Muscle weakness and mitochondrial stress occur before metastasis in a novel mouse model of ovarian cancer cachexia.

Objectives: A high proportion of women with advanced epithelial ovarian cancer (EOC) experience weakness and cachexia. This relationship is associated with increased morbidity and mortality. EOC is the most lethal gynecological cancer, yet no preclinical cachexia model has demonstrated the combined hallmark features of metastasis, ascites development, muscle loss and weakness in adult immunocompetent mice. Methods: Here, we evaluated a new model of ovarian cancer-induced cachexia with the advantages of inducing cancer in adult immunocompetent C57BL/6J mice through orthotopic injections of EOC cells in the ovarian bursa. We characterized the development of metastasis, ascites, muscle atrophy, muscle weakness, markers of inflammation, and mitochondrial stress in the tibialis anterior (TA) and diaphragm ~45, ~75 and ~90 days after EOC injection. Results: Primary ovarian tumour sizes were progressively larger at each time point while robust metastasis, ascites development, and reductions in body, fat and muscle weights occurred by 90 Days. There were no changes in certain inflammatory (TNFα), atrogene (MURF1 and Atrogin) or GDF15 markers within both muscles whereas IL-6 was increased at 45 and 90 Day groups in the diaphragm. TA weakness in 45 Day preceded atrophy and metastasis that were observed later (75 and 90 Day, respectively). The diaphragm demonstrated both weakness and atrophy in 45 Day. In both muscles, this pre-metastatic muscle weakness corresponded with considerable reprogramming of gene pathways related to mitochondrial bioenergetics as well as reduced functional measures of mitochondrial pyruvate oxidation and creatine-dependent ADP/ATP cycling as well as increased reactive oxygen species emission (hydrogen peroxide). Remarkably, muscle force per unit mass at 90 days was partially restored in the TA despite the presence of atrophy and metastasis. In contrast, the diaphragm demonstrated progressive weakness. At this advanced stage, mitochondrial pyruvate oxidation in both muscles exceeded control mice suggesting an apparent metabolic super-compensation corresponding with restored indices of creatine-dependent adenylate cycling. Conclusion: This mouse model demonstrates the concurrent development of cachexia and metastasis that occurs in women with EOC. The model provides physiologically relevant advantages of inducing tumour development within the ovarian bursa in immunocompetent adult mice. Moreover, the model reveals that muscle weakness in both TA and diaphragm precedes metastasis while weakness also precedes atrophy in the TA. An underlying mitochondrial bioenergetic stress corresponded with this early weakness. Collectively, these discoveries can direct new research towards the development of therapies that target pre-atrophy and pre-metastatic weakness during EOC in addition to therapies targeting cachexia.

Therapy-naïve malignancy causes cardiovascular disease: A state-of-the-art cardio-oncology perspective.

Cardiovascular disease (CVD) and cancer are the leading causes of mortality worldwide. Although generally thought of as distinct diseases, the intersectional overlap between CVD and cancer is increasingly evident in both causal and mechanistic relationships. The field of cardio-oncology is largely focused on cardiotoxic effects of cancer therapies (e.g., chemotherapy, radiation). Further, the cumulative effects of cardiotoxic therapy exposure and the prevalence of CVD risk factors in cancer patients leads to long-term morbidity and poor quality of life in this patient population-even when patients are cancer-free. Evidence from cancer patients and animal models demonstrates that the presence of malignancy itself, independent of cardiotoxic therapy exposure or CVD risk factors, negatively impacts cardiac structure and function. As such, the primary focus of this review is the cardiac pathophysiologic and molecular features of therapy-naïve cancer. We also summarize the strengths and limitations of preclinical cancer models for cardio-oncology research and discuss therapeutic strategies that have been tested experimentally for the treatment of cancer-induced cardiac atrophy and dysfunction. Finally, we explore an adjacent area of interest, called 'reverse cardio-oncology', where the sequelae of heart failure augment cancer progression. Here, we emphasize the cross-disease communication between malignancy and the injured heart, and discuss the importance of chronic low-grade inflammation and endocrine factors in the progression of both diseases.

Mutant p53 murine oviductal epithelial cells induce progression of high-grade serous carcinoma and are most sensitive to simvastatin therapy in vitro and in vivo.

High-grade serous carcinoma (HGSC) is the most common and aggressive subtype of epithelial ovarian cancer, characterized by gain-of-function TP53 mutations originating in the fallopian tube epithelium. Therapeutic intervention occurs at advanced metastatic disease, due to challenges in early-stage diagnosis, with common disease recurrence and therapy resistance despite initial therapy success. The mevalonate pathway is exploited by many cancers and is potently inhibited by statin drugs. Statins have shown anti-cancer activity in many, but not all cancers. Here, we investigated the role of p53 status in relation to mevalonate pathway signaling in murine oviductal epithelial (OVE) cells and identified OVE cell sensitivity to statin inhibition. We found that p53R175H mutant and Trp53 knockout OVE cells have increased mevalonate pathway signaling compared to p53 wild-type OVE cells. Through orthotopic implantation to replicate the fallopian tube origin of HGSC, p53R175H mutant cells upregulated the mevalonate pathway to drive progression to advanced-stage ovarian cancer, and simvastatin treatment abrogated this effect. Additionally, simvastatin was more efficacious at inhibiting cell metabolic activity in OVE cells than atorvastatin, rosuvastatin and pravastatin. In vitro, simvastatin demonstrated potent effects on cell proliferation, apoptosis, invasion and migration in OVE cells regardless of p53 status. In vivo, simvastatin induced ovarian cancer disease regression through decreased primary ovarian tumor weight and increased apoptosis. Simvastatin also significantly increased cytoplasmic localization of HMG-CoA reductase in ovarian tumors. Downstream of the mevalonate pathway, simvastatin had no effect on YAP or small GTPase activity. This study suggests that simvastatin can induce anti-tumor effects and could be an important inhibitor of ovarian cancer progression.

Liposomal delivery of gene therapy for ovarian cancer: a systematic review.

OBJECTIVE: To systematically identify and narratively synthesize the evidence surrounding liposomal delivery of gene therapy and the outcome for ovarian cancer. METHODS: An electronic database search of the Embase, MEDLINE and Web of Science from inception until July 7, 2023, was conducted to identify primary studies that investigated the effect of liposomal delivery of gene therapy on ovarian cancer outcomes. Retrieved studies were assessed against the eligibility criteria for inclusion. RESULTS: The search yielded 564 studies, of which 75 met the inclusion criteria. Four major types of liposomes were identified: cationic, neutral, polymer-coated, and ligand-targeted liposomes. The liposome with the most evidence involved cationic liposomes which are characterized by their positively charged phospholipids (n = 37, 49.3%). Similarly, those with neutrally charged phospholipids, such as 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, were highly researched as well (n = 25, 33.3%). Eight areas of gene therapy research were identified, evaluating either target proteins/transcripts or molecular pathways: microRNAs, ephrin type-A receptor 2 (EphA2), interleukins, mitogen-activated protein kinase (MAPK), human-telomerase reverse transcriptase/E1A (hTERT/EA1), suicide gene, p53, and multidrug resistance mutation 1 (MDR1). CONCLUSION: Liposomal delivery of gene therapy for ovarian cancer shows promise in many in vivo studies. Emerging polymer-coated and ligand-targeted liposomes have been gaining interest as they have been shown to have more stability and specificity. We found that gene therapy involving microRNAs was the most frequently studied. Overall, liposomal genetic therapy has been shown to reduce tumor size and weight and improve survivability. More research involving the delivery and targets of gene therapy for ovarian cancer may be a promising avenue to improve patient outcomes.

Mutant p53, the Mevalonate Pathway and the Tumor Microenvironment Regulate Tumor Response to Statin Therapy.

Tumor cells have the ability to co-opt multiple metabolic pathways, enhance glucose uptake and utilize aerobic glycolysis to promote tumorigenesis, which are characteristics constituting an emerging hallmark of cancer. Mutated tumor suppressor and proto-oncogenes are frequently responsible for enhanced metabolic pathway signaling. The link between mutant p53 and the mevalonate (MVA) pathway has been implicated in the advancement of various malignancies, with tumor cells relying heavily on increased MVA signaling to fuel their rapid growth, metastatic spread and development of therapy resistance. Statin drugs inhibit HMG-CoA reductase, the pathway's rate-limiting enzyme, and as such, have long been studied as a potential anti-cancer therapy. However, whether statins provide additional anti-cancer properties is worthy of debate. Here, we examine retrospective, prospective and pre-clinical studies involving the use of statins in various cancer types, as well as potential issues with statins' lack of efficacy observed in clinical trials and future considerations for upcoming clinical trials.

Paternal obesity induces placental hypoxia and sex-specific impairments in placental vascularization and offspring metabolism†.

Paternal obesity predisposes offspring to metabolic dysfunction, but the underlying mechanisms remain unclear. We investigated whether this metabolic dysfunction is associated with changes in placental vascular development and is fueled by endoplasmic reticulum (ER) stress-mediated changes in fetal hepatic development. We also determined whether paternal obesity indirectly affects the in utero environment by disrupting maternal metabolic adaptations to pregnancy. Male mice fed a standard chow or high fat diet (60%kcal fat) for 8-10 weeks were time-mated with female mice to generate pregnancies and offspring. Glucose tolerance was evaluated in dams at mid-gestation (embryonic day (E) 14.5) and late gestation (E18.5). Hypoxia, angiogenesis, endocrine function, macronutrient transport, and ER stress markers were evaluated in E14.5 and E18.5 placentae and/or fetal livers. Maternal glucose tolerance was assessed at E14.5 and E18.5. Metabolic parameters were assessed in offspring at ~60 days of age. Paternal obesity did not alter maternal glucose tolerance but induced placental hypoxia and altered placental angiogenic markers, with the most pronounced effects in female placentae. Paternal obesity increased ER stress-related protein levels (ATF6 and PERK) in the fetal liver and altered hepatic expression of gluconeogenic factors at E18.5. Offspring of obese fathers were glucose intolerant and had impaired whole-body energy metabolism, with more pronounced effects in female offspring. Metabolic deficits in offspring due to paternal obesity may be mediated by sex-specific changes in placental vessel structure and integrity that contribute to placental hypoxia and may lead to poor fetal oxygenation and impairments in fetal metabolic signaling pathways in the liver.

Addition of an Fc-IgG induces receptor clustering and increases the in vitro efficacy and in vivo anti-tumor properties of the thrombospondin-1 type I repeats (3TSR) in a mouse model of advanced stage ovarian cancer.

OBJECTIVES: Tumor vasculature is structurally abnormal, with anatomical deformities, reduced pericyte coverage and low tissue perfusion. As a result of this vascular dysfunction, tumors are often hypoxic, which is associated with an aggressive tumor phenotype, and reduced delivery of therapeutic compounds to the tumor. We have previously shown that a peptide containing the thrombospondin-1 type I repeats (3TSR) specifically targets tumor vessels and induces vascular normalization in a mouse model of epithelial ovarian cancer (EOC). However, due to its small size, 3TSR is rapidly cleared from circulation. We now introduce a novel construct with the 3TSR peptide fused to the C-terminus of each of the two heavy chains of the Fc region of human IgG1 (Fc3TSR). We hypothesize that Fc3TSR will have greater anti-tumor activity in vitro and in vivo compared to the native compound. METHODS: Fc3TSR was evaluated in vitro using proliferation and apoptosis assays to investigate differences in efficacy compared to native 3TSR. In light of the multivalency of Fc3TSR, we also investigate whether it induces greater clustering of its functional receptor, CD36. We also compare the compounds in vivo using an orthotopic, syngeneic mouse model of advanced stage EOC. The impact of the two compounds on changes to tumor vasculature morphology was also investigated. RESULTS: Fc3TSR significantly decreased the viability and proliferative potential of EOC cells and endothelial cells in vitro compared to native 3TSR. High-resolution imaging followed by image correlation spectroscopy demonstrated enhanced clustering of the CD36 receptor in cells treated with Fc3TSR. This was associated with enhanced downstream signaling and greater in vitro and in vivo cellular responses. Fc3TSR induced greater vascular normalization and disease regression compared to native 3TSR in an orthotopic, syngeneic mouse model of advanced stage ovarian cancer. CONCLUSION: The development of Fc3TSR which is greater in size, stable in circulation and enhances receptor activation compared to 3TSR, facilitates its translational potential as a therapy in the treatment of metastatic advanced stage ovarian cancer.

Characterizing Endocrine Status, Tumor Hypoxia and Immunogenicity for Therapy Success in Epithelial Ovarian Cancer.

Epithelial ovarian cancer is predominantly diagnosed at advanced stages which creates significant therapeutic challenges. As a result, the 5-year survival rate is low. Within ovarian cancer, significant tumor heterogeneity exists, and the tumor microenvironment is diverse. Tumor heterogeneity leads to diversity in therapy response within the tumor, which can lead to resistance or recurrence. Advancements in therapy development and tumor profiling have initiated a shift from a "one-size-fits-all" approach towards precision patient-based therapies. Here, we review aspects of ovarian tumor heterogeneity that facilitate tumorigenesis and contribute to treatment failure. These tumor characteristics should be considered when designing novel therapies or characterizing mechanisms of treatment resistance. Individual patients vary considerably in terms of age, fertility and contraceptive use which innately affects the endocrine milieu in the ovary. Similarly, individual tumors differ significantly in their immune profile, which can impact the efficacy of immunotherapies. Tumor size, presence of malignant ascites and vascular density further alters the tumor microenvironment, creating areas of significant hypoxia that is notorious for increasing tumorigenesis, resistance to standard of care therapies and promoting stemness and metastases. We further expand on strategies aimed at improving oxygenation status in tumors to dampen downstream effects of hypoxia and set the stage for better response to therapy.

Quantitative and Semi-quantitative Methods for Assessing the Degree of Methylene Blue Staining in Sentinel Lymph Nodes in Dogs.

Background: To develop a digital algorithm for quantitative assessment of surface methylene blue staining in whole lymph nodes and validate a semi-quantitative visual scoring method for patient-side use. Methods: Lymph nodes from canine patients with spontaneous tumors undergoing sentinel lymph node mapping were prospectively assessed ex vivo and photographed. Using an open-source computer-based imaging software, an algorithm was developed for quantification of staining based on a signal-to-background ratio. Next, two blinded observers evaluated images and assigned a semi-quantitative visual score based on surface staining (0-no blue stain, 1-1-50% stained, and 2-51-100% stained) and those results were compared to the established quantitative standard. Results: Forty-three lymph nodes were included. Image analysis successfully quantified blue staining and differentiated from normal lymph node tissue in all cases. Agreement between observers using the Kappa coefficient demonstrated strong agreement (k = 0.8581, p < 0.0001) between semi-quantitative visual scoring and image analysis. There was substantial interobserver and intraobserver agreement for the scoring system (k = 0.7340, p < 0.0001 and k = 0.8983, p < 0.0001, respectively). Conclusion: A digital algorithm using an open-source software was simple and straightforward to use for quantification of blue staining. The use of a semi-quantitative visual scoring system shows promise for a simple, objective, repeatable assessment of methylene blue staining at the time of surgery. This study demonstrates reliable and repeatable methods for blue staining quantification thereby providing a novel and objective reporting mechanism in scientific research involving sentinel lymph node mapping.

Normalizing Tumor Vasculature to Reduce Hypoxia, Enhance Perfusion, and Optimize Therapy Uptake.

A basic requirement of tumorigenesis is the development of a vascular network to support the metabolic requirements of tumor growth and metastasis. Tumor vascular formation is regulated by a balance between promoters and inhibitors of angiogenesis. Typically, the pro-angiogenic environment created by the tumor is extremely aggressive, resulting in the rapid vessel formation with abnormal, dysfunctional morphology. The altered morphology and function of tumor blood and lymphatic vessels has numerous implications including poor perfusion, tissue hypoxia, and reduced therapy uptake. Targeting tumor angiogenesis as a therapeutic approach has been pursued in a host of different cancers. Although some preclinical success was seen, there has been a general lack of clinical success with traditional anti-angiogenic therapeutics as single agents. Typically, following anti-angiogenic therapy, there is remodeling of the tumor microenvironment and widespread tumor hypoxia, which is associated with development of therapy resistance. A more comprehensive understanding of the biology of tumor angiogenesis and insights into new clinical approaches, including combinations with immunotherapy, are needed to advance vascular targeting as a therapeutic area.

Endothelial Cell Behavior Is Determined by Receptor Clustering Induced by Thrombospondin-1.

The thrombospondins (TSPs) are a family of multimeric extracellular matrix proteins that dynamically regulate cellular behavior and response to stimuli. In so doing, the TSPs directly and indirectly affect biological processes such as embryonic development, wound healing, immune response, angiogenesis, and cancer progression. Many of the direct effects of Thrombospondin 1 (TSP-1) result from the engagement of a wide range of cell surface receptors including syndecans, low density lipoprotein receptor-related protein 1 (LRP1), CD36, integrins, and CD47. Different or even opposing outcomes of TSP-1 actions in certain pathologic contexts may occur, depending on the structural/functional domain involved. To expedite response to external stimuli, these receptors, along with vascular endothelial growth factor receptor 2 (VEGFR2) and Src family kinases, are present in specific membrane microdomains, such as lipid rafts or tetraspanin-enriched microdomains. The molecular organization of these membrane microdomains and their constituents is modulated by TSP-1. In this review, we will describe how the presence of TSP-1 at the plasma membrane affects endothelial cell signal transduction and angiogenesis.

Obesity during pregnancy results in maternal intestinal inflammation, placental hypoxia, and alters fetal glucose metabolism at mid-gestation.

We investigated whether diet-induced changes in the maternal intestinal microbiota were associated with changes in bacterial metabolites and their receptors, intestinal inflammation, and placental inflammation at mid-gestation (E14.5) in female mice fed a control (17% kcal fat, n = 7) or a high-fat diet (HFD 60% kcal fat, n = 9; ad libitum) before and during pregnancy. Maternal diet-induced obesity (mDIO) resulted in a reduction in maternal fecal short-chain fatty acid producing Lachnospiraceae, lower cecal butyrate, intestinal antimicrobial peptide levels, and intestinal SCFA receptor Ffar3, Ffar2 and Hcar2 transcript levels. mDIO increased maternal intestinal pro-inflammatory NFκB activity, colonic CD3+ T cell number, and placental inflammation. Maternal obesity was associated with placental hypoxia, increased angiogenesis, and increased transcript levels of glucose and amino acid transporters. Maternal and fetal markers of gluconeogenic capacity were decreased in pregnancies complicated by obesity. We show that mDIO impairs bacterial metabolite signaling pathways in the mother at mid-gestation, which was associated with significant structural changes in placental blood vessels, likely as a result of placental hypoxia. It is likely that maternal intestinal changes contribute to adverse maternal and placental adaptations that, via alterations in fetal hepatic glucose handling, may impart increased risk of metabolic dysfunction in offspring.

High-fat diet intake modulates maternal intestinal adaptations to pregnancy and results in placental hypoxia, as well as altered fetal gut barrier proteins and immune markers.

KEY POINTS: Maternal obesity has been associated with shifts in intestinal microbiota, which may contribute to impaired barrier function Impaired barrier function may expose the placenta and fetus to pro-inflammatory mediators We investigated the impacts of diet-induced obesity in mice on maternal and fetal intestinal structure and placental vascularization Diet-induced obesity decreased maternal intestinal short chain fatty acids and their receptors, impaired gut barrier integrity and was associated with fetal intestinal inflammation. Placenta from obese mothers showed blood vessel immaturity, hypoxia, increased transcript levels of inflammation, autophagy and altered levels of endoplasmic reticulum stress markers. These data suggest that maternal intestinal changes probably contribute to adverse placental adaptations and also impart an increased risk of obesity in the offspring via alterations in fetal gut development. ABSTRACT: Shifts in maternal intestinal microbiota have been implicated in metabolic adaptations to pregnancy. In the present study, we generated cohorts of female C57BL/6J mice fed a control (17% kcal fat, n = 10-14) or a high-fat diet (HFD 60% kcal from fat, n = 10-14; ad libitum) aiming to investigate the impact on the maternal gut microbiota, intestinal inflammation and gut barrier integrity, placental inflammation and fetal intestinal development at embryonic day 18.5. HFD was associated with decreased relative abundances of short-chain fatty acid (SCFA) producing genera during pregnancy. These diet-induced shifts paralleled decreased maternal intestinal mRNA levels of SCFA receptor Gpr41, modestly decreased cecal butyrate, and altered mRNA levels of inflammatory cytokines and immune cell markers in the maternal intestine. Maternal HFD resulted in impaired gut barrier integrity, with corresponding increases in circulating maternal levels of lipopolysaccharide (LPS) and tumour necrosis factor. Placentas from HFD dams demonstrated blood vessel immaturity and hypoxia; decreased free carnitine, acylcarnitine derivatives and trimethylamine-N-oxide; and altered mRNA levels of inflammation, autophagy, and ER stress markers. HFD exposed fetuses had increased activation of nuclear factor-kappa B and inhibition of the unfolded protein response in the developing intestine. Taken together, these data suggest that HFD intake prior to and during pregnancy shifts the composition of the maternal gut microbiota and impairs gut barrier integrity, resulting in increased maternal circulating LPS, which may ultimate contribute to changes in placental vascularization and fetal gut development.

Pro-apoptotic and anti-angiogenic actions of 2-methoxyestradiol and docosahexaenoic acid, the biologically derived active compounds from flaxseed diet, in preventing ovarian cancer.

BACKGROUND: We have previously shown that a whole flaxseed supplemented diet decreased the onset and severity of ovarian cancer in the laying hen, the only known animal model of spontaneous ovarian cancer. Flaxseed is rich in omega-3 fatty acids (OM3FA), mostly α-Linoleic acid (ALA), which gets converted to Docosahexaenoic acid (DHA) by the action of delta-6 desaturase enzyme. Ingestion of flaxseed also causes an increase in production of 2-methoxyestradiol (2MeOE2) via the induction of the CYP1A1 pathway of estrogen metabolism. We have previously reported that the flaxseed diet induces apoptosis via p38-MAPK pathway in chicken tumors. The objective of this study was to investigate the effect of the flaxseed diet on ovarian cancer in chickens, focusing on two hallmarks of cancer, apoptosis and angiogenesis. RESULTS: The anti-cancer effects of two active biologically derived compounds of flax diet, 2MeOE2 and DHA, were individually tested on human ovarian cancer cells and in vivo by the Chick Chorioallantoic Membrane (CAM) assay. Our results indicate that a flaxseed-supplemented diet promotes apoptosis and inhibits angiogenesis in chicken tumors but not in normal ovaries. 2MeOE2 promotes apoptosis in human ovarian cancer cells, inhibits angiogenesis on CAM and its actions are dependent on the p38-MAPK pathway. DHA does not have any pro-apoptotic effect on human ovarian cancer cells but has strong anti-angiogenic effects as seen on CAM, but not dependent on the p38-MAPK pathway. CONCLUSIONS: Dietary flaxseed supplementation promotes a pro-apoptotic and anti-angiogenic effect in ovarian tumors, not in normal ovaries. The biologically derived active compounds from flaxseed diet act through different pathways to elicit their respective anti-cancer effects. A flaxseed-supplemented diet is a promising approach for prevention of ovarian cancer as well as having a significant potential as an adjuvant treatment to supplement chemotherapeutic agents for treatment of advanced stages of ovarian cancer.

Combining Vascular Normalization with an Oncolytic Virus Enhances Immunotherapy in a Preclinical Model of Advanced-Stage Ovarian Cancer.

PURPOSE: Intravenous delivery of oncolytic viruses often leads to tumor vascular shutdown, resulting in decreased tumor perfusion and elevated tumor hypoxia. We hypothesized that using 3TSR to normalize tumor vasculature prior to administration of an oncolytic Newcastle disease virus (NDV) would enhance virus delivery and trafficking of immunologic cell subsets to the tumor core, resulting in systemically enhanced immunotherapy and regression of advanced-stage epithelial ovarian cancer (EOC). EXPERIMENTAL DESIGN: Using an orthotopic, syngeneic mouse model of advanced-stage EOC, we pretreated mice with 3TSR (4 mg/kg per day) alone or followed by combination with fusogenic NDV(F3aa) (1.0 × 108 plaque-forming units). RESULTS: Treatment with 3TSR normalized tumor vasculature, enhanced blood perfusion of primary EOC tumors, and induced disease regression. Animals treated with combination therapy had the greatest reduction in primary tumor mass, ascites accumulation, and secondary lesions (50% of mice were completely devoid of peritoneal metastases). Combining 3TSR + NDV(F3aa) led to enhanced trafficking of immunologic cells into the primary tumor core. CONCLUSIONS: We have shown, for the first time, that NDV, like other oncolytic viruses, is a potent mediator of acute vascular shutdown and that preventing this through vascular normalization can promote regression in a preclinical model of advanced-stage ovarian cancer. This challenges the current focus on induction of intravascular thrombosis as a requisite for successful oncolytic virotherapy.See related commentary by Bykov and Zamarin, p. 1446.

Production and Purification of High-Titer Newcastle Disease Virus for Use in Preclinical Mouse Models of Cancer.

Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus in the Paramyxoviridae family. Although primarily an avian pathogen, NDV is a potent oncolytic virus that has been shown to be safe and effective in a variety of preclinical cancer models and human clinical trials. To produce virus for oncolytic trials, NDV is commonly amplified in embryonated chicken eggs and purified from the allantoic fluid. Conventional methods for purifying virus from allantoic fluid often result in relatively low-titer preparations containing high levels of impurities, including immunogenic chicken host cell proteins from allantoic fluid. However, large quantities of virus need to be delivered intravenously to administer oncolytic NDV systemically to mice. This route of administration requires virus preparations that are both highly concentrated (to enable delivery of small volumes) and highly pure (to limit toxic effects from contaminants). Given the accumulation of promising preclinical and clinical data demonstrating the efficacy of NDV as an oncolytic agent, strategies for increasing the titer and purity of NDV preparations are sorely needed to allow for effective intravenous administration in mice. Here, we describe an optimized protocol for the rescue, production, and purification of high-titer in vivo-grade NDV for preclinical studies in mouse models.

Hyperglycemia promotes insulin-independent ovarian tumor growth.

INTRODUCTION: Epithelial ovarian cancer (EOC) is notoriously difficult to diagnose in its earlier and more treatable stages, making it one of the deadliest cancers in women. Comorbid diabetes is associated with poor prognosis in EOC and pro-growth insulin signalling is often considered to be the driving factor. However, EOC cells are also highly glycolytic and insulin-independent glucose uptake is essential to their metabolism. Evidence of gluconeogenesis in cancer in vivo suggests that the normal concentration of circulating glucose does not meet the energy demands of the tumor and may therefore be a limiting factor in cancer cell metabolism. Diabetics have elevated blood glucose that has the potential to meet these energy demands and facilitate cancer progression. METHODS: To determine whether hyperglycemia is a potentially modifiable factor independent of insulin, orthotopic ovarian tumors were induced in mice with acute Type 1 (hypo-insulinemic) or Type 2 (hyper-insulinemic) diabetes. RESULTS: Hyperglycemia accelerated the growth of ovarian tumors in a glucose concentration-dependent manner and significantly shortened overall survival. Reciprocally, the presence of a tumor improved impaired glucose tolerance in both Type 1 and Type 2 diabetes. In mice with chronic Type 1 diabetes, hyperglycemia limited tumor growth without changing overall survival, indicating that systemic metabolic stress can accelerate time to death independent of primary tumor size. When modeled in vitro, long-term culture in 25mM vs 6mM glucose resulted in significantly different growth and metabolism. CONCLUSIONS: Taken together, this study shows that systemic metabolic disturbances can have a profound impact on both the growth of ovarian tumors and on overall survival.

Maternal nutrient restriction impairs young adult offspring ovarian signaling resulting in reproductive dysfunction and follicle loss.

Reproductive abnormalities are included as health complications in offspring exposed to poor prenatal nutrition. We have previously shown in a rodent model that offspring born to nutrient restriction during pregnancy are born small, enter puberty early, and display characteristics of early ovarian aging as adults. The present study investigated whether key proteins involved in follicle recruitment and growth mediate ovarian follicle loss. Pregnant rats were randomized to a standard diet throughout pregnancy and lactation (CON), or a calorie-restricted (50% of control) diet (UN) during pregnancy. Offspring reproductive phenotype was investigated at postnatal days 4, 27, and 65. Maternal UN resulted in young adult (P65) irregular estrous cyclicity due to persistent estrus, a significant loss of antral follicles, corpora lutea, and an increase in atretic follicles. This decrease in growing follicles in UN offspring appears to be due to increased apoptosis as seen by immunopositive staining of pro-apoptotic factor CASP3 (caspase 3) in ovaries of young adult offspring. UN prepubertal offspring had reduced expression levels of Fshr in antral follicles, which may contribute to a decrease in PI3K/AKT activation evident as a decrease in pAKT immunolocalization in prepubertal antral follicles. Moreover, neonatal ovaries of UN offspring show decreased levels of immunopositive staining for AMHR2 (anti-mullerian hormone receptor 2). Collectively, these data demonstrate that maternal UN during pregnancy impacts ovarian function in offspring as early as P65 and provides a model for understanding the mechanisms driving early life UN-induced follicle loss and reproductive dysfunction.

Low-dose metronomic delivery of cyclophosphamide is less detrimental to granulosa cell viability, ovarian function, and fertility than maximum tolerated dose delivery in the mouse.

Chemotherapy can cause early menopause or infertility in women and have a profound negative impact on the quality of life of young female cancer survivors. Various factors are known to influence the risk of chemotherapy-induced ovarian failure, including the drug dose and treatment duration; however, the scheduling of dose administration has not yet been evaluated as an independent risk factor. We hypothesized that low-dose metronomic (LDM) chemotherapy scheduling would be less detrimental to ovarian function than the traditional maximum tolerated dose (MTD) strategy. In vitro, MTD cyclophosphamide exposure resulted in decreased proliferation and increased granulosa cell apoptosis, while cells treated with LDM cyclophosphamide were not different from untreated controls. Treatments of MTD cyclophosphamide induced high levels of follicle atresia and enhanced follicle recruitment in mice. In contrast, LDM delivery of an equivalent dose of cyclophosphamide reduced growing follicle numbers, but was not associated with higher levels of follicle atresia or recruitment. MTD cyclophosphamide induced significant vascular disruption and DNA damage in vivo, while LDM chemotherapy with equal cumulative amounts of cyclophosphamide was not different from controls. MTD chemotherapy also had a negative effect on mouse-fertility outcomes. Our findings suggest that LDM scheduling could potentially minimize the long-term effects of cyclophosphamide on female fertility by preventing follicle depletion from enhanced activation.

Akt isoform specific effects in ovarian cancer progression.

Ovarian cancer remains a significant therapeutic problem and novel, effective therapies are needed. Akt is a serine-threonine kinase that is overexpressed in numerous cancers, including ovarian. Mammalian cells express three Akt isoforms which are encoded by distinct genes. Although there are several Akt inhibitors in clinical trials, most indiscriminately target all isoforms. Current in vitro data and animal knockout experiments suggest that the Akt isoforms may have divergent roles. In this paper, we determined the isoform-specific functions of Akt in ovarian cancer cell proliferation in vitro and in ovarian cancer progression in vivo. For in vitro experiments, murine and human ovarian cancer cells were treated with Akt inhibitors and cell viability was assessed. We used two different in vivo approaches to identify the roles of Akt isoforms in ovarian cancer progression and their influence on the primary tumor and tumor microenvironment. In one experiment, wild-type C57Bl6 mice were orthotopically injected with ID8 cells with stable knockdown of Akt isoforms. In a separate experiment, mice null for Akt 1-3 were orthotopically injected with WT ID8 cells (Figure 1). Our data show that inhibition of Akt1 significantly reduced ovarian cancer cell proliferation and inhibited tumor progression in vivo. Conversely, disruption of Akt2 increased tumor growth. Inhibition of Akt3 had an intermediate phenotype, but also increased growth of ovarian cancer cells. These data suggest that there is minimal redundancy between the Akt isoforms in ovarian cancer progression. These findings have important implications in the design of Akt inhibitors for the effective treatment of ovarian cancer.