunohistochemical (IHC) and H&E-staining procedures, tumors were fixed in formalin and embedded in paraffin. For immunofluorescence staining, tumors were embedded in OCT compound (Miles, Inc., Elkhart, IN), frozen rapidly in liquid nitrogen, and stored at −80°C. For western blot analysis, tumors were snap frozen rapidly in liquid nitrogen and stored at −80°C.
Treatment and data collection
For therapy experiments, mice were injected i.p. with HeyA8 and SKOV3ip1 cells as detailed above and randomized into 4 groups (n = 10/group). Seven days after tumor cells injection, the following treatments were initiated: (1) vehicle control; (2) bevacizumab (6.25 mg/kg, i.p. twice per week) alone; (3) AX102 (50 mg/kg, i.p., daily) alone; (4) bevacizumab (6.25 mg/kg, i.p. twice per week) plus AX102 (50 mg/kg, i.p., daily). Following 3 weeks of therapy for the HeyA8 model and 5 weeks of therapy for the SKOV3ip1 model, mice were sacrificed when mice in the control group became moribund.
For longitudinal assessment, mice were injected i.p. with the HeyA8-Luc cells at 2.5 × 105
cells/mouse. Seven days later, mice were randomized into one of the following four groups (n
= 10/group): (1) vehicle control; (2) bevacizumab (6.25 mg/kg, i.p. twice per week) alone; (3) bevacizumab (6.25 mg/kg, i.p. twice per week) plus docetaxel (2 mg/kg, i.p. weekly); (4) bevacizumab (6.25 mg/kg, i.p. twice per week) plus AX102 (50 mg/kg, i.p., daily) plus docetaxel (2 mg/kg, i.p. weekly). In vivo bioluminescence imaging was conducted twice a week on a cryogenically cooled IVIS 100 imaging system (Xenogen Corp., Alameda, CA), as described previously,14
and the data were analyzed with Living Image software coupled to the IVIS system. The experiment was concluded when mice in any group became moribund.
Sections were fixed in cold acetone for 30 minutes, and blocked with protein blocker (4% of fish gel) for 1 hour at room temperature. For dual immunofluorescence staining for CD31 and desmin, the sections were probed with CD31 antibody (1:500, BD Pharmingen, San Diego, CA) at 4°C overnight, after washing with phosphate-bufferred saline (PBS), the sections were then incubated with Alexa 594-conjugated anti-rat antibody (1:1,000, Invitrogen, Eugene, OR) for 1 hour at room temperature. After extensive washing with PBS, samples were next probed with anti-desmin antibody (1:400, DakoCytomation, Denmark) for 2 hours, followed by washing with PBS and incubation with Alexa 488-conjugated anti-rabbit antibody (1:1,000, Invitrogen) for 1 hour at room temperature.
Immunohistochemical (IHC) staining for proliferating cell nuclear antigen (PCNA) and CD31
For using formalin-fixed, paraffin-embedded tissue, sections were deparaffinized in xylene, rehydrated in graded alcohol, and transferred to PBS. After antigen retrieval with citrate buffer (pH 6.0) for PCNA, and proteinase K for CD31, the endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 15 minutes, and the nonspecific epitopes were blocked with fragment block (1:10, Jackson ImmunoResearch Laboratories) overnight at 4°C. Sections were next incubated with protein blocker (4% of fish gel) for 1 hour at room temperature, followed by incubation with the anti-PCNA PC10 antibody (1:50; DAKO) or anti CD31 (1:200, PharMingen) overnight at 4°C. After washing with PBS, for PCNA staining, sections were incubated with horseradish peroxidase (HRP)-conjugated rat anti-mouse IgG2a (1:100, Serotec, Harlan Bioproducts for Science, Inc.,) for 1 hour, for CD31 staining, sections were incubated with rat probe (BioCare) for 20 minutes, followed by incubation with rat HRP polymer (BioCare) for 20 minutes at room temperature. Finally, Visualization was attained with 3,3′-diaminobenzidine (Research Genetics) and counter-staining with Gil’s hematoxylin (BioGenex Laboratories). For using frozen sections for IHC staining, sections were fixed in cold acetone for 30 minutes, washed with PBS, blocked with protein blocker (4% of fish gel), and then were incubated with rat monoclonal anti-mouse CD31 (1:800, PharMingen) overnight at 4°C, washed with PBS and then incubated with HRP-conjugated goat anti-rat IgG (1:200, Jackson ImmunoResearch Laboratories) for 1 hour. Visualization was attained with 3,3′-diaminobenzidine (Research Genetics) and counterstaining with Gil’s hematoxylin (BioGenex Laboratories).
Quantification of pericyte coverage, microvessel density (MVD) and PCNA
For quantification, 5 samples from each group were examined. To quantify pericyte coverage, the percentage of vessels with at least 50% coverage of associated desminpositive cells was determined in 5 random 0.159-mm2 fields at x200 magnification for each sample. MVD was quantified by the number of lumen-like structures adjacent to CD31-positive endothelial cells within 5 randomly selected 0.159 mm2 fields at x200 magnification. PCNA was determined by the percentage of PCNA-positive cells in 5 randomly selected 0.159 mm2 fields at x200 magnification.
All measurements were represented as the average ± S.E. of the mean. Continuous variables were analyzed by two-tailed Student’s t test or analysis of variance (ANOVA) (for all groups) if normally distributed, and the nonparametric Mann-Whitney rank sum test (for all groups) if the distribution is unkonwn. A Bonferroni adjustment to α (default value 0.05) was made based on the number of pairwise comparisons within a treatment experiment using the formula: a (α) = 0.05/k, where K = number of comparisons against control. A p-value of <0.05 was considered statistically significant.