Xenograft Model Of Ovarian Carcinoma Research Articles

Abstract 5085: Preclinical development of NaPi2b-PL2202, a novel camptothecin-based antibody-drug conjugate targeting solid tumors expressing NaPi2b

Abstract NaPi2b, encoded by the SLC34A2 gene, is a multi-transmembrane, sodium-dependent phosphate transporter expressed at high levels in tumors and low levels in normal tissues. Functionally, it is involved in trans-cellular flux of phosphate in the small intestine and in the synthesis of surfactant in lung alveoli. NaPi2b is reported to be expressed at a high frequency in ovarian carcinoma as well as in NSCLC, bladder, endometrial and papillary thyroid carcinoma1, and its expression is associated with poor prognosis 2, 3. The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity and tolerability of NaPi2b-PL2202, an antibody-drug conjugate (ADC) composed of a humanized, Fc-silenced IgG1 antibody directed against human NaPi2b, to which PL2202, a novel payload containing a valine-alanine cleavable linker and a camptothecin warhead, has been site-specifically conjugated with a drug to antibody ratio of 6. In addition, the level of NaPi2b expression in selected human tumor specimens was evaluated by immunohistochemistry (IHC). In vitro, NaPi2b-PL2202 showed good binding affinity for mouse, rat, cynomolgus monkey and human NaPi2b, and displayed highly potent, targeted cytotoxicity against various NaPi2b-expressing tumor cell lines. NaPi2b-PL2202 also exhibited strong in vitro bystander activity. In vivo, intravenous (IV) administration of a single dose of NaPi2b-PL2202 at 6.6 mg/kg resulted in substantial tumor regression in the OVCAR-3 ovarian carcinoma xenograft model, with 3/10 partial responders (PR) and 7/10 complete responders (CR), and all CRs being tumor-free at the end of the study (Day 44). In the G-402 renal leiomyoblastoma xenograft model, administration of a single IV dose of NaPi2b-PL2202 at 3.3 mg/kg was associated with durable antitumor activity and resulted in 1/10 CR, with this animal remaining tumor-free by the end of the study (Day 42). With respect to toxicity, in single dose studies in male rats, NaPi2b-PL2202 was tolerated up to 300 mg/kg IV. Furthermore, in a dose-range finding study in male cynomolgus monkeys, NaPi2b-PL2202 was well tolerated up to 40 mg/kg IV given on Day 1 and 22. Finally, cell membranous NaPi2b expression was confirmed by IHC in a panel of tissue microarrays including ovarian, lung, bladder and endometrial cancers. In conclusion, NaPi2b-PL2202 demonstrated specific and potent in vitro and in vivo anti-tumor activity, and was tolerated at high doses in toxicity studies in rats and cynomolgus monkeys, indicating a good therapeutic index. Together, these observations support future clinical development of NaPi2b-PL2202 for the treatment of NaPi2b-expressing cancers. 1- Bodyak N. D. et al., 2021 2- Valsenkova R. et al., 2021 3- Hakim S. A. et al., 2021 Citation Format: Elizabeth Horsley, Asma Jabeen, Nicolas Veillard, Karin Havenith, Narinder Janghra, Pedro Alves, Cecile Oblette, Ian Kirby, Paul W. Hogg, Francesca Zammarchi, Lolke de Haan, Patrick van Berkel. Preclinical development of NaPi2b-PL2202, a novel camptothecin-based antibody-drug conjugate targeting solid tumors expressing NaPi2b [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5085.

Adenosine Deaminase 1 Overexpression Enhances the Antitumor Efficacy of Chimeric Antigen Receptor-Engineered T Cells.

Chimeric antigen receptor (CAR) T cell therapy mediates unprecedented benefit in certain leukemias and lymphomas, but has yet to achieve similar success in combating solid tumors. A substantial body of work indicates that the accumulation of adenosine in the solid tumor microenvironment (TME) plays a crucial role in abrogating immunotherapies. Adenosine deaminase 1 (ADA) catabolizes adenosine into inosine and is indispensable for a functional immune system. We have, for the first time, engineered CAR T cells to overexpress ADA. To potentially improve the pharmacokinetic profile of ADA, we have modified the overexpressed ADA in two ways, through the incorporation of a (1) albumin-binding domain or (2) collagen-binding domain. ADA and modified ADA were successfully expressed by CAR T cells and augmented CAR T cell exhaustion resistance. In a preclinical engineered ovarian carcinoma xenograft model, ADA and collagen-binding ADA overexpression significantly enhanced CAR T cell expansion, tumor tissue infiltration, tumor growth control, and overall survival, whereas albumin-binding ADA overexpression did not. Furthermore, in a syngeneic colon cancer solid tumor model, the overexpression of mouse ADA by cancer cells significantly reduced tumor burden and remodeled the TME to favor antitumor immunity. The overexpression of ADA for enhanced cell therapy is a safe, straightforward, reproducible genetic modification that can be utilized in current CAR T cell constructs to result in an armored CAR T product with superior therapeutic potential.

Abstract A21: Digital vivarium cloud platform facilitates nonclinical endpoint assessment in an ovarian carcinoma xenograft model with ascites

Abstract Background: Successful translation of nonclinical data is reliant upon the validity of measures for assessing disease morbidity and endpoint in mouse oncology models. Conventional means of endpoint evaluation (i.e., tumor weight, volume of ascites, or clinical signs) can be subjective and unreliable. Here we demonstrate continuous automated measurement of motion reliably predicts endpoint; is correlative with efficacy in an ovarian cancer xenograft model with ascites; and can be used to improve adherence to the three Rs, the guiding principles utilized to ensure animal welfare when conducting nonclinical research. Methods: Six groups of athymic mice were intraperitoneally inoculated with increasing numbers of ES-2 ovarian cancer cells or control (PBS) and housed in a homecage on a continuous monitoring platform. Leveraging the platform’s videographic and electronic records, we retrospectively analyzed night-time motion data. Based on these analyses, we established a Motion Threshold for noticeable decline in the motion metric. Relative to the Motion Threshold, the following parameters were defined: motion loss post induction (MLPI) and motion loss from endpoint (MLFE). These were in turn analyzed for their ability to predict endpoint in comparison to conventional parameters. Upon validation, the Motion Threshold was applied to a subsequent study to analyze its ability to predict efficacy. Results: In the first study, MLPI showed significant correlation with endpoint when graphed on a linear regression plot (R2 = 0.87; p < 0.0001), while conventional metrics showed low correlations with endpoint: number of tumor nodules (R2 = 0.1334; p = 0.1133), volume of ascites (R2 = 0.2524; p = 0.0240), tumor weight (R2 = 0.1162; p = 0.1413). Additionally, we found the MLFE predicted endpoint earlier than the first onset of clinical signs (p < 0.0001). A subsequent study was performed utilizing the Motion Threshold to analyze the efficacy of cisplatin in combination with OS2966 versus vehicle or both therapies alone. OS2966 + cisplatin significantly extended normal activity of mice (according to the Motion Threshold) compared to vehicle (p = 0.003). The Motion Threshold was also shown to correlate with standard survival curve (R2 = 0.86; p < 0.0001) and was superior to conventional parameters for determining intergroup differences. Conclusion: The results demonstrate the high sensitivity, reproducibility, and objectivity of continuous collection of the motion metric. These results are relevant as they enhance study interpretation and reduce time to humane endpoint. The use of digital metrics also increases statistical power, which can be used to potentially reduce the number of animals required for cancer research, hence facilitating faithfulness to the three Rs. Citation Format: Chibueze D. Nwagwu, Erwin Defensor, Anne-Marie Carbonell, Shawn Carbonell. Digital vivarium cloud platform facilitates nonclinical endpoint assessment in an ovarian carcinoma xenograft model with ascites [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A21.

Abstract C095: Antitumor activity of STRO-002, a novel anti-folate receptor-α (FRα) antibody drug conjugate (ADC), in patient-derived xenograft (PDX) models and preliminary phase I dose escalation safety outcomes in patients with ovarian carcinoma (OC)

Abstract Introduction: FRα is overexpressed in 80% of OC and endometrial carcinoma (EC), with minimal expression in normal tissues. STRO-002 is a novel FRα-targeting ADC designed to circumvent limitations of current ADCs. STRO-002 was generated with Sutro’s cell-free antibody production system (XpressCF™) and site-specific conjugation (XpressCF+™) platform, which uses the non-natural amino acid pAMF. STRO-002 has a drug-antibody ratio (DAR) of 4 and contains the proprietary tubulin-targeting 3-aminophenyl hemiasterlin warhead SC209, a potent cytotoxin that is rapidly cleared and a weak substrate for efflux pumps. STRO-002 demonstrated potent cytotoxic activity in multiple FRα-positive tumor cell lines and significant therapeutic efficacy in OC xenograft models. Toxicology studies demonstrate dose-dependent and reversible neutropenia when STRO-002 is administered to cynomolgus monkeys. No ocular toxicity was observed. Herein we report additional preclinical efficacy in PDX models and preliminary safety experience of STRO-002 in patients (pts) with OC. PDX model methods: FRα expression was evaluated in EC PDX models by IHC. Single agent efficacy of 10 mg/kg STRO-002 was assessed in models with negative, low (+), medium (++) and high (+++) FRα expression. Phase 1 trial methods: STRO-002-GM1 is a first-in-human Phase I, open-label, multicenter, dose escalation (Part 1) study with dose expansion (Part 2) to identify the maximum tolerated dose (MTD), and recommended Phase II dose (RP2D) in pts with advanced, relapsed or refractory OC and EC. STRO-002 is given IV over an hour on Day 1 of each 21-day cycle until disease progression. Results: Statistically significant efficacy was observed in 60% of the FRα-positive PDX models tested, with STRO-002 response appearing to positively correlate with FRα expression levels. Though high FRα PDX models showed the highest response rates, some models with low and medium FRα also exhibited good responses. 5 pts with OC have completed at least one cycle (21 days) of STRO-002 at 3 dose levels: 0.5, 1.0, and 1.8 mg/kg and are evaluable for safety and toxicity. Median age is 63 (r, 52-64). Median ECOG performance status is 0 (r, 0-1). Median # of prior therapies is 6 (r, 2-8). Four pts have received PARP inhibitors. Median number of STRO-002 doses administered is 2 (r, 1-5). 3 additional pts have been treated at the 2.9 mg/kg dose level and continue in the first cycle. No grade ≥ 3 treatment-related adverse events (TRAEs) have been observed. 93% of AEs are grade 1 or 2. The most common TRAEs in ≥20 % of pts includes nausea, vomiting, abdominal pain, fatigue and insomnia. Four grade 3 events occurred in 2 pts after completion of the first cycle and were related to disease progression, including small intestine obstruction, gross hematuria, dehydration and vomiting. 2 pts have discontinued due to disease progression. 6 pts remain on treatment and dose escalation is ongoing. Conclusions: STRO-002 is the first ADC generated with cell-free protein synthesis technology and site-specific conjugation of a novel hemiasterlin linker-warhead to be tested in pts with solid tumors. The potent anti-tumor activity of STRO-002 in PDX models further supports the clinical development of this agent. The preliminary safety profile in 5 OC pts treated with STRO-002 is encouraging. MTD has not been reached, no DLTs have been observed and dose escalation is continuing. Updated results will be presented. This study is registered with clinicaltrials.gov identifier NCT03748186. Citation Format: Denise Uyar, Russell J Schilder, R Wendel Naumann, Fadi S Braiteh, Erika Hamilton, Sami Diab, John Moroney, Richard T Penson, Jennifer Smith, Cristina Abrahams, Michael Palumbo, Venita DeAlmeida, Shannon Matheny, Arturo Molina. Antitumor activity of STRO-002, a novel anti-folate receptor-α (FRα) antibody drug conjugate (ADC), in patient-derived xenograft (PDX) models and preliminary phase I dose escalation safety outcomes in patients with ovarian carcinoma (OC) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C095. doi:10.1158/1535-7163.TARG-19-C095

FTY720 enhances the anti-tumor activity of carboplatin and tamoxifen in a patient-derived xenograft model of ovarian cancer

Ovarian cancer is the fifth leading cause of cancer-related deaths among women in the United States. Although most patients respond to frontline therapy, virtually all patients relapse with chemoresistant disease. This study addresses the hypothesis that carboplatin or tamoxifen + FTY720, a sphingosine analogue, will minimize or circumvent drug-resistance in ovarian cancer cells and tumor models.In vitro data demonstrate that FTY720 sensitized two drug-resistant (A2780. cp20, HeyA8. MDR) and two high-grade serous ovarian cancer cell lines (COV362, CAOV3) to carboplatin, a standard of care for patients with ovarian cancer, and to the selective estrogen receptor modulator tamoxifen. FTY720 + tamoxifen was synergistic in vitro, and combinations of FTY720 + carboplatin or + tamoxifen were more effective than each single agent in a patient-derived xenograft model of ovarian carcinoma. FTY720 + tamoxifen arrested tumor growth. FTY720 + carboplatin induced tumor regressions, with tumor volumes reduced by ∼86% compared to initial tumor volumes. Anti-tumor efficacy was concomitant with increases in intracellular proapoptotic lipid ceramide. The data suggest that FTY720 + tamoxifen or carboplatin may be effective in treating ovarian tumors.

Abstract 5625: Myeloid derived suppressor cells (MDSCs) express Sialyl Tn (STn) and are a therapeutic target for anti-STn antibody drug conjugates

Abstract Introduction: Myeloid derived suppressor cells (MDSCs) are functionally defined by their capacity to suppress T cell immunity; therefore inhibiting these cells is of great interest for immuno-oncology applications. STn, the sialylated version of the carbohydrate Tn antigen, is broadly expressed in human cancers such as breast, ovarian, bladder, cervical, colon, and lung and is rarely expressed in normal adult human tissues. The presence of STn in tumors is associated with metastatic disease, poor prognosis, and reduced overall survival. Tumor STn expression is well established and can be leveraged for targeted cancer therapy, however its expression on immuno-suppressive tumor infiltrating lymphocytes such as MDSCs has not been established. Methods: We generated humanized anti-STn mAbs having no cross-reactivity to the asialylated form of STn (Tn) or other glycan antigens. The epitope targeted by these mAbs is the STn glycan itself, not a particular glycopeptide or carrier protein, which offers the broadest potential to bind to multiple STn-glycosylated proteins on cell surfaces. We used these mAbs to quantify specifically the expression of tumor and MDSC STn expression across a set of patient tumor samples. We further treated mice with a humanized anti-STn-MMAE antibody-drug conjugate to evaluate targeted depletion of STn+ MDSCs. In order to evaluate the therapeutic potential of targeting this MDSC phenotype, we further evaluated the immunosuppressive environment of tumors containing STn+ and STn- MDSCs. Results: For the first time, we report the detection of STn on the surface of MDSCs in a growing body of patient tumor samples. Our data in patients as well as xenograft models additionally links STn expression on MDSCs to tumor cell STn expression. We have also demonstrated depletion of STn+ MDSCs after treatment with an anti-STn antibody-drug conjugate in an ovarian carcinoma xenograft model. Conclusions: Our data demonstrated that STn provides a uniquely glycan-specific and potent target for treatment of solid tumors. Anti-STn therapeutics offer the potential to go beyond tumor targeting to also directly target and deplete immune-suppressive MDSCs, thus fostering immune re-engagement and increasing the potential for better patient outcomes. Citation Format: David A. Eavarone, Patricia E. Rao, Jillian M. Prendergast, Adam D. Curtis, Rong Zhang, Lindsay S. Shopland, Jenna N. Stein, Jeff Behrens, Daniel T. Dransfield. Myeloid derived suppressor cells (MDSCs) express Sialyl Tn (STn) and are a therapeutic target for anti-STn antibody drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5625.

Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand from a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen.

Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography.

A novel far-red fluorescent xenograft model of ovarian carcinoma for preclinical evaluation of HER2-targeted immunotoxins.

We have created a novel fluorescent model of a human ovarian carcinoma xenograft overexpressing receptor HER2, a promising molecular target of solid tumors. The model is based on a newly generated SKOV-kat cell line stably expressing far-red fluorescent protein Katushka. Katushka is most suitable for the in vivo imaging due to an optimal combination of high brightness and emission in the "window of tissue transparency". The relevance of the fluorescent model for the in vivo monitoring of tumor growth and response to treatment was demonstrated using a newly created HER2-targeted recombinant immunotoxin based on the 4D5scFv antibody and a fragment of the Pseudomonas exotoxin A.

Abstract 666: Activity of the MEK inhibitor Binimetinib (MEK162) in combination with paclitaxel in patient-derived xenograft models of high-grade serous ovarian carcinoma

Abstract High-grade serous ovarian carcinoma (HGSOC) has the poorest prognosis amongst all gynecological cancers with a median survival of approximately 5 years. While most patients (pts) initially respond to therapy, few achieve long-term remission or cure; thus, new treatment strategies are needed. Paclitaxel (Pac), an agent commonly used in the treatment of HGSOC, induces the Ras/Raf/MEK/ERK pathway and resistance may be partially mediated through activation of MEK/ERK, suggesting a rationale for combining a MEK inhibitor with Pac. Binimetinib (MEK162) is an oral, potent, selective, allosteric small-molecule MEK1/2 inhibitor. The combination of binimetinib and Pac was studied in a panel of in vivo ovarian carcinoma xenograft models derived from either cell lines (A2780, SK-OV-3) or primary patient tumor resections pathologically confirmed to be HGSOC (OVC38B1, OVC604, OVC629, OVC104) and propagated solely in mice (pt-derived xenograft [PDX]). Three of these PDX models were from newly diagnosed pts, while 1 (OVC38B1) was from a pt who had relapsed following a prior response to a platinum/taxane regimen. All models were sequenced for cancer-specific genes by next-generation sequencing; no mutations were found in Ras, Raf, MEK or ERK. To evaluate single-agent and combination activity, tumor-bearing mice received 25 mg/kg Pac IP (days 1,5,9) and/or 30 mg/kg binimetinib PO (days 2-15). Single-agent binimetinib was modestly effective in 3/6 models (tumor growth inhibition [TGI] ≥ 50%), which is consistent with the lack of MAPK pathway mutations in these models. Single-agent Pac was highly effective in 3/6 models (TGI ≥ 90%) including OVC38B1 and 2 PDX models from newly diagnosed pts; the remaining models were taxane-resistant. This is consistent with expected high rates of response in the setting of newly diagnosed pts and/or prior responders. Binimetinib plus Pac had superior efficacy to either agent alone in 4/6 models including all taxane-resistant models. In 2 PDX models from newly diagnosed pts, combination activity was not improved, but Pac was highly effective as a single agent (72-82% regression) leaving little room for enhancement. Encouragingly, the combination induced tumor regressions in 2 taxane-resistant models in which no single-agent regressions were observed. In conclusion, in vivo drug sensitivity studies with binimetinib and Pac recapitulate the response expected in HGSOC based on stage, genetic background and clinical history. In models of taxane-resistant disease, combination with binimetinib had improved activity over taxane alone warranting further study of this approach in HGSOC. Citation Format: Shannon L. Winski, Karyn Bouhana, Susan Rhodes, LouAnn Cable, Deborah Anderson, Lance Williams, Brian Tunquest, Tiffany Logan, Guy Vigers, Patrice Lee. Activity of the MEK inhibitor Binimetinib (MEK162) in combination with paclitaxel in patient-derived xenograft models of high-grade serous ovarian carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 666. doi:10.1158/1538-7445.AM2015-666

Abstract 3916: HSP90 mediates tumor-associated matrix metalloproteinase 2 and Cathepsin L protease activities in ovarian carcinoma

Abstract Ovarian cancer is the fifth leading cause of cancer-related deaths among women in the United States. At diagnosis, disease has commonly spread beyond the ovary. Ovarian cancer cells dislodge from primary tumor and spread locally throughout the abdominal cavity and lymphatics. Cancer cell shedding and colonization at other sites involves the degradation of extra cellular matrix (ECM) by tumor-associated proteases, such as matrix metalloproteinase (MMPs) and cathepsin proteases. Heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone that interacts with and stabilizes MMPs in some solid tumors. Whether HSP90 regulates cathepsin activity is currently unknown. To determine the role of HSP90 on tumor-associated MMP and cathepsin protease activities in ovarian carcinoma, HSP90 activity was inhibited using pharmacologic and RNA interference (RNAi) approaches. A small molecule inhibitor, ganetespib, and two HSP90 targeting siRNAs were used to determine the effects of HSP90 inhibition on ovarian carcinoma cell line invasion using transwell and spheroid invasion assays. The effects of ganetespib or HSP90 targeting siRNA treatment on MMP and cathepsin protease activities were assayed by gelatin zymography (MMPs), enzyme assay (cathepsins) and by dynamic imaging using protease cleavable imaging probes. The effects of ganetespib treatment on in vivo orthotopic tumor growth, dissemination and tumor associated protease activities were determined using human ovarian carcinoma xenograft models. In vitro, ganetespib treatment or expression of HSP90-targeted siRNAs inhibited transwell and spheroid invasion, and MMP-2 and cathepsin L protease activities. In vivo, ganetespib treatment resulted in decreased growth of primary tumors and disseminated tumor nodules and decreased tumor-associated MMP and cathepsin activities. Importantly, significant inhibition of MMP and cathepsin activities was detected prior to differences in tumor volume, demonstrating rapid functional response to the drug. These results show that HSP90 regulates ovarian carcinoma-associated proteolytic activities that are essential to tumor progression and dissemination. They also point to the potential utility of HSP90 inhibition as a therapeutic strategy for the treatment of ovarian cancer. Citation Format: Shane W. O'Brien, Fang Xiao, Marsia A. Maglaty, Joshua S. Trinadad, Lainie P. Martin, David A. Proia, Denise C. Connolly. HSP90 mediates tumor-associated matrix metalloproteinase 2 and Cathepsin L protease activities in ovarian carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3916. doi:10.1158/1538-7445.AM2014-3916

First In-Mouse Development and Application of a Surgically Relevant Xenograft Model of Ovarian Carcinoma

PurposePreclinical models of epithelial ovarian cancer have not been exploited to evaluate the clinical standard combination therapy of surgical debulking with follow-up chemotherapy. As surgery is critical to patient survival, here we establish a combined surgical/chemotherapy xenograft model of epithelial ovarian cancer and demonstrate its translational relevance.Experimental DesignSKOV-3luc+ ovary cancer cells were injected topically into the ovaries of immunodeficient mice. Disease development and effect of clinical standard treatment including hysterectomy, bilateral salpingoophorectomy and removal of metastasis with follow up chemotherapy (carboplatin 12 mg/kg + paclitaxel 15 mg/kg) was evaluated by clinical parameters. Tumor burden was quantified by bioluminescence imaging (BLI).ResultsThe xenograft ovarian tumors developed were poorly differentiated and multicystic and the disease disseminated into the peritoneal cavity. When compared to the controls with a mean survival time of 4.9 weeks, mice treated with surgery and chemotherapy, surgery or chemotherapy demonstrated significantly improved mean survival of 16.1 weeks (p = 0.0008), 12.7 weeks (p = 0.0008), or 10.4 weeks (p = 0.008), respectively.ConclusionCombined surgical intervention and adjuvant chemotherapy was demonstrated for the first time in an orthotopic xenograft model of ovarian cancer. Similar to observation in human studies the combined approach resulted in the longest medial survival time, advocating application of this strategy in future preclinical therapeutic development for this disease.

VEGF-D-induced draining lymphatic enlargement and tumor lymphangiogenesis promote lymph node metastasis in a xenograft model of ovarian carcinoma

BackgroundVascular endothelial growth factor (VEGF)-D has been shown to promote lymph node metastasis in several cancers. Although generally overexpressed in ovarian carcinoma, its role in nodal dissemination of this cancer is unclear. To clarify the role of VEGF-D and the underlying molecular mechanisms, we investigated the function of VEGF-D using a mouse xenograft model of ovarian cancer.MethodsHuman ovarian serous adenocarcinoma SKOV3 cells were transfected with VEGF-D recombinant plasmid DNA, or with control vectors. The cells were injected subcutaneously into the footpads of nude mice. Tumor growth was evaluated weekly. Draining lymphatics were observed grossly with Evan’s blue lymphangiography. Tumoral lymphatics were delineated with both Evan’s blue and LYVE-1 immunostaining. Tumor metastases to lymph nodes were evaluated by H&E and CA125/CD40 staining. Expression of VEGF-D in primary tumors and levels of CA125 in involved lymph nodes were examined by immunohistochemistry. Tumor cell apoptosis was analyzed by Hoechst dyeing.ResultsMice bearing VEGF-D overexpressing xenografts showed a significantly higher rate of lymph node metastasis and markedly greater tumor volume compared with the controls. The functional lymphatic vessels were denser and enlarged in marginal and central tumor portions. Additionally, higher CA125 expression was observed in the involved lymph nodes. Mice bearing VEGF-D overexpressing xenografts also exhibited a markedly lower apoptotic index compared with the controls.ConclusionsOur data demonstrate the important role of VEGF-D in promoting lymph node metastasis by increasing tumor lymphangiogenesis, stimulating draining lymphatic vessel formation, and enhancing tumor invasiveness. Our findings show that VEGF-D can be a promising therapeutic target for ovarian cancer.

Vascular Endothelial Growth Factor C Promotes Ovarian Carcinoma Progression through Paracrine and Autocrine Mechanisms

Vascular endothelial growth factor C (VEGFC) has been reported to promote tumor progression in several tumor types, mainly through the stimulation of lymphangiogenesis and lymphatic metastasis. However, the expression and biological significance of the VEGFC/VEGF receptor (VEGFR)-3 pathway in ovarian cancer growth and dissemination are unclear, and have been investigated in this study. Soluble VEGFC was detected in the plasma and ascites of patients with ovarian carcinoma, and VEGFR3 expression was found in their tumor tissues. In human ovarian carcinoma xenograft models, high levels of soluble VEGFC in ascites and serum were detected, in association with disease progression, tumor burden, and volume of ascites. Peak VEGFC expression preceded para-aortic lymph node infiltration by HOC8 neoplastic cells. Histological detection of tumor cells in blood and lymphatic vessels indicated both hematogenous and lymphatic dissemination. Overexpression of VEGFC in the VEGFR3-positive and luciferase-expressing IGROV1 cells promoted carcinoma dissemination after orthotopic transplantation in the ovary of immunodeficient mice. In vitro, VEGFC released by the tumor cells stimulated tumor cell migration in an autocrine manner. Cediranib, an inhibitor of VEGFR1-3 and c-kit, inhibited in vivo metastasis of VEGFC-overexpressing IGROV1 and in vitro autocrine effects. These findings suggest that the VEGFC/VEGFR3 pathway acts as an enhancer of ovarian cancer progression through autocrine and paracrine mechanisms, hence offering a potential target for therapy.

A novel antibody-like TCRγδ-Ig fusion protein exhibits antitumor activity against human ovarian carcinoma

TCRγ9δ2(OT3) is a tumor-specific TCR with an unique complementarity-determining region 3 (CDR3) sequence, referred to as OT3, in its δ2 chain. This region was identified in tumor-infiltrating lymphocytes (TILs) from human ovarian epithelial carcinoma. We demonstrated that TCRγ9δ2(OT3)-Fc, a fusion protein composed of the complete extracellular domains of the γ9 and δ2 chains linked to the Fc domains of human IgG1, exhibited successful binding to multiple human carcinoma cell lines. In vitro, TCRγ9δ2(OT3)-Fc mediated cell killing via antibody-dependent cellular cytotoxicity (ADCC) in a dose-dependent manner. In vivo, TCRγ9δ2(OT3)-Fc significantly inhibited tumor growth and enhanced survival in human ovarian carcinoma xenograft models. Our findings suggest that the TCRγ9δ2(OT3)-Fc fusion protein possesses both the antigen-recognition properties of TCR γδ and the Fc-mediated effector functions of the antibody.

Avirulent Toxoplasma gondii Generates Therapeutic Antitumor Immunity by Reversing Immunosuppression in the Ovarian Cancer Microenvironment

Reversing tumor-associated immunosuppression seems necessary to stimulate effective therapeutic immunity against lethal epithelial tumors. Here, we show this goal can be addressed using cps, an avirulent, nonreplicating uracil auxotroph strain of the parasite Toxoplasma gondii (T. gondii), which preferentially invades immunosuppressive CD11c(+) antigen-presenting cells in the ovarian carcinoma microenvironment. Tumor-associated CD11c(+) cells invaded by cps were converted to immunostimulatory phenotypes, which expressed increased levels of the T-cell receptor costimulatory molecules CD80 and CD86. In response to cps treatment of the immunosuppressive ovarian tumor environment, CD11c(+) cells regained the ability to efficiently cross-present antigen and prime CD8(+) T-cell responses. Correspondingly, cps treatment markedly increased tumor antigen-specific responses by CD8(+) T cells. Adoptive transfer experiments showed that these antitumor T-cell responses were effective in suppressing solid tumor development. Indeed, intraperitoneal cps treatment triggered rejection of established ID8-VegfA tumors, an aggressive xenograft model of ovarian carcinoma, also conferring a survival benefit in a related aggressive model (ID8-Defb29/Vegf-A). The therapeutic benefit of cps treatment relied on expression of IL-12, but it was unexpectedly independent of MyD88 signaling as well as immune experience with T. gondii. Taken together, our results establish that cps preferentially invades tumor-associated antigen-presenting cells and restores their ability to trigger potent antitumor CD8(+) T-cell responses. Immunochemotherapeutic applications of cps might be broadly useful to reawaken natural immunity in the highly immunosuppressive microenvironment of most solid tumors.

Effects of Cyclooxygenase Inhibitors in Combination with Taxol on Expression of Cyclin D1 and Ki-67 in a Xenograft Model of Ovarian Carcinoma

The present study was designed to investigate the effects of cyclooxygenase (COX) inhibitors in combination with taxol on the expression of cyclin D1 and Ki-67 in human ovarian SKOV-3 carcinoma cells xenograft-bearing mice. The animals were treated with 100 mg/kg celecoxib (a COX-2 selective inhibitor) alone, 3 mg/kg SC-560 (a COX-1 selective inhibitor) alone by gavage twice a day, 20 mg/kg taxol alone by intraperitoneally (i.p.) once a week, or celecoxib/taxol, SC-560/celecoxib, SC-560/taxol or SC-560/celecoxib/taxol, for three weeks. To test the mechanism of the combination treatment, the index of cell proliferation and expression of cyclin D1 in tumor tissues were determined by immunohistochemistry. The mean tumor volume in the treated groups was significantly lower than control (p < 0.05), and in the three-drug combination group, tumor volume was reduced by 58.27% (p < 0.01); downregulated cell proliferation and cyclin D1 expression were statistically significant compared with those of the control group (both p < 0.01). This study suggests that the effects of COX selective inhibitors on the growth of tumors and decreased cell proliferation in a SKOV-3 cells mouse xenograft model were similar to taxol. The three-drug combination showing a better decreasing tendency in growth-inhibitory effect during the experiment may have been caused by suppressing cyclin D1 expression.

Cisplatin plus paclitaxel and maintenance of bevacizumab on tumour progression, dissemination, and survival of ovarian carcinoma xenograft models

Background:Bevacizumab is being incorporated as first-line therapy with standard-of-care chemotherapy on epithelial ovarian carcinoma (EOC). We investigated bevacizumab combined with chemotherapy on tumour progression and mouse survival in EOC xenograft models.Methods:Bevacizumab was administered concomitantly with cisplatin plus paclitaxel (DDP+PTX), continued after induction (maintenance) or started after chemotherapy. The effect on tumour progression was monitored by bioluminescence imaging (BLI) (1A9-luc xenograft). Tumour dissemination into the peritoneal organs and ascites formation (HOC22 xenograft) was evaluated by histological analysis at the end of treatment (interim) and at euthanasia (survival). The effects on overall survival (OS) were investigated in both EOC models.Results:Bevacizumab with PTX+DDP delayed tumour progression in mice bearing EOC xenografts. OS was significantly extended, with complete responses, by bevacizumab continued after stopping chemotherapy in the HOC22 xenograft. Bevacizumab alone inhibited ascites formation, with only limited effect on tumour burden, but combined with PTX+DDP reduced ascites and metastases. Bevacizumab started after induction with PTX+DDP and maintained was equally effective on tumour progression and survival on 1A9-luc xenograft.Conclusion:Bevacizumab combined with chemotherapy not only affected tumour progression, but when administered as maintenance regimen significantly prolonged survival, reducing ascites, and tumour dissemination. We believe our findings are consistent with the clinical results and shed light on the potential effects of this kind of treatment on tumour progression.

Abstract 2756: Combination therapy of doxorubicin and the acid-sensitive albumin-binding prodrug of doxorubicin INNO-206 induces complete regressions in a xenograft pancreatic carcinoma model demonstrating excellent tolerability

Abstract Combination therapy of macromolecular prodrugs with clinically established low-molecular weight anticancer drugs is a field of research that has been insufficiently explored to date. Recently, we investigated the anticancer effect of a combination of doxorubicin with the clinically assessed albumin-binding prodrug INNO-206, the (6-maleimidocaproyl)hydrazone of doxorubicin, in an ovarian carcinoma xenograft model (A2780). The combination achieved complete remissions as did therapy with INNO-206 alone, but was far better tolerated. Due to these encouraging results, we extended our combination trials to a more chemoresistant tumor indication, i.e. pancreatic cancer. Thus, we compared the following weekly schedules against the MiaPaca-2 xenograft model (i.v. administration): 3 x 6 mg doxorubicin (MTD), 3 x 24 mg/kg INNO-206 (doxorubicin equivalents, MTD), 3 x 3 mg/kg doxorubicin followed 6 h later by 3 x 12 mg/kg INNO-206, and 3 x 12 mg/kg INNO-206 followed 6 h later by 3 x 3 mg/kg doxorubicin. Whereas therapy with doxorubicin only produced a moderate tumor inhibition, all other therapy arms induced complete long-term remissions until the end of the experiment on day 44. However, there were significant differences in the tolerability as assessed by the body weight changes: whereas therapy at the MTD of INNO-206 (3 x 24 mg/kg) produced a body weight loss of −16 %, therapy with 3 x 12 mg/kg INNO-206 followed 6 h later by 3 x 3 mg/kg doxorubicin produced −7 % body weight loss, but 3 x 3 mg/kg doxorubicin followed 6 h later by 3 x 12 mg/kg INNO-206 produced a body weight gain of + 2 % as a clear indication of minimal systemic toxicity. This animal study is to best of our knowledge the first investigation demonstrating that a combination of an albumin-binding prodrug with its free parent drug not only has clear therapeutic advantages over the free drug but that the time-dependent schedule has a critical influence on the overall tolerability and results in its best case in a 100 % cure rate without causing any body weight loss. The current working hypothesis to explain this surprising effect is that the combination of doxorubicin with an albumin-binding prodrug of doxorubicin with acid-sensitive properties enables a more homogenous distribution of the drugs in the tumor due to different uptake mechanisms in a heterogeneous solid tumor (EPR effect for INNO-206 and free diffusion into the tumor interstitium for doxorubicin). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2756. doi:1538-7445.AM2012-2756

Differential Effect of Intraperitoneal Albendazole and Paclitaxel on Ascites Formation and Expression of Vascular Endothelial Growth Factor in Ovarian Cancer Cell-Bearing Athymic Nude Mice

The purposes of our study were to evaluate the effect of intraperitoneal albendazole on tumor growth, ascites formation, and vascular endothelial growth factor (VEGF) mRNA expression, and to assess the synergistic effect of paclitaxel in OVCAR-3-bearing nude mice. In all, 4 groups of mice were injected intraperitoneally with weekly albendazole (450 mg/kg per week), paclitaxel (30 mg/kg per week), albendazole plus paclitaxel, or normal saline for 4 weeks. Ascitic fluid accumulation (2.47, 2.65, 2.88, and 5.90 mL, respectively) and in ascitic VEGF levels were significantly reduced in the 3 treatment groups compared to the control group (170.83, 229.16, 267, and 1625 pg/mL, respectively). However, complete tumor suppression was more prominent in the paclitaxel group, and VEGF mRNA expression was more strongly inhibited in the albendazole group (P < .05). No synergistic effect of albendazole and paclitaxel was observed. We demonstrated a differential effect of albendazole and paclitaxel in a xenograft model of ovarian carcinoma; albendazole suppressed ascites formation by inhibiting VEGF secretion, and paclitaxel exerted its effects by direct cytotoxicity.

Non-invasive monitoring of hypoxia-inducible factor activation by optical imaging during antiangiogenic treatment in a xenograft model of ovarian carcinoma

Targeting the hypoxia response pathway and angiogenesis are two promising therapeutic strategies for cancer treatment. Their use as single strategies has important limitations. Thus, development of combined regimens has become an important step toward improving therapeutic efficacy. Also, non-invasive monitoring of the response to targeted biological therapies, as well as determination of the optimal schedule for combination regimens has become an active field of research over the last five years, with relevance for both preclinical and clinical settings. Here, we used an optical imaging method to non-invasively monitor the functional changes in HIF activity in response to antiangiogenic treatment in a xenograft model of human ovarian carcinoma. A bioluminescent reporter construct containing nine copies of the hypoxia response element upstream of the luciferase gene (9xHRE-luciferase) was characterized in vitro in a panel of tumor cell lines and in vivo in a subcutaneous xenograft model of ovarian carcinoma by means of optical imaging. We showed that in OVCAR-3 subcutaneous xenografts, the most abrupt change in the HIF functional reporter occurs before the onset of massive tumor growth. However, this system failed to detect hypoxia induced upon antiangiogenic treatment due to the compensating effects of increased hypoxia and decreased tumor cell viability caused by imbalanced neovascularization vs. tumor expansion. Therefore, the readout based on HIF functional reporter could be conditioned by the dynamics of tumor growth and angiogenesis, which is highly variable depending on the tumor type, tumor model and stage of progression.