Human ovarian cancer specimens
Following approval by the Institutional Review Board, 84 paraffin-embedded epithelial ovarian cancer specimens with available clinical outcome data and confirmed diagnosis by a board-certified gynecologic pathologist were obtained from the Gynecologic Oncology tumor bank of The University of Texas M. D. Anderson Cancer Center. All 84 cases were diagnosed between 1986 and 2003 following primary cytoreductive surgery. Slides of tumor samples were obtained for Dll4 expression analysis. Clinical variables obtained for correlative analyses included age at diagnosis, tumor stage and grade, and vital status of patients relative to disease-specific survival at the time of chart review. An additional 24 paraffin-embedded epithelial ovarian cancer specimens from patients who were treated with an anti-VEGF agent (aflibercept or bevacizumab) were obtained from the Gynecologic Oncology tumor bank.
Immunohistochemical staining for Dll4 (1:200 dilution) was quantified by two investigators in a blinded fashion on the basis of percentage of positively stained tumor cells and staining intensity. The Dll4 antibody, which reacts with both mouse and human Dll4, was obtained from Rockland Immunochemicals for Research (Gilbertsville, PA). For the negative control, we used PBS instead of primary antibody. Dll4 expression in the endothelial cell compartment was defined as more than 25% of cells staining positive. The endothelial Dll4 score was dichotomized, with a positive score consisting of the presence of staining with moderate or strong intensity. In short, an overall score (OS 0-3) was generated based on the percentage of positively stained cells plus the intensity of staining (13
Cell lines and culture
A2780, SKOV3ip, OVCAR3, IGROV, and 2774 human epithelial ovarian cancer cells and pericyte-like cells (10T1/2) were maintained as described previously (14
). The derivation and characterization of murine ovarian endothelial cells (MOEC) have been described previously (15
). Human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (Walkersville, MD) and maintained with heparin and gentamicin/amphotericin B.
Dll4 gene silencing in MOEC and A2780 ovarian cancer cells
Nonsilencing control small interfering RNA (siRNA) and human and mouse Dll4 siRNA sequences were obtained from Sigma-Aldrich (St. Louis, MO). The nonsilencing siRNA did not share sequence homology with any known human mRNA (based on a BLAST search). The sequences are included in supplementary Table 3
. For in vitro
transfection studies, N-TER transfection kit (Sigma-Aldrich, St. Louis, MO) was used per the manufacturer’s guidelines.
Reverse transcriptase polymerase chain reaction
Relative expression of Jag 1, Jag2, Dll1, Dll3, Dll4, Notch1, Notch2, Notch3, and Notch4 in cells representing ovarian cancer (HeyA8, SKOV3ip1, OVAR3, A2774, IGROV), endothelium (HUVEC, MOEC), and pericyte-like (10T1/2) and nontransformed ovarian surface epithelial cells (HIO180) was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Each RT-PCR reaction used 5 μg total RNA isolated from treated cells using the RNeasy Mini Kit (Qiagen, Valencia, CA). Primer sequences, size of PCR products, and annealing temperature are given in supplementary Tables S1
. Real time quantitative RT-PCR was performed in an ABI 7500 Sequence Detection system (Applied Biosystems, Austin, TX). The SensiMix™ SYBR Low-ROX Kit was used (Bioline USA Inc, MA, USA).The relative quantification (RQ) was calculated by 2−ΔΔCT
Western Blot Analysis
Western blot analysis was performed as previously reported (16
DNA extraction and methylation analysis
DNA was extracted from MOEC using standard phenol-chloroform methods. MOEC were treated with immobilized Dll4 (1, 10, or 20 μg/mL). Other MOEC were treated with the demethylating agent azacytidine (AZA; 25 μM), immobilized Dll4 (10 μg/mL), or a combination of the two for 48 hours. Methylation status was determined by methylation-specific PCR using a methylation kit (EZ-96 gold; Zymo Research, Orange, CA). MethPrimer software was used for prediction of the CpG island of VEGF receptor 2 (VEGFR2; NM-002253.2) and design of methylation-specific primers. The sequences of primers for methylated VEGFR2 at the promoter region were TGTTTTTA-GATGCGATTTGTCGTTC (forward) and AAAATAAAAACTCCCTACGTCCGAC (reverse); for unmethlyated VEGFR2 promoter, TTTTTAGATGTGATTTGTTGTTTGG (forward) and AAAATAAAAACTCCCTACATCCAAC (reverse). The PCR conditions were 94°C for 5 minutes with hot start, then 94°C for 45 seconds, 58°C and 60°C for 45 seconds, and 72°C for 45 seconds, repeated for 40 cycles. Image analysis (Scion Image for Windows) was used for semiquantitative measurement of methylated and unmethylated VEGFR2. Methylated VEGFR2 was normalized by comparison with unmethylated VEGFR2. The experiments were repeated three times.
Cell proliferation and migration
Cells were seeded in 12-well plates at 8 × 104
cells/well in replicates of two. After 48 hours, cell growth was arrested, and specific mediators were added to untreated cells. Proliferation was assessed by the BrdU proliferation kit (BD Biosciences, San Jose, CA). The membrane invasion culture system chamber was used to measure the in vitro
migration ability of cells, as previously described by our group (16
Female athymic nude mice (NCr-nu) were purchased from the Animal Production Area of the National Cancer Institute–Frederick Cancer Research and Development Center (Frederick, MD). The animals 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 United States Department of Health and Human Services, the United States Department of Agriculture, and the National Institutes of Health. Mice used in these experiments were aged 8–12 weeks. Tissue specimens were fixed with either formalin or optimum cutting temperature (OCT; Miles, Inc., Elkhart, IN) or were snap frozen.
Orthotopic implantation of tumor cells
To produce tumors in nude mice, subconfluent cultures of A2780 and SKOV3ip1 cells were lifted with trypsin, mixed with medium containing 10% fetal bovine serum, subjected to centrifugation at 1000 rpm for 5 minutes, and washed in PBS. Mice were injected intraperitoneally (i.p.) with one cell type at a concentration of 2 × 106
cells/0.2 ml for A2780 cells or 1 × 106
cells/0.2 ml for SKOV3ip1cells. Mice were killed 40 to 50 days after tumor cell injection. We have used the intraperitoneal (i.p.) injection model for therapeutic studies since it reflects a typical pattern of ovarian cancer spread in patients with recurrent disease(16
Treatment and data collection
For systemic delivery of siRNA into both tumor cells and tumor-associated vasculature, we developed and characterized chitosan (CH) nanoparticles and demonstrated that nanoparticles with a 3:1 chitosan:triphosphate ratio (CH3) showed the greatest (75%) incorporation efficiency (16
). For all subsequent experiments, therefore, we used the siRNA/CH3 nanoparticles because of their small size, slight positive charge, and high efficiency in incorporating siRNA. To assess tumor growth for long-term therapy experiments, A2780 and SKOV3ip1 cells were injected i.p.; 7 days later, mice were randomized into eight groups (n
= 10/group) and underwent the following treatments including bevacizumab, i.p, twice per week or human/murine Dll4siRNA, i.v. twice per week: (1) control siRNA 150 μg/kg; (2) mouse Dll4 siRNA 150 μg/kg; (3) human Dll4 siRNA 150 μg/kg; (4) mouse Dll4 siRNA plus human Dll4 siRNA; (5) bevacizumab 6.25mg/kg; (6) bevacizumab plus mouse Dll4 siRNA; (7) bevacizumab plus human Dll4 siRNA; or (8) bevacizumab plus human Dll4 siRNA and mouse Dll4 siRNA. Mice were killed after 4–6 weeks of therapy, when animals in the control group became moribund. Fifteen minutes before sacrifice, the mice were i.v. injected with 100μl of Hypoxyprobe ™-1
(pimonidazole HCl, 100mg/kg, NPI, Inc, Burlington, MA) through the tail vein, At the time of death, mouse weight, tumor weight, number of nodules, and distribution of tumors were recorded. The individuals who performed the necropsies, tumor collections, and tissue processing were blinded to the treatment group assignments.
Immunofluorescence double staining for CD31 and desmin
Sections were fixed in cold acetone for 15 minutes, blocked with protein blocker for 30 minutes, and then incubated with anti-CD31 antibody (1:500; BD Pharmingen, San Diego, CA) overnight at 4°C, after which they were incubated with Alexa 594-conjugated anti-rat antibody (1:1000; Invitrogen, Eugene, OR) for 1 hour. After being washed with PBS, samples were incubated with anti-desmin (1:400; DakoCytomation, Glostrup, Denmark) antibody for 1 hour and then with Alexa 488-conjugated anti-rabbit antibody (1:1000; Invitrogen) for 1 hour. Samples were then counterstained with Hoechst 33342 for 2 minutes and mounted.
Paraffin-embedded tissues were used for detection of proliferating cell nuclear antigen (PCNA, i.e., cell proliferation) and pimonidazole (i.e., hypoxic area). Sections were deparaffinized, rehydrated, and transferred to PBS. After antigen retrieval with citrate buffer (pH 6.0), the sections were blocked with 3% hydrogen peroxide in methanol and protein blocker at room temperature. The sections were then incubated with the monoclonal mouse anti-PCNA PC10 antibody (1:50; DAKO) or Hypoxyprobe-1-Mab 1 (1:50; Natural Pharmacia International, Inc., Burlington, MA) overnight at 4°C. After being washed with PBS, sections were incubated with horseradish peroxidase (HRP)–conjugated rat anti-mouse IgG2a (1:100; Serotec, Harlan Bioproducts for Science, Inc., Madison, WI) for PCNA staining or anti-fluorescein isothiocyanate HRP IgG (1:500; Natural Pharmacia International, Inc.) for 1 hour. CD31 staining was performed on frozen sections. Sections were fixed in cold acetone for 15 minutes, washed with PBS, blocked with protein blocker (4% fish gel), and then incubated with rat monoclonal anti-mouse CD31 (1:800, PharMingen, San Diego, CA) overnight at 4°C. They were then washed with PBS and incubated with HRP-conjugated goat anti-rat IgG (1:200, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour. Reactive tissues were visualized by staining with 3,3′-diaminobenzidine (Research Genetics, Huntsville, AL) and counterstaining with Gil’s hematoxylin (BioGenex Laboratories, San Ramon, CA).
Quantification of microvessel density, PCNA, pericyte coverage, and hypoxic area
For quantification, five samples from each group were examined. To quantify microvessel density (MVD) for each sample, the microvessels within five randomly selected 0.159-mm2 fields at ×200 were counted. A single microvessel was defined as a discrete cluster or single cell stained positive for CD31 (CD31+). To quantify PCNA expression, the percentage of positive cells was determined in five random 0.159-mm2 fields at ×200 magnification. For pericyte coverage, the percentage of vessels with at least 50% coverage of associated desmin-positive cells was determined in five random 0.159-mm2 fields at ×200 magnification. Hypoxic area was measured using image J at magnification ×20. The percentage of hypoxic area was determined by subtracting areas of necrosis from total pimonidazole-positive areas and then normalizing by whole section areas.
Differences in continuous variables such as mean body weight, tumor weight, MVD and vessel maturation, tumor cell proliferation, and intratumoral hypoxia were analyzed using the Mann-Whitney rank sum test. Statistical analyses were performed using SPSS 12.0 for Windows (SPSS Inc., Chicago, IL). A two-tailed p<0.05 was considered statistically significant. Kaplan-Meier survival plots were generated and comparisons between survival curves were made using the log-rank statistic.