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.

Kinetic analysis of oncolytic OrfV-induced innate and adaptive immune responses in a murine model of late-stage ovarian cancer.

Immunotherapies revive host immune responses against tumors by stimulating innate and adaptive immune effector cells with antitumor functions. Thus, detailed studies of immunological cell phenotypes and functions within the tumor microenvironment (TME) following immunotherapy treatments is critical to identifying the determinants of therapeutic success, optimizing treatment regimens, and driving curative outcomes. Oncolytic viruses such as Orf virus (OrfV) are multifunctional biologics that preferentially infect and kill cancer cells while simultaneously causing inflammation that drives anticancer immune responses. Here, we describe the immunological impact of OrfV on the ascites TME in a preclinical model of advanced-stage epithelial ovarian cancer. OrfV promoted the infiltration of several immune effector cells with increased expression of activation markers and effector cytokines into the ascites TME, which correlated with reduced ascites tumor burden and improved survival. The kinetics of the immune response and change in tumor burden following OrfV therapy revealed an optimal re-administration time to sustain antitumor immunity, further extending survival. The data presented highlight the importance of investigating immune response kinetics following immunotherapy and demonstrate that detailed kinetic profiling of immune responses can reveal novel insights into mechanisms of action that can guide the development of more effective therapies.

Comparative analysis of syngeneic mouse models of high-grade serous ovarian cancer.

Ovarian cancers exhibit high rates of recurrence and poor treatment response. Preclinical models that recapitulate human disease are critical to develop new therapeutic approaches. Syngeneic mouse models allow for the generation of tumours comprising the full repertoire of non-malignant cell types but have expanded in number, varying in the cell type of origin, method for transformation, and ultimately, the properties of the tumours they produce. Here we have performed a comparative analysis of high-grade serous ovarian cancer models based on transcriptomic profiling of 22 cell line models, and intrabursal and intraperitoneal tumours from 12. Among cell lines, we identify distinct signalling activity, such as elevated inflammatory signalling in STOSE and OVE16 models, and MAPK/ERK signalling in ID8 and OVE4 models; metabolic differences, such as reduced glycolysis-associated expression in several engineered ID8 subclones; and relevant functional properties, including differences in EMT activation, PD-L1 and MHC class I expression, and predicted chemosensitivity. Among tumour samples, we observe increased variability and stromal content among intrabursal tumours. Finally, we predict differences in the microenvironment of ID8 models engineered with clinically relevant mutations. We anticipate that this work will serve as a valuable resource, providing new insight to help select models for specific experimental objectives.

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.

Oncolytic Orf virus licenses NK cells via cDC1 to activate innate and adaptive antitumor mechanisms and extends survival in a murine model of late-stage ovarian cancer.

BACKGROUND: Novel therapies are needed to improve outcomes for women diagnosed with ovarian cancer. Oncolytic viruses are multifunctional immunotherapeutic biologics that preferentially infect cancer cells and stimulate inflammation with the potential to generate antitumor immunity. Herein we describe Parapoxvirus ovis (Orf virus (OrfV)), an oncolytic poxvirus, as a viral immunotherapy for ovarian cancer. METHODS: The immunotherapeutic potential of OrfV was tested in the ID8 orthotopic mouse model of end-stage epithelial ovarian carcinoma. Immune cell profiling, impact on secondary lesion development and survival were evaluated in OrfV-treated mice as well as in Batf3 knockout, mice depleted of specific immune cell subsets and in mice where the primary tumor was removed. Finally, we interrogated gene expression datasets from primary human ovarian tumors from the International Cancer Genome Consortium database to determine whether the interplay we observed between natural killer (NK) cells, classical type 1 dendritic cells (cDC1s) and T cells exists and influences outcomes in human ovarian cancer. RESULTS: OrfV was an effective monotherapy in a murine model of advanced-stage epithelial ovarian cancer. OrfV intervention relied on NK cells, which when depleted abrogated antitumor CD8+ T-cell responses. OrfV therapy was shown to require cDC1s in experiments with BATF3 knockout mice, which do not have mature cDC1s. Furthermore, cDC1s governed antitumor NK and T-cell responses to mediate antitumor efficacy following OrfV. Primary tumor removal, a common treatment option in human patients, was effectively combined with OrfV for optimal therapeutic outcome. Analysis of human RNA sequencing datasets revealed that cDC1s correlate with NK cells in human ovarian cancer and that intratumoral NK cells correlate positively with survival. CONCLUSIONS: The data herein support the translational potential of OrfV as an NK stimulating immunotherapeutic for the treatment of advanced-stage ovarian cancer.

AAV-Vectored Expression of the Vascular Normalizing Agents 3TSR and Fc3TSR, and the Anti-Angiogenic Bevacizumab Extends Survival in a Murine Model of End-Stage Epithelial Ovarian Carcinoma.

Epithelial ovarian cancer is the deadliest gynecological malignancy. The lack of effective treatments highlights the need for novel therapeutic interventions. The aim of this study was to investigate whether sustained adeno-associated virus (AAV) vector-mediated expression of vascular normalizing agents 3TSR and Fc3TSR and the antiangiogenic monoclonal antibody, Bevacizumab, with or without oncolytic virus treatment would improve survival in an orthotopic syngeneic mouse model of epithelial ovarian carcinoma. AAV vectors were administered 40 days post-tumor implantation and combined with oncolytic avian orthoavulavirus-1 (AOaV-1) 20 days later, at the peak of AAV-transgene expression, to ascertain whether survival could be extended. Flow cytometry conducted on blood samples, taken at an acute time point post-AOaV-1 administration (36 h), revealed a significant increase in activated NK cells in the blood of all mice that received AOaV-1. T cell analysis revealed a significant increase in CD8+ tumor specific T cells in the blood of AAV-Bevacizumab+AOaV-1 treated mice compared to control mice 10 days post AOaV-1 administration. Immunohistochemical staining of primary tumors harvested from a subset of mice euthanized 90 days post tumor implantation, when mice typically have large primary tumors, secondary peritoneal lesions, and extensive ascites fluid production, revealed that AAV-3TSR, AAV-Fc3TSR+AOaV-1, or AAV-Bevacizumab+AOaV-1 treated mice had significantly more tumor-infiltrating CD8+ T cells than PBS controls. Despite AAV-mediated transgene expression waning faster in tumor-bearing mice than in non-tumor bearing mice, all three of the AAV therapies significantly extended survival compared to control mice; with AAV-Bevacizumab performing the best in this model. However, combining AAV therapies with a single dose of AOaV-1 did not lead to significant extensions in survival compared to AAV therapies on their own, suggesting that additional doses of AOaV-1 may be required to improve efficacy in this model. These results suggest that vectorizing anti-angiogenic and vascular normalizing agents is a viable therapeutic option that warrants further investigation, including optimizing combination therapies.

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.

Tumour vasculature: Friend or foe of oncolytic viruses?

In the past two decades there have been substantial advances in understanding the anti-cancer mechanisms of oncolytic viruses (OVs). OVs can mediate their effects directly, by preferentially infecting and killing tumour cells. Additionally, OVs can indirectly generate anti-tumour immune responses. These differing mechanisms have led to a paradoxical divergence in strategies employed to further increase the potency of oncolytic virotherapies. On one hand, the tumour neovasculature is seen as a vital lifeline to the survival of the tumour, leading some to use OVs to target the tumour vasculature in hopes to starve cancers. Therapeutics causing vascular collapse can potentiate tumour hypoxia, nutrient restriction and pro-inflammatory cytokine release, which has shown promise in oncological studies. On the other hand, the same vasculature plays an important role for the dissemination of OVs, trafficking of effector cells and other therapeutics, which has prompted researchers to find ways of normalizing the vasculature to enhance infiltration of leukocytes and delivery of therapeutic agents. This article describes the recent developments of therapies aimed to shut down versus normalize tumour vasculature in order to inform researchers striving to optimize OV-based therapies.

AAV-mediated expression of 3TSR inhibits tumor and metastatic lesion development and extends survival in a murine model of epithelial ovarian carcinoma.

An integral step in the development of solid tumors is the recruitment of blood vessels to fuel tumor growth. Antiangiogenic therapies can inhibit this process and control solid tumor growth. Thrombospondin-1 is an antiangiogenic protein possessing three type I repeats (3TSR) near the center of the protein and a CD47-binding peptide (CD47) in its C-terminus. Previously, we showed that treatment with recombinant 3TSR induces tumor regression, normalizes tumor vasculature, and improves uptake of chemotherapy drugs in an orthotopic, syngeneic mouse model of advanced stage epithelial ovarian cancer (EOC). While effective, this intervention required daily intraperitoneal injections. To circumvent this, here we employ adeno-associated virus (AAV) gene therapy vectors to express 3TSR alone or in combination with the CD47-binding peptide of TSP-1 and evaluate the impact on tumor development and survival in a mouse model of EOC. A single intraperitoneal injection of 1 × 1011 vg of AAV expressing 3TSR, CD47-binding peptide, or 3TSR + CD47 effectively suppressed primary tumor growth; however, only AAV-3TSR was able to inhibit development of secondary lesions at 90-days post-tumor implantation and significantly improve survival. Taken together, AAV-mediated expression of 3TSR appears safe and effective at inhibiting tumor development and represents a novel, less invasive approach for treating ovarian carcinoma.

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.