Immunocompetent C57BL/6 syngeneic mouse ovarian cancer models with defined genetic alterations

Background: Ovarian cancer is the most lethal gynecologic malignancy. While women with BRCA-deficient tumors show sensitivity to PARP inhibitors (PARPi), new treatment options are needed for PARPi-resistant tumors. An emerging strategy to improve PARPi response is combination therapy with epigenetic drugs. A newly recognized epigenetic drug target in ovarian cancer is the bromodomain and extraterminal (BET) protein family. BET proteins such as BRD4 promote oncogenic transcription of progrowth and survival genes, including the established link between inflammation and cancer, nuclear factor-kappaB (NF-κB). A complementary strategy to targeting cancer cells with cytotoxic drugs is to activate normal immune processes in the tumor microenvironment (TME). In syngeneic mouse ovarian cancer models, we have shown that M2-like protumor macrophages are a prominent component of the TME, and that NF-κB inhibition reduces the M2 population. Thus, BET inhibitors (BETi) have the potential to induce transcriptional reprogramming in both tumors and macrophages for therapeutic benefit. Objective: To determine the cellular and molecular effects of combining BETi and PARPi in mouse ovarian cancer and peritoneal macrophage cell lines. Methods: Cultured wild-type and CRISPR-modified (TP53 and TP53/BRCA2 knockout) ID8 mouse ovarian cancer cells, and PMJ2-PC mouse peritoneal macrophages, were treated with vehicle, the PARPi olaparib, the first-in-class BETi JQ1 or the JQ1/olaparib combination for 24-72h. Sulforhodamine B (SRB) assays assessed cell growth. Immunofluorescence assays assessed adherent cell number, DNA damage (pH2AX) and cell cycle indices. Protein levels of pH2AX and the apoptosis marker cleaved PARP were assessed by Western blot. NF-κB activity was measured by luciferase assays of a transiently transfected reporter plasmid. Results: Combined JQ1 and olaparib treatment synergistically reduced cell growth in SRB assays in wild-type and TP53 knockout ID8 cells. TP53/BRCA2 knockout cells showed greater responses to PARPi alone and no synergism was observed. Consistent with these results, the JQ1/olaparib combination cooperatively reduced the number of adherent cells and cells in S phase, and increased the G0/G1 population, DNA damage and apoptosis. In contrast, the drug combination had minimal effects on DNA damage or apoptosis in PMJ2-PC macrophages, while NF-κB activity was reduced in both cancer cells and macrophages. Conclusions: BETi sensitize mouse ovarian cancer cells to the cytotoxic effects of PARPi. Combined drug treatment also has potential to inhibit NF-κB in both cancer cells and macrophages. Our novel immunomodulatory strategy will be tested in ID8 syngeneic ovarian cancer models. We believe BETi combination treatment could expand the use of PARPi in ovarian cancer patients, with the potential to benefit a substantial number of women with this devastating disease. Citation Format: Andrew J. Wilson, Alyssa Hoover, Whitney Harris, Esther Liu, Dineo Khabele, Fiona Yull. Bromodomain inhibition in ovarian cancer and the tumor microenvironment to improve PARP inhibitor response [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 626.

Survival of cancer patients with high-grade serous carcinoma of the ovary (HGSCs) remains less than 30% five years after diagnosis, despite initial chemosensitivity of most HGSCs. We hypothesized that a tumor antigen-targeted vaccine capable of eliciting a robust cell-mediated immune response may help prevent ovarian cancer. Mesothelin (meso) is overexpressed by ovarian, lung and pancreatic cancers and is 57% conserved at the protein level between mouse and human. To elicit cell-mediated immune responses directed against mesothelin, we combined a human mesothelin recombinant protein with various adjuvants including: aluminum hydroxide gel (alum, for Th2 immune response); TLR4 ligand (synthetic lipid MPL, for Th1 response); squalene-oil-in-water (AddaVaxTM, for both Th1 and Th2 responses); and STING (stimulator of interferon genes) ligand (CDN) that activates innate immunity and triggers type 1 IFN response and NF-κB-dependent cytokine secretion. We detected robust anti-meso antibody (Ab) responses after 1 prime and 2 boosts with meso/alum/MPL or meso/CDN/AddaVax. The highest and most stable Ab titers were obtained with CDN/AddaVax-based adjuvants. Four groups of female C57BL/6 mice were immunized with alum/MPL or CDN/AddaVax with or without meso (n = 12 per group). Six weeks after the prime immunization, cells from a luciferase-transduced syngeneic mouse ovarian cancer cell line, ID8 (Luc-ID8) were injected orthotopically in the left ovary of the mice. In vivo bioluminescent imaging (BLI) suggested a 92% take rate of tumors 3 weeks after ID8-Luc injections in mice immunized with meso/alum/MPL or adjuvants only. However, in the group immunized with meso/CDN/Addavax only 30% of the mice showed a detectable signal 3 weeks after ID8-Luc implantation. After 10 weeks the average BLI signal was still significantly lower than in all the other groups. Furthermore, the majority of mice immunized with alum/MPL +/- meso developed ascites by 12 weeks; no significant difference of peritoneal leukocyte numbers or phenotype was observed between the groups immunized with alum/MPL vs. meso/alum/MPL. In contrast, 7 out 11 mice immunized with meso/CDN/Addavax did not develop ascites through 14 weeks post tumor challenge; most of the tumors were unilateral, smaller, with more T and B cells present in the peritoneal lavages compared to the other groups. Immunizations with CDN/AddaVax or meso/CDN/AddaVax strongly increased the frequency of CD62L-CD44+ effector memory T cells, stimulated innate immunity with M1 polarization, profoundly decreased MDSC levels in peritoneal lavages, and correlated with a less aggressive tumor phenotype (EpCAM+ PD-L1-). H&E staining of the harvested tumors and fallopian tubes showed that tumors in animals immunized with meso/CDN/AddaVax were confined to the left ovary and/or fad pad for the majority of the animals as compared to metastatic disease for most animals of the other groups. In addition, tumor-infiltrating lymphocytes (TILs) were visible by H&E in animals immunized with meso/CDN/AddaVax. Further characterization is ongoing. These results support the hypothesis that a vaccine regimen combining mesothelin with CDN and Addavax can protect against aggressive forms of ovarian cancer. A similar vaccination study is in progress implementing a new model of inducible ovarian cancer in mice carrying floxed alleles of Dicer and PTEN. Prevention of ovarian cancer may be possible by vaccination against mesothelin tumor antigen in combination with adjuvants that stimulate type I IFN response. Citation Format: Nathalie Scholler, Paul Stein, Khushboo Sharma, Claire Repellin, Kalika Kamat, Travis Harrison, Robert H. Shoemarker, Shizuko Sei, Lidia Sambucetti. Preclinical development of a preventive vaccine against ovarian cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A041.

Background: Rucaparib (CO-338) is an oral small molecule inhibitor of poly(ADP-ribose) polymerase (PARP)-1, PARP-2 and PARP-3 that has shown clinical activity in patients with BRCA1 and BRCA2 mutated advanced ovarian cancer. Monoclonal antibodies against programmed death receptor-1 (PD-1) and programmed death-ligand (PD-L1) have also shown efficacy in advanced ovarian cancer patients. It has been reported that BRCA1 and BRCA2 mutated tumors have a higher mutational load and increased CD8+ T cell infiltration, suggesting that the combination of rucaparib and immune checkpoint inhibition may be complementary. However, PARP inhibition has also been reported to have an immunosuppressive effect in preclinical studies. Methods: Subcutaneous syngeneic models using the BRCA1 wild-type C2Km (P53-/-, myc, Kras-G12D, Akt-myr) and BRCA1 mutant BrKras (BRCA1-/-; P53-/-; myc; Kras-G12D; Akt-myr) murine ovarian cell lines were developed in the murine FVB/N background. Antibodies targeting PD-1 (RMP1-14) and PD-L1 (10F.9G2) were dosed by intraperitoneal injection at 5-10 mg/kg twice weekly, while rucaparib was administered by oral gavage at 150 mg/kg twice daily. Treatment was initiated at a tumor volume of ~150mm3 (n=15/group). Animals were dosed for 21 days, and tumors were allowed to regrow to day 76. Results: In vitro cytotoxicity assays demonstrated that rucaparib was 155-fold more potent in the BRCA1 deficient BrKras cell line (IC50 = 84 nM) than the isogenic BRCA1 wild-type C2Km cell line (IC50 = 13 μM). An in vivo study using the syngeneic BrKras model was performed in mice treated with: vehicle, rucaparib, PD-1, PD-L1, rucaparib+PD-1, and rucaparib+PD-L1. All monotherapy and combination groups resulted in significant tumor growth inhibition and were followed for survival analysis. The median survival time (MST) and % cures (defined as undetectable growth at Day 76 post-tumor implantation) for vehicle, PD-L1, PD-1 and rucaparib monotherapy treated animals was 34 days (0%), 41 days (13%), 76 days (40%) and >76 days (56%), respectively. The rucaparib+PD-1 and rucaparib+PD-L1 combination groups demonstrated greater efficacy than the monotherapies, with a MST of >76 days (100%) and >76 days (88%), respectively. Dose response and immune profiling studies are ongoing. In vivo studies were also performed in the BRCA1/2 wild-type models EMT-6, Pan02, and MC38. As expected, as a single agent, rucaparib showed limited activity in these homologous recombination competent models, whereas a range of tumor growth inhibition was observed with monotherapy PD-L1 treatment. No impact on anti-tumor activity was observed in animals treated with rucaparib+PD-L1 as compared to PD-L1 monotherapy in the BRCA1/2 wild-type syngeneic models examined. Conclusions: The combination of rucaparib with PD-1 and PD-L1 inhibition improved survival in a BRCA1 mutant syngeneic model. Citation Format: Liliane Robillard, Minh Nguyen, Andrea Loehr, Sandra Orsulic, Rebecca S. Kristeleit, Kevin Lin, Mitch Raponi, Thomas C. Harding, Andrew D. Simmons. Preclinical evaluation of the PARP inhibitor rucaparib in combination with PD-1 and PD-L1 inhibition in a syngeneic BRCA1 mutant ovarian cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3650. doi:10.1158/1538-7445.AM2017-3650

Introduction: High grade serous cancer (HGSC) is the most common and lethal subtype of epithelial ovarian cancer, with only ∼25% survival rates at 5 years post diagnosis. Transforming Growth Factor-beta (TGF-β) is an immunosuppressive cytokine found in high levels in ascites fluid that hinders the efficacy of PD-(L)1 immune checkpoint blockade. Specifically, TGF-β signaling promotes the recruitment and activation of immunosuppressive cells, including regulatory T cells (Tregs) and tumor-associated macrophages (TAMs). Here, we use single cell RNA sequencing (scRNAseq), macrophage depletion studies and ex vivo co-culture models to define molecular and cellular mechanisms of action of bintrafusp alfa (BA), a dual TGF-β/PD-L1 inhibitor, in ovarian cancer models. Methods: Murine HGSC syngeneic engraftment models were used to model metastatic disease using BR5-Luc (Trp53-/-:Brca1-/-:MycO/E) cells injected in female FVB mice, or ID8-Trp53-/-:Nf1-/--Luc cells injected in female C57BL/6 mice (i.p.). Mice were randomized between treatment groups: control IgG, anti-PD-L1, TGF-β Trap, or BA (treated in final week of endpoint studies). Some experiments included macrophage depletion using clodronate liposomes prior to treatments. Tumors and ascites were collected for cytokines and immunophenotyping. ScRNAseq was performed on dissociated ovarian tumors (WT Parse Evercode) and compared with publicly available human HGSC data. Interactions between HGSC, macrophages and other effector cells were visualized using ex vivo co-culture models using OT-1 T cells and ID8-Nf1 cells transduced with chicken ovalbumin antigen and a Granzyme B-cleavable forster resonance energy transfer (FRET) reporter. Results: In the BR5/FVB model, flow cytometry analysis of tumors from BA-treated mice showed significantly more CTLs expressing IFN-γ, CD8 Teff/memory cells, naive NK cells, and activated cytolytic NK cells expressing CD107a compared to controls. Also, distinct clusters of anti-tumor and proinflammatory immune populations were detected in the acites of BA-treated mice, especially upregulated M1 macrophages, activated CD69+ NK cells, and reduced Tregs. Differential immune transcriptome signatures and rare immune populations were explored using scRNAseq, and compared to publicly available human HGSC scRNAseq data. Flow cytometry on tumor & ascites showed a shift in immune balance and response in HGSC TIME when macrophages were depleted, compared to wildtype. Testing of BA in a HGSC/T cell co-culture model showed improved Granzyme B-mediated killing of HGSC cells compared to controls & delineated roles for macrophages, T cells and NK cells in BA immune response. Conclusion: These findings underscore the critical roles of T cells, NK cells, and macrophages in mediating the anti-tumor effects of BA in HGSC models. Translation of these findings should involve patient stratification for TGF-β-induced biomarkers to include in future trials of this new immunotherapy regime for ovarian cancer. Citation Format: Jacob LK Kment, Sai Agash Surendran, Stephanie Young, Ken Huang, Paul Kubes, Andrew W Craig. Single cell analyses of syngeneic ovarian cancer models implicates macrophage skewing as key to evoking anti-tumor immunity via coordinated TGF-β and PD-L1 blockade [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A116.

MUC16, a subunit of CA125 that is overexpressed on epithelial ovarian cancers, can be targeted by T cells via the incorporation of the 4h11m28m chimeric antigen receptor (CAR) into the T-cell surface. However, the microenvironment of ovarian cancers consists of regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages that act to immunosuppress CAR T-cell function, thus abrogating CAR T-cell efficacy. To overcome this immunosuppression, our lab has developed IL-12 and IL-18 secreting CAR T cells, which have shown enhanced efficacy in a syngeneic ovarian cancer model when compared to second-generation CAR T cells. While IL-18 secreting CAR T cells have proven to be superior to IL-12 secreting CAR T cells in various leukemia and lymphoma mouse models, this observation does not translate to the ovarian cancer model. Mechanistic studies into the effects of IL-18 and IL-12 in a syngeneic ovarian cancer model are currently under way in order to better understand what each cytokine is doing to the tumor microenvironment as well as to the CAR T cell. With better understanding of the effects of IL-18 and IL-12, we can contribute to the immunobiology of these cytokines as well as harness their efficacy to apply to other tumor models. Citation Format: Christina E. Bebernitz, Renier J. Brentjens. Comparing IL-12 and IL-18 secreting CAR T-cell efficacy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A31.

A growing body of literature supports the role of apolipoproteins present in HDL in the treatment of pro-inflammatory diseases including cancer. We examined whether bovine HDL (bHDL) and three dual-domain peptides, namely AEM-28 and its analog AEM-28–2, and HM-10/10, affect tumor growth and development in mouse models of ovarian and colon cancer. We demonstrate that bHDL inhibits mouse colorectal cancer cell line CT26-mediated lung tumor development, and mouse ovarian cancer cell line ID8-mediated tumor burden. We also demonstrate that, although to different degrees, dual-domain peptides inhibit cell viability of mouse and human ovarian and colon cancer cell lines, but not that of normal human colonic epithelial cells or NIH3T3 mouse fibroblasts. Dual-domain peptides administered subcutaneously or in a chow diet decrease CT26 cell-mediated tumor burden, tumor growth, and tumor dissemination in BALB/c mice. Plasma levels of lysophosphatidic acid (LPA) are significantly reduced in mice that received bHDL and the dual-domain peptides, suggesting that reduction by effecting accumulation and/or synthesis of pro-inflammatory lipids may be one of the mechanisms for the inhibition of tumor development by bHDL and the dual-domain peptides. Our studies suggest that therapeutics based on apolipoproteins present in HDL may be novel agents for the treatment of epithelial adenocarcinomas of the ovary and colon.

ObjectivesThe aim of this study is to evaluate the antitumor immune responses of mouse stem cell-derived dendritic cells (stem-DCs), corresponding to human CD141+ DCs, derived from bone marrow hematopoietic stem...

BackgroundNew treatment options for ovarian cancer are urgently required. Tumor-associated macrophages (TAMs) are an attractive target for therapy; repolarizing TAMs from M2 (pro-tumor) to M1 (anti-tumor) phenotypes represents an important therapeutic goal. We have previously shown that upregulated NF-kappaB (NF-κB) signaling in macrophages promotes M1 polarization, but effects in the context of ovarian cancer are unknown. Therefore, we aimed to investigate the therapeutic potential of increasing macrophage NF-κB activity in immunocompetent mouse models of ovarian cancer.MethodsWe have generated a transgenic mouse model, termed IKFM, which allows doxycycline-inducible overexpression of a constitutively active form of IKK2 (cIKK2) specifically within macrophages. The IKFM model was used to evaluate effects of increasing macrophage NF-κB activity in syngeneic murine TBR5 and ID8-Luc models of ovarian cancer in two temporal windows: 1) in established tumors, and 2) during tumor implantation and early tumor growth. Tumor weight, ascites volume, ascites supernatant and cells, and solid tumor were collected at sacrifice. Populations of macrophages and T cells within solid tumor and/or ascites were analyzed by immunofluorescent staining and qPCR, and soluble factors in ascitic fluid were analyzed by ELISA. Comparisons of control versus IKFM groups were performed by 2-tailed Mann-Whitney test, and a P-value < 0.05 was considered statistically significant.ResultsIncreased expression of the cIKK2 transgene in TAMs from IKFM mice was confirmed at the mRNA and protein levels. Tumors from IKFM mice, regardless of the timing of doxycycline (dox) administration, demonstrated greater necrosis and immune infiltration than control tumors. Analysis of IKFM ascites and tumors showed sustained shifts in macrophage populations away from the M2 and towards the anti-tumor M1 phenotype. There were also increased tumor-infiltrating CD3+/CD8+ T cells in IKFM mice, accompanied by higher levels of CXCL9, a T cell activating factor secreted by macrophages, in IKFM ascitic fluid.ConclusionsIn syngeneic ovarian cancer models, increased canonical NF-κB signaling in macrophages promoted anti-tumor TAM phenotypes and increased cytotoxic T cell infiltration, which was sufficient to limit tumor progression. This may present a novel translational approach for ovarian cancer treatment, with the potential to increase responses to T cell-directed therapy in future studies.

Src tyrosine kinase promotes survival and resistance to chemotherapeutics in a mouse ovarian cancer cell line

We have previously reported the tumor suppressor effect of SPARC in ovarian cancer through inhibiting cancer cell growth and their interactions within the peritoneal milieu. In the present study, we sought to determine the effect of SPARC on metabolic perturbations associated with ovarian cancer using a syngeneic murine ovarian cancer model in SPARC knockout (KO) mice and their wildtype (WT) counterparts. Murine ID8 ovarian cancer cell line were injected intraperitoneally in SPARC KO and WT mice. Matched intraperitoneal tumor tissue, and ascitic fluid were harvested 6 weeks post-injection of ID8 cells and were subjected to global biochemical metabolomic profiling. In tandem, WT and KO tumors were dissected by LCM-microdissected and were subjected to gene expression profiling. Metabolomic profiling of SPARC KO tumors and ascites revealed significantly altered metabolites associated with glucose, energy, amino acid, and lipid metabolism, as well as redox homeostasis. Integrated transcriptome and metabolome analysis of the top significantly upregulated genes and metabolites in the KO compared to WT tumors and ascitic fluid revealed that the major pathways upregulated in absence of SPARC are those involved in: conversion of glucose to acetyl CoA and entry into the citric acid (TCA) cycle, respiratory electron transport and oxidative phosphorylation (SUCLG2; NDUFAB1; ATP5F1; UQCRH; ATP5H; COX5B; NDUFV1; ATP5O; IDH3B/ succinate, fumarate and pyruvate), protein metabolism (EIF5A; PREB; EIF4H; DAD1; STT3A; RPL4; SUMO1; CCT4; CCT2; IGFBP5; HSPA5; EIF3A; EIF4B; RPS19; EIF3E/ mannose; N-acetylneuraminate; N-glycolylneuraminate; glutamate; spermidine), fatty acid beta oxidation (HADH; TPI1; LPL/glycerol), purine nucleotides de novo biosynthesis and salvage (ATP5F1; ATP5H; PAICS; ATP6V1G1/ aspartate; glycine; glutamate; fumarate). Together, these data provide novel insight on the molecular mechanisms of the SPARC-induced metabolic perturbation associated with ovarian cancer that can be translated into diagnostic and prognostic biomarkers of the disease. Citation Format: Sherine Taylor, Christian Sanchez, Amna Adrees, Hale Nur Ozbek, Harjapjit Sahni, Neveen A. Said. Global profiling of SPARC-regulated metabolic pathways in ovarian cancer. [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 1146. doi:10.1158/1538-7445.AM2015-1146

Simple SummaryWe developed a new labeled mouse cell line to study ovarian cancer. The STOSE mouse cell line was engineered to express a luciferase label to enable real-time tumor monitoring by imaging. We characterized the growth of the cell line in mice and identified the immune cells within the formed tumors. We treated the mice with immunotherapy, which had no effect on tumor growth. Overall, the STOSE.M1 luc model closely resembles ovarian cancer in humans and will further aid in our understanding and treatment of this disease.Despite advances in surgery and targeted therapies, the prognosis for women with high-grade serous ovarian cancer remains poor. Moreover, unlike other cancers, immunotherapy has minimally impacted outcomes in patients with ovarian cancer. Progress in this regard has been hindered by the lack of relevant syngeneic ovarian cancer models to study tumor immunity and evaluate immunotherapies. To address this problem, we developed a luciferase labeled murine model of high-grade serous ovarian cancer, STOSE.M1 luc. We defined its growth characteristics, immune cell repertoire, and response to anti PD-L1 immunotherapy. As with human ovarian cancer, we demonstrated that this model is poorly sensitive to immune checkpoint modulators. By developing the STOSE.M1 luc model, it will be possible to probe the mechanisms underlying resistance to immunotherapies and evaluate new therapeutic approaches to treat ovarian cancer.

Ovarian cancer is the most fatal gynecological cancer. Epithelial ovarian cancer (OvCa), the most common subtype, usually goes undetected until metastatic and often fatal stages of the disease. OvCa follows a unique form of metastasis, spreading through the peritoneal cavity and forming metastatic sites on the peritoneum. Epidemiologic data suggest that child-bearing, or parity, reduces a woman's risk of developing ovarian cancer, with more births providing greater protection. Despite the association of parity with a decreased incidence of ovarian cancer, very few studies have explored the relationship between parity and metastatic success. A recent study compared metastatic success to the omentum in 12-month-old C57Bl/6 retired breeders and 5-month-old virgin mice, reporting that parous mice are less susceptible to metastasis due to the parity-associated differences in the immune compositional profile in the omental fat band (Cohen et al. 2013). This tumor study compared mice of different ages and did not report specific numbers of pregnancies. To further investigate the role of parity number in OvCa metastasis, we designed a study that controls for age and compares mice with specific parity number. Three age-matched C57Bl/6 groups were evaluated: nulliparous (V), parous 1 (P1), and parous 3 (P3) mice. We tested the effect of parity on metastatic success in vivo with an allograft study using the C57Bl/6 syngeneic ID8 mouse ovarian surface epithelial cell line. ID8 ovarian cancer cells (106) were injected into the peritoneal cavity of V, P1 and P3 mice. In contrast to the results of Cohen at al. that utilized a different syngeneic ovarian cancer cell line, we found no significant difference in metastasis to the omentum in the parous animals, but significantly reduced metastasis to the fat-enveloped ovaries and visceral fat pads in the P3 mice. This suggests that the visceral fat adjacent to the uterus and ovaries in multi-parous animals is a unique environment, resilient to metastasis. Factors in fat tissue responsible for this phenomenon are being investigated. Citation Format: Elizabeth A. Loughran, Ryan Phan, Annemarie K. Leonard, Laura Tarwater, Marwa Asem, Yuliya Klymenko, Yueying Liu, Jing Yang, Jeff Johnson, Matthew Ravosa, M. Sharon Stack. The impact of parity on the metastatic success of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B67.

e17550 Background: With a prevalence of more than 2 million cases, a 5-year survival below 20% in stage 3 and 4, and limited treatment options, ovarian cancer represents a significant unmet medical need. We assessed the efficacy of AUP-55, a genetically engineered Lactococcus cremoris for the treatment of ovarian cancer and peritoneal carcinomatosis. We evaluated the efficacy of two AUP-55 leads producing human therapeutic proteins in situ; namely interleukin 18 (IL-18), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-12 (IL-12) and interferon alpha (IFN-A) in syngeneic intraperitoneal ID8 ovarian cancer and B16F1 melanoma models in mice. Methods: ID8-Luc-mCherry-Puro ovarian cancer model was used for survival and tumor burden by bioluminescent imaging with post-mortem pathology for all animals, whereas the B16F1 melanoma model was used solely for survival. Animals were treated daily or every other day with i.p injections of the AUP-55 leads (1e8 CFU/dose for 12-22 times). Results: In the ID8 model treatment was started 14 days after tumor implantation. Most untreated mice were terminated due to the disease progress. Treatment with AUP-55 resulted in 91.3 % survival and lower tumor load during the study duration of 81 days compared to the 0 % survival and massively increased tumor load in untreated mice. When compared to the available data from the CRO, the survival advantage was superior to, for example anti-PD1 and cisplatin treatments in the same model. In the B16F1 model, treatment with AUP-55 led to 90 % survival in comparison to 10 % untreated mice. All AUP-55 treatments were well tolerated. In the pathology post-mortem, all untreated animals had typical findings for ovarian cancer, such as ascites and high number of tumor nodules present in abdominal cavity, liver, spleen, kidney and pancreas. In the group treated with AUP-55 there were no macroscopic tumor deposits detected. Conclusions: Cytokine armed Lactococcus cremoris AUP-55 enables multimodal action. Treatment with AUP-55 suggests anti-tumor activity as a single therapeutic entity for the treatment of ovarian cancer and peritoneal carcinomatosis.

Carrier Cell–mediated Delivery of a Replication-competent Adenovirus for Cancer Gene Therapy

Sequential Molecular and Cellular Events during Neoplastic Progression: A Mouse Syngeneic Ovarian Cancer Model