Human Ovarian Cancer Model Research Articles

A Xenograft Model of Human Ovarian Cancer in Immunodeficient Mice that Can Be Used to Assay for Ovarian Cancer Stem Cells

Objective. Cancer stem cells have been identified in several human cancers including solid tumors, such as breast tumors, and head and neck squamous cell carcinomas. The search for stem cells in ovarian cancer is hampered by the lack of an efficient assay system. The identification of human cancer stem cells requires a xenograft model of tumor formation in order to assay for tumorigenic potential. We established a xenograft model of ovarian cancer from primary human epithelial ovarian carcinomas using immunodeficient mice that can be used to detect human ovarian cancer stem cells.

A NOD/SCID tumor model for human ovarian cancer that allows tracking of tumor progression through the biomarker Sp17

No experimental animal model employing a primary human ovarian carcinoma (OC) cell line is presently available that tracks the progression of this cell line with an identifiable marker. This hinders investigations related to developing new approaches for treating OC. Here, we describe the development of a tumor model in NOD/SCID mice for human OC that makes use of the endogenously expressed tumor specific sperm protein 17 (Sp17) cancer testis antigen. In this model, human SKOV-3 OC cell lines were intra-peritoneally seeded. Subsequently viable SKOV-3 cells were recovered from primary organ cell cultures from the liver ovaries, abdomen, and ascitic fluid, and their presence was confirmed by the detection of Sp17 mRNA by RT-PCR and Sp17 protein by immunocytochemistry and FACS analysis. When SKOV-3 tumor cells were administered intravenously the mice developed primarily lung tumor foci. This model makes it possible to evaluate new immunotherapeutic strategies for the treatment of human OC based on the biomarker Sp17.

Up-regulation of Tumor Susceptibility Gene 101 Protein in Ovarian Carcinomas Revealed by Proteomics Analyses

Small GTPase RAS plays a critical role in cellular signaling and oncogenic transformation. Proteomics analysis of genetically defined human ovarian cancer models identified the tumor susceptibility gene 101 (TSG101) as a downstream target of RAS oncogene. Mechanistic studies revealed a novel post-translational regulation of TSG101 through the RAS/RAF/MEK/MAPK signaling pathway and downstream molecules p14(ARF)/HDM2. Immunoanalysis using ovarian cancer samples and microtissue array revealed elevated TSG101 levels in human ovarian carcinomas. Silencing of TSG101 by short interfering RNA in ovarian cancer cells led to growth inhibition and cell death. Concurrent with the apparent growth-inhibitory effect, the levels of the CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) and hypoxia-inducible factor 1alpha (HIF-1alpha), as well as its cellular activity, were markedly reduced after TSG101 knockdown. These results demonstrate that TSG101 is important for CITED2- and HIF-1alpha-mediated cellular regulation in ovarian carcinomas.

Proteomics analyses of ovarian caner using genetically defined human ovarian cancer models

Using a genetically defined human ovarian cancer model, we have analyzed the protein expression profile of human ovarian cancer cells established by oncogenic RAS transformation. Cells were immortalized by retroviral transfection of SV40 t/T antigens and the catalytic subunit of telomerase (hTERT). Careful analyses of protein targets associated with oncogenic transformation have enabled us to identify several novel signaling pathways that play important roles in oncogenic transformation of human ovarian epithelial cells.

The Use of a Tropism-Modified Measles Virus in Folate Receptor–Targeted Virotherapy of Ovarian Cancer

Attenuated measles viruses are promising experimental anticancer agents currently being evaluated in a phase I dose escalation trial for ovarian cancer patients. Virus attachment, entry, and subsequent intercellular fusion between infected and uninfected neighboring cells are mediated via the two measles receptors (CD46 and SLAM). To minimize potential toxicity due to measles virus-associated immunosuppression and infection of nontarget tissues, we sought to develop an ovarian cancer exclusive fully retargeted measles virus. Interactions of measles virus with its natural receptors were ablated, and a single-chain antibody (scFv) specific for alpha-folate receptor (FRalpha), a target overexpressed on 90% of nonmucinous ovarian cancer, was genetically engineered on the viral attachment protein (MV-alphaFR). Specificity of virus tropism was tested on tumor and normal cells. Biodistribution of measles virus infection was evaluated in measles-susceptible CD46 transgenic mice, whereas antitumor activity was monitored noninvasively by bioluminescence imaging in xenograft models. Tropism and fusogenic activity of MV-alphaFR was redirected exclusively to FRalpha without compromise to virus infectivity. In contrast to the parental virus, MV-alphaFR has no background infectivity on normal human cells. The antitumor activity of MV-alphaFR, as assessed by tumor volume reduction and overall survival increase, was equal to the parental virus in two models of human ovarian cancer (s.c. and i.p.). A FR-exclusive ovarian cancer targeted oncolytic virus was generated and shown to be therapeutically effective, thus introducing a new modality for FR targeting and a candidate measles virus for clinical testing.

CA125 expression in spontaneous ovarian adenocarcinomas from laying hens

Currently, there is not a fully characterized model for human ovarian cancer; however, 2- to 4-year-old laying hens spontaneously develop ovarian tumors. CA125 expression is a hallmark of ovarian cancer in women. The major objective of this study was to characterize the in vitro growth of avian ovarian tumor cells, and CA125 expression in avian ovarian tumors. Immunohistochemistry was employed to evaluate CA125 expression in avian ovarian tumor tissue. A high temperature antigen retrieval step was an essential part of the CA125 staining procedure. In vitro growth curves were constructed for avian ovarian cancer cells. Western blotting was used to estimate the size of the CA125 reactive protein and to confirm CA125 expression. The growth of avian tumors in culture fits a sigmoidal curve for cell growth and suggests a cell cycle time of 28 h. The tumors taken from the chicken stained positive for CA125. Approximately 90% of cells isolated from avian ovarian tumors also stained positive for CA125. Western blots show a band of approximately 25 kDa when immunodetected with CA125. Similar to human ovarian tumors, chicken ovarian tumors express CA125. Cultured chicken ovarian cancer cells express CA125 and CA125 expression does not appear to change with time in culture.

Cyclooxygenase 1 and 2 mRNA and protein expression in the Gallus domesticus model of ovarian cancer

Objective. Our purpose was to determine the mRNA and protein expression of cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) in ovarian tumors and normal ovaries of the hen, which is an excellent model for human ovarian cancer. Tissue concentrations of prostaglandin E 2 (PGE 2) and PGE 2 metabolites were also determined. Methods. Tissue was obtained from ovarian tumor ( n = 18) and normal ovary ( n = 29) of 2- to 4-year old Single-comb White Leghorn hens. Quantitative real-time PCR with Sybr Green was used to quantify the mRNA expression of COX-1 and COX-2, using 18S expression as an internal control for COX normalization. Immunohistochemistry using antibodies for COX-1 and COX-2 was used to localize protein expression of each isoform in a subset of tumor ( n = 5) and normal samples ( n = 6). For determination of tissue prostaglandin concentration, tissue was obtained from ovarian tumor ( n = 8) and normal ovary ( n = 8). PGE 2 and PGE 2 metabolites were measured using competitive enzyme immunoassays (EIAs). Results. Our results indicate that COX-1 mRNA expression is significantly higher ( P < 0.05) in ovarian tumor samples compared to normal ovaries while there is no significant difference in expression of COX-2 between the samples. Immunohistochemistry results support this finding and show COX-1 expression only in tumor samples and COX-2 expression unchanged between normal ovary and tumor samples. PGE 2 levels are significantly higher ( P < 0.05) in tumor samples compared to normal ovaries, and there is no significant difference in PGE 2 metabolite levels between the samples. Conclusion. These findings may implicate COX-1 as a suitable target for the prevention or treatment of ovarian cancer.

MORAb-003 inhibits folate-driven growth mediated by the human folate receptor alpha

10108 Background: MORAb-003 is a therapeutic humanized antibody currently in Phase I clinical trials for advanced ovarian cancer, and is directed against the human folate receptor alpha (FRα), an antigen which is highly overexpressed on the surface of the majority of ovarian cancers. Overexpression of FRα has been shown to confer a growth advantage to tumorigenic cells in vitro, under conditions of reduced folate availability. We have previously determined that MORAb-003 elicits robust antibody-dependent cellular cytotoxicity (ADCC) and complement-directed cytotoxicity (CDC) in vitro, and is effective in mouse xenograft models of human ovarian cancer. We now show that MORAb-003 possesses novel, growth-inhibitory functions distinct from its immune effector properties. This activity can be recapitulated, using a cell-based assay to score inhibition of folate-dependent growth. Methods: Chinese Hamster Ovary (CHO) cells were engineered to express surface FRα. The cells were cultured under a low folate (&lt; 20 nM) regimen, then re-fed medium supplemented with various concentrations (0–10 μM) of 5-methyltetrahydrofolate (5-MTF), a folate preferentially internalized by the FRα. Cellular proliferation was measured as a function of added 5-MTF, in the presence or absence of MORAb-003. Results: FRα-expressing CHO cells were found to require approximately 100-fold lower 5-MTF than a matched, non-expressing cell line. This increased proliferation could be reduced in a concentration-dependent manner by MORAb-003. Conclusions: MORAb-003 can antagonize the activity of human FRα, resulting in the loss of growth advantage conferred by overexpression of FRα under conditions which bracket physiological (10–100 nM) folate concentrations. [Table: see text]

A highly reproducible xenograft model for human ovarian carcinoma and application of MRI and ultrasound in longitudinal follow-up

Objective. Typically, human ovarian cancer is widely disseminated at the time of diagnosis and shows extremely poor prognosis. Experimental animal models that mimic human ovarian cancer are often incomplete when compared to the full spectrum of the human disease with regard to its histological hallmarks such as the spread of carcinoma, its ability to seed the peritoneal cavity and formation of ascites. We have established and characterized a new animal model for human ovarian cancer in nude mouse. Methods. A new cell line SKOV-3m was injected intraperitoneally in nude mice. Mice were divided in two groups A and B, which received 1 × 10 7 and 2 × 10 7 cells, respectively. Histology, immunohistochemistry, magnetic resonance imaging and ultrasound were used to analyze tumors. Results. All mice had tumors within 18 days and histologically they resembled poorly differentiated human cystadenocarcinomas with bloody ascites. Mean survival of the mice was 42 ± 14 and 21 ± 2 days in groups A and B, respectively. Magnetic resonance imaging and ultrasound were used to confirm the presence of tumors and to monitor their growth without sacrificing the animals. Conclusion. This new xenograft model accompanied by noninvasive imaging is highly reproducible and likely to be very useful in testing new treatment strategies for ovarian cancer.

Synergistic effects of combined therapy using paclitaxel and [ 90Y-DOTA]776.1 on growth of OVCAR-3 ovarian carcinoma xenografts

Objective. 776.1 is a monoclonal antibody prepared against the human ovarian cancer antigen CA 125 that demonstrates preferential binding to the cell-associated form of the antigen and has shown promising results as an yttrium-90-labeled antibody in pre-clinical studies examining the effects on tumor growth in a murine xenograft model of human ovarian cancer. The purpose of the present study was to examine the effects of combined therapy with [ 90Y-DOTA]776.1 and paclitaxel compared with monotherapy with either agent on the growth of OVCAR-3 xenografts in nude mice. Methods. Mice bearing OVCAR-3 xenografts were treated with paclitaxel alone, 50 μCi or 150 μCi [ 90Y-DOTA]776.1 alone, or a combination of both treatments. Control groups were included which consisted of a nonspecific antibody, MOPC-21, labeled to a similar degree, administered as monotherapy or in combination with paclitaxel. The effects of administration of radioimmunotherapy prior to or following chemotherapy were also examined. Results. Treatment with paclitaxel and [ 90Y-DOTA]776.1 had a synergistic anti-tumor effect on the growth of OVCAR-3 xenografts. Synergy was only observed when a tumor-specific antibody was used in radioimmunotherapy. While no difference in tumor growth was observed with order of dosing, reduced toxicity was seen when paclitaxel was administered prior to radioimmunotherapy. Conclusion. The combination of radioimmunotherapy using an anti-CA 125 monoclonal antibody and chemotherapy with paclitaxel was shown to be effective in an in vivo model of ovarian cancer and may hold promise as a treatment regimen for patients with ovarian cancer.

301. Safe and Effective Treatment of Ovarian Cancer Models Using Targeted Adenovirus Vector Delivery and Selective Expression of Tumor Necrosis Factor

Ovarian cancer is the fourth most common cancer among women, and despite advances in therapy most patients still die as a consequence of disease that is disseminated within the peritoneal cavity. Delivery of hTNFa gene to tumors using direct intra-tumoral injection of a non-replicating adenovector is a promising strategy for treatment for a variety of diseases. The relative abundance of the coxsackie and adenovirus receptor (CAR) in the mesothelial lining of the peritoneal space and paucity of CAR in tumors may limit on the use of un-targeted adenovectors administered to the peritoneal cavity. To enhance selectivity of TNFa exposure to the tumor we combined two approaches. We modified adneovector capsids to limit transduction of normal cells. We analyzed the ability of Ad5 based vectors in which the fiber and penton base binding interactions were deleted. To allow tumor uptake we incorporated a RGD-4C motif in the fiber knob (Ad|[ast]||[ast]|RGD) to target |[alpha]|v|[beta]|3/5 integrins receptors that are highly expressed in tumor and sparsely expressed in the epithelial layer of peritoneum. Ad|[ast]||[ast]|RGD vector exhibits effective gene transfer in a variety of ovarian cancer cell lines that we examined in vitro and in vivo. Importantly, the Ad|[ast]||[ast]|RGD vector has a capability to target ovarian tumor nodules and avoid infecting the normal mesothelial cells that line the intraperitoneal space of athymic nude mice following intraperitoneal (ip) administration. We have now added an additional level of control by placing the TNFa expression under the control of tumor selective promoters. These promoters were incorporated into the capsid-modified vector to confer the expression of human tumor necrosis factor alpha (hTNFa) therapeutic gene. We compared both constitutive (CMV or RSV) and tumor selective promoters (DF3, E2F or hTERT) for safety and efficacy in human ovarian cancer models. Vector containing capsid targeting combined with the DF3 promoter expression control was shown to reduce tumor burden in the human ovarian cancer models (Caov3 and Igrov1) following ip administration. The DF3 vector was well tolerated with no evidence of body weight changes, and limited exposure to systemic TNFa at the effective dose (1.2e11 pu/20g). Together, our data support a strategy of combining receptor targeting combined with expression control of TNFa as a potential gene delivery mechanism for ovarian cancer treatment.

RAS‐Mediated epigenetic inactivation of OPCML in oncogenic transformation of human ovarian surface epithelial cells

Opioid binding protein/cell adhesion molecule-like gene (OPCML), a recently identified tumor-suppressor, is frequently inactivated by allele loss and CpG island promoter methylation in epithelial ovarian cancer. Since elevated activation of the RAS signaling pathway, including overexpression of HER-2/neu and mutations of RAS and BRAF, is common in human ovarian carcinoma, we examined the cellular effect of oncogenic RAS on the expression status of OPCML in a genetically defined human ovarian cancer model. Our study revealed that RAS(V12)-mediated oncogenic transformation was accompanied by a concomitant loss of OPCML expression. Methylation-sensitive PCR analysis showed that the OPCML promoter was hypermethylated in RAS-transformed human ovarian epithelial cells (T29H) and that treatment with the DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine promoted demethylation of the OPCML promoter and restored OPCML expression in T29H cells. Furthermore, suppression of oncogenic RAS activity by stable siRNA specific for HRAS(V12) led to the demethylation and re-expression of OPCML in T29H cells, demonstrating that oncogenic RAS activity is directly responsible for the observed OPCML promoter hypermethylation and epigenetic gene silencing of OPCML. Taken together, our study suggests that elevation of the RAS signaling pathway may play an important role in epigenetic inactivation of OPCML in human epithelial ovarian cancer.

Pharmacokinetics, Biodistribution, and Radioimmunotherapy with Monoclonal Antibody 776.1 in a Murine Model of Human Ovarian Cancer

776.1 is a murine IgG1 monoclonal antibody to the human ovarian cancer antigen CA 125 that has the unique property of having a clear preference for binding to the cell-associated form of the antigen. We have examined the tumor localization properties and efficacy of 776.1 in a subcutaneous OVCAR-3 xenograft mouse model of human ovarian cancer. Biodistribution experiments using (125)I-labeled 776.1 demonstrated a peak uptake in tumors at 72 hours postinjection, with an average of 17.7% of injected dose per gram localized to the tumor. Little uptake in other organs was observed. Further experiments using CA 125-transfected syngeneic tumors, as well as an immunoprecipitation assay using human chimeric 776.1, both clearly demonstrated that 776.1 localizes to the tumor in a CA 125-dependent manner. DOTA-776.1 (1,4,7,10-tetraazacyclododecane-N,N',N",N'" tetraacetic acid-conjugated 776.1) was labeled with (90)Y and used in efficacy studies. [(90)Y-DOTA]776.1 at a single dose of 150 microCi was able to mediate efficient reduction of tumor growth, with regression observed in a subset of animals for a period ranging from 3 to 48 days, equivalent to 3 weekly administrations of cisplatin at 6 mg/kg. No significant regression was observed in groups receiving [(90)Y-DOTA]MOPC-21 control antibody at any dose. These results suggest that 776.1 may be a promising radioimmunotherapeutic agent for the treatment of human ovarian cancer.

Fully Retargeted Oncolytic Measles Virus Specific for the WUE-Ag Results in Specific Lysis of Multiple Myeloma Cells.

Effective cancer therapy with a minimum of undesired side effects in multiple myeloma (MM) requires specifity of the therapeutic agent against the neoplastic cells. One approach to reach this aim is virotherapy using oncolytic measles virus. Attenuated replication-competent Edmonston lineage strains of measles virus (MV-Edm) have proven anti-tumor activity against xenograft models of human multiple myeloma, ovarian cancer, lymphoma and glioma. The virus is selectively oncolytic, causing extensive lethal cell to cell fusion via CD46, which is more highly expressed on tumor cells than on normal cells. However, MV-Edm retains the capacity to infect a variety of nontransformed cell types via its native receptors, CD46 and SLAM which are present on many different cell types. One approach to avoid this risk is to engineer the viral attachment protein to ablate its natural tropisms and at the same time, redirect its specificity to interact with alternative tumor specific receptors. The native measles Hemagglutinin (H) protein recognizes CD46 or SLAM resulting in membrane fusion and syncytia formation of cells. In this work the H protein was engineered to restrict and retarget membrane fusion through the display of a single-chain antibody (scFv) recognizing the Wue-1-antigen known to be highly specific for MM cells and abrogation of native measles binding domains for CD46 and SLAM by mutation. This modified H protein (chimeric H) was cloned in a full-length viral backbone including EGFP enabling to detect infected cells and syncytia formation under the UV light emitting green fluorescence (EGFP). On the basis of the parental measles virus expressing EGFP (MV-GFP) two different viruses were generated:non-ablated virus expressing the chimeric H protein including the scFv Wue-1 still competent to infect cells expressing CD46 and SLAM (MV-W1)with the chimeric H protein but ablated for the interactions with CD46 and SLAM (MV-W2).The genetically modified viruses propagated as recently described (Nat. Biotech., Vol.23, Nr.2, Feb.2005, pp.209–214.). To determine if the fully retargeted MV-Edm would be able to infect MM cell lines selectively a first array of infection assays was performed using the MM cell lines RPMI 8226 and ARH-77 expressing the Wue-1 antigen as expected targets and K562 and healthy CD40L activated CD19 positive B cells as controls. 24 to 96 hours after infection with MV-GFP, MV-W1 and MV-W2 we observed syncytia formation and expression of EGFP with MV-GFP and MV-W1 in all cells indicating that the modification of the virus with the scFv-Wue-1 doesn't alter the potential to infect and kill cells compared to the parental virus. In contrast MV-W2 was able to form only EGFP positive plaques with the Wue-1 antigen positive cells RPMI 8226 and ARH-77 but did not infect K562 or CD19 positve B cells both negative for Wue-1 antigen.These results indicate that the measles virus vaccine strain Edmonston B can be modified to express a scFv recognizing the Wue-1 antigen. In addition the native H protein can be mutated resulting in ablation of the natural tropism towards CD46 and SLAM positive cells. Viruses with these modifications can be rescued and propagated in vitro and selectively infected Multiple Myeloma cells without causing damage to normal B-cell progenitors.

Phosphatidylinostol 3-Kinase Mediates Angiogenesis and Vascular Permeability Associated with Ovarian Carcinoma

To assess the role of phosphatidylinositol-3 kinase (PI3K) inhibition in vascular permeability, angiogenesis, and vascular remodeling in tumor vessels and peritoneal lining in an athymic mouse model of i.p. human ovarian carcinoma. Mice were inoculated i.p. with cells from the human ovarian cancer cell line, OVCAR-3. Fourteen days after inoculation, mice were treated with or without the PI3K inhibitor LY294002, 3 days weekly for 4 weeks. At the end of the experiment, some mice were anesthetized and injected via the tail vein with FITC-labeled lycopersicon lectin and perfused through the aorta before sacrifice. The peritoneal wall and tumor from all mice were removed and embedded in 10% agarose. Tumor sections were visualized by fluorescence microscopy. Ascites in the LY294002-treated group (0.69 +/- 0.27 mL) was reduced by 72.4% compared with the control group (2.5 +/- 1.2 mL). Tumor burden in the LY294002-treated group (0.62 +/- 0.32 g) was reduced by 47.3% compared with the control group (1.18 +/- 0.41 g). LY294002 inhibited peritoneal and tumor vascularization resulting in numerous leaky, irregular, tortuous vessels in scant, straight, relatively impermeable vessels. The data indicate that LY294002 inhibits ascites formation in our mouse model of human ovarian cancer by inhibiting tumor and peritoneal neovascularization as well as vascular permeability. The data also show that LY294002 directly inhibits vascular endothelial growth factor (VEGF) protein expression and release from ovarian carcinoma and suggest that LY294002 blocks the VEGF signaling pathway involved in angiogenesis and vascular permeability.

Synergism of Epidermal Growth Factor Receptor–Targeted Immunotherapy With Photodynamic Treatment of Ovarian Cancer In Vivo

Epithelial ovarian cancer often develops resistance to standard treatments, which is a major reason for the high mortality associated with the disease. We examined the efficacy of a treatment regimen that combines immunotherapy to block the activity of epidermal growth factor receptor (EGFR), overexpression of which is associated with the development of resistant ovarian cancer, and photodynamic therapy (PDT), a mechanistically distinct photochemistry-based modality that is effective against chemo- and radioresistant ovarian tumors. We tested a combination regimen consisting of C225, a monoclonal antibody that inhibits the receptor tyrosine kinase activity of EGFR, and benzoporphyrin derivative monoacid A (BPD)-based PDT in a mouse model of human ovarian cancer. Therapeutic efficacy was evaluated in acute treatment response and survival studies that used 9-19 mice per group. Analysis of variance and Wilcoxon statistics were used to analyze the data. All statistical tests were two-sided. Mice treated with PDT + C225 had the lowest mean tumor burden compared with that in the no-treatment control mice (mean percent tumor burden = 9.8%, 95% confidence interval [CI] = 2.3% to 17.3%, P < .001). Mean percent tumor burden for mice treated with C225 only or PDT only was 66.6% (95% CI = 58.7% to 74.4%, P < .001) and 38.2% (95% CI = 29.3% to 47.0%, P < .001), respectively. When compared with PDT only or C225 only, PDT + C225 produced synergistic reductions in mean tumor burden (P < .001, analysis of variance) and improvements in survival (P = .0269, Wilcoxon test). Median survival was approximately threefold greater for mice in the PDT + C225 group than for mice in the no-treatment control group (80 days versus 28 days), and more mice in the PDT + C225 group were alive at 180 days (3/9; 33% [95% CI = 7% to 70%]) than mice in the C225-only (0/12; 0% [95% CI = 0% to 22%]) or PDT-only (1/10; 10% [95% CI = 0.2% to 44%]) groups. A mechanistically nonoverlapping combination modality consisting of receptor tyrosine kinase inhibition with C225 and BPD-PDT is well tolerated, effective, and synergistic in mice.

Vascular Endothelial Growth Factor Trap Combined with Paclitaxel Strikingly Inhibits Tumor and Ascites, Prolonging Survival in a Human Ovarian Cancer Model

Ovarian cancer is characterized by i.p. carcinomatosis and massive ascites. Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis and vascular leakage leading to ascites. We assessed the efficacy of a soluble decoy receptor (VEGF Trap) combined with paclitaxel, in a mouse model of human ovarian cancer. Tumor burden after VEGF Trap plus paclitaxel was reduced by approximately 98% versus controls. No measurable ascites developed in the treated group. Morphologic studies showed that most residual tumor had degenerative changes. Diaphragmatic and hepatic tumors were not found in the VEGF Trap plus paclitaxel group in contrast to controls, indicating lack of metastasis. In vivo FITC-lectin tumor vessel imaging showed sparse, short, straight vessels in treated mice as compared to controls, in which vessels were numerous, irregular, tortuous, and leaky. In a survival study, all controls underwent euthanasia between 29 and 58 days after tumor cell inoculation (cachexia, extensive ascites, and tumor masses). In the VEGF Trap plus paclitaxel group, mice were ambulating and eating normally with no signs of disease for at least 81 days after tumor cell inoculation, and survival occurred for 129.9 +/- 38.88 days with no further treatment. We conclude that combination therapy with VEGF Trap plus paclitaxel may provide a novel, long-lasting therapeutic strategy for treatment of patients with ovarian cancer associated with ascites.

A clinically relevant orthotopic implantation nude mouse model of human epithelial ovarian cancer—based on consecutive observation

A clinically relevant orthotopic implantation nude mouse model of human epithelial ovarian cancer—based on consecutive observation

Proteomics analysis of H-RAS-mediated oncogenic transformation in a genetically defined human ovarian cancer model

RAS is a small GTP binding protein mutated in approximately 30% human cancer. Despite its important role in the initiation and progression of human cancer, the underlying mechanism of RAS-induced human epithelial transformation remains elusive. In this study, we probe the cellular and molecular mechanisms of RAS-mediated transformation, by profiling two human ovarian epithelial cell lines. One cell line was immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase (T29), while the second cell line was transformed with an additional oncogenic ras(V12) allele (T29H). In total, 32 proteins associated with RAS-mediated transformation have been identified using peptide mass fingerprinting. These protein targets are involved in several cellular pathways, including metabolism, redox balance, calcium signaling, apoptosis, and cellular methylation. One such target, the 40 kDa procaspase 4 is significantly upregulated at the protein level in RAS-transformed T29H cells, related directly to signaling through MEK, but not PI3 kinase. Cellular caspase 4 activity is, however, suppressed in the T29H cells, suggesting that the maturation process of caspase 4 is abrogated in RAS-transformed T29H cells. Consistent with this notion, transformed T29H cells were less susceptible to the toxic effects of anti-Fas antibody than were immortalized, nontransformed T29 cells, associated with less activation of caspase 4. This study demonstrates that functional proteomic analysis of a genetically defined cancer model provides a powerful approach toward systematically identifying cellular targets associated with oncogenic transformation.

73. Fully Retargeted Oncolytic Measles Viruses for Cancer Therapy

Attenuated replication-competent Edmonston lineage strains of measles virus (MV-Edm) have proven anti-tumor activity against xenograft models of human multiple myeloma, ovarian cancer and glioma. The virus is selectively oncolytic, causing extensive cell to cell fusion via CD46, which is more highly expressed on tumor cells than normal cells. However, MV-Edm retains the capacity to infect a variety of nontransformed cell types via its native receptors, CD46 and SLAM. CD46 is found on all human nucleated cells. SLAM (signaling lymphocyte activation molecule) is only expressed on activated T and B cells, dendritic cells and macrophages. Therefore, the native tropism has possibility to cause immunosupression, unwanted damage to normal tissues, and toxicity of oncolytic measles virus.