Oncolytic myxoma virus is effective in murine models of triple negative breast cancer despite poor rates of infection.

Oncolytic viruses are being heavily investigated as novel methods to treat cancers; however, predicting their therapeutic efficacy remains challenging. The most commonly used predictive tests involve determining the in vitro susceptibility of a tumor's malignant cells to infection with an oncolytic agent. Whether these tests are truly predictive of in vivo efficacy, however, remains unclear. Here we demonstrate that a recombinant, oncolytic myxoma virus shows efficacy in two murine models of triple negative breast cancer despite extremely low permissivity of these models to viral infection. These data demonstrate that in vitro infectivity studies are not an accurate surrogate for therapeutic efficacy and suggest that other tests need to be developed.

Defects in intratumoral arginine metabolism attenuate the replication and therapeutic efficacy of oncolytic myxoma virus.

BACKGROUND: Arginine (Arg) is a semiessential amino acid whose bioavailability is required for the in vitro replication of several oncolytic viruses. In vivo, Arg bioavailability is regulated by a combination of dietary intake, protein catabolism, and limited biosynthesis through portions of the urea cycle. Interestingly, despite the importance of bioavailable Arg to support cellular proliferation, many forms of cancer are functionally auxotrophic for this amino acid due to the epigenetic silencing of argininosuccinate synthetase 1 (ASS1), an enzyme responsible for the conversion of citrulline and aspartate into the Arg precursor argininosuccinate. The impact of this silencing on oncolytic virotherapy (OV), however, has never been examined. METHODS: To address this gap in knowledge, we generated tumor cells lacking ASS1 and examined how loss of this enzyme impacted the in vivo replication and therapeutic efficacy of oncolytic myxoma virus (MYXV). We also generated a series of recombinant MYXV constructs expressing exogenous ASS1 to evaluate the therapeutic benefit of virally reconstituting Arg biosynthesis in ASS1-/- tumors. RESULTS: Our results show that the in vitro replication of oncolytic MYXV is dependent on the presence of bioavailable Arg. This dependence can be overcome by the addition of the metabolic precursor citrulline, however, this rescue requires expression of ASS1. Because of this, tumors formed from functionally ASS1-/- cells display significantly reduced MYXV replication as well as poorer therapeutic responses. Critically, both defects could be partially rescued by expressing exogenous ASS1 from recombinant oncolytic MYXVs. CONCLUSIONS: These results demonstrate that intratumoral defects to Arg metabolism can serve as a novel barrier to virally induced immunotherapy and that the exogenous expression of ASS1 can improve the efficacy of OV in Arg-auxotrophic tumors.

Saturated fatty acids dampen the immunogenicity of cancer by suppressing STING.

Oncogenes destabilize STING in epithelial cell-derived cancer cells, such as head and neck squamous cell carcinomas (HNSCCs), to promote immune escape. Despite the abundance of tumor-infiltrating myeloid cells, HNSCC presents notable resistance to STING stimulation. Here, we show how saturated fatty acids in the microenvironment dampen tumor response to STING stimulation. Using single-cell analysis, we found that obesity creates an IFN-I-deprived tumor microenvironment with a massive expansion of suppressive myeloid cell clusters and contraction of effector T cells. Saturated fatty acids, but not unsaturated fatty acids, potently inhibit the STING-IFN-I pathway in HNSCC cells. Myeloid cells from obese mice show dampened responses to STING stimulation and are more suppressive of T cell activation. In agreement, obese hosts exhibited increased tumor burden and lower responsiveness to STING agonist. As a mechanism, saturated fatty acids induce the expression of NLRC3, depletion of which results in a T cell inflamed tumor microenvironment and IFN-I-dependent tumor control.

High Levels of Extracellular Potassium Can Delay Myxoma Virus Replication by Preventing Release of Virions from the Endosomes.

Potassium (K+) is one of the most abundant cations in the human body. Under normal conditions, the vast majority of K+ is found within cells, and the extracellular [K+] is tightly regulated to within 3.0 to 5.0 mM. However, it has recently been shown that high levels of localized necrosis can increase the extracellular concentration of K+ to above 50 mM. This raises the possibility that elevated extracellular K+ might influence a variety of biological processes that occur within regions of necrotic tissue. For example, K+ has been shown to play a central role in the replication cycles of numerous viral families, and in cases of lytic infection, localized regions containing large numbers of necrotic cells can be formed. Here, we show that the replication of the model poxvirus myxoma virus (MYXV) is delayed by elevated levels of extracellular K+. These increased K+ concentrations alter the cellular endocytic pathway, leading to increased phagocytosis but a loss of endosomal/lysosomal segregation. This slows the release of myxoma virus particles from the endosomes, resulting in delays in genome synthesis and infectious particle formation as well as reduced viral spread. Additionally, mathematical modeling predicts that the extracellular K+ concentrations required to impact myxoma virus replication can be reached in viral lesions under a variety of conditions. Taken together, these data suggest that the extracellular [K+] plays a role in determining the outcomes of myxoma infection and that this effect could be physiologically relevant during pathogenic infection. IMPORTANCE Intracellular K+ homeostasis has been shown to play a major role in the replication of numerous viral families. However, the potential impact of altered extracellular K+ concentrations is less well understood. Our work demonstrates that increased concentrations of extracellular K+ can delay the replication cycle of the model poxvirus MYXV by inhibiting virion release from the endosomes. Additionally, mathematical modeling predicts that the levels of extracellular K+ required to impact MYXV replication can likely be reached during pathogenic infection. These results suggest that localized viral infection can alter K+ homeostasis and that these alterations might directly affect viral pathogenesis.

The Combination of TIM3-Based Checkpoint Blockade and Oncolytic Virotherapy Regresses Established Solid Tumors.

T-cell immunoglobulin and mucin domain 3 (TIM3) is emerging as a potential target for antibody-based checkpoint blockade. However, the efficacy of TIM3 blockade in combination with other treatment modalities, has not been extensively studied. In the current work we combined TIM3 blockade with myxoma virus-based oncolytic virotherapy (OV). Our results demonstrate that myxoma virus's ability to initiate an immense antitumor immune response complements the ability of TIM3 blockade to shift the tumor microenvironment to a more proinflammatory state. As a result, the combination of TIM3 blockade and OV is able to completely eradicate established disease, while neither monotherapy is effective. These data represent the first demonstration that OV can enhance the efficacy of TIM3 blockade and suggest that this treatment may need to be incorporated into more aggressive, combinatorial regimens in order to fulfill its potential as an immunotherapeutic.

Monitoring Therapeutic Responses to Silicified Cancer Cell Immunotherapy Using PET/MRI in a Mouse Model of Disseminated Ovarian Cancer.

Current imaging approaches used to monitor tumor progression can lack the ability to distinguish true progression from pseudoprogression. Simultaneous metabolic 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET) and magnetic resonance imaging (MRI) offers new opportunities to overcome this challenge by refining tumor identification and monitoring therapeutic responses to cancer immunotherapy. In the current work, spatial and quantitative analysis of tumor burden were performed using simultaneous [18F]FDG-PET/MRI to monitor therapeutic responses to a novel silicified cancer cell immunotherapy in a mouse model of disseminated serous epithelial ovarian cancer. Tumor progression was validated by bioluminescence imaging of luciferase expressing tumor cells, flow cytometric analysis of immune cells in the tumor microenvironment, and histopathology. While PET demonstrated the presence of metabolically active cancer cells through [18F]FDG uptake, MRI confirmed cancer-related accumulation of ascites and tissue anatomy. This approach provides complementary information on disease status without a confounding signal from treatment-induced inflammation. This work provides a possible roadmap to facilitate accurate monitoring of therapeutic responses to cancer immunotherapies.

TNF blockade enhances the efficacy of myxoma virus-based oncolytic virotherapy.

BACKGROUND: Oncolytic virotherapy (OV) represents a method to treat a variety of solid tumors by inducing antitumor immune responses. While this therapy has been extremely efficacious in preclinical models, translating these successes into human patients has proven challenging. One of the major reasons for these failures is the existence of immune-regulatory mechanisms, which dampen the efficacy of virally induced antitumor immunity. Unfortunately, the full extent of these immune-regulatory pathways remains unclear. METHODS: To address this issue, we generated a doubly recombinant, oncolytic myxoma virus which expresses both a soluble fragment of programmed cell death protein 1 (PD1) and an interleukin 12 (IL-12) fusion protein (vPD1/IL-12 (virus-expressing PD1 and IL-12)). We then tested the molecular impact and therapeutic efficacy of this construct in multiple models of disseminated disease to identify novel pathways, which are associated with poor therapeutic outcomes. RESULTS: Our results demonstrate that vPD1/IL-12 causes robust inflammation during therapy including inducing high levels of tumor necrosis factor (TNF). Surprisingly, although expression of TNF has generally been assumed to be beneficial to OV, the presence of this TNF appears to inhibit therapeutic efficacy by reducing intratumoral T-cell viability. Likely because of this, disruption of the TNF pathway, either through genetic knockout or antibody-based blockade, significantly enhances the overall outcomes of vPD1/IL-12-based therapy that allows for the generation of complete cures in normally non-responsive models. CONCLUSIONS: These data suggest that some aspects of OV-induced inflammation might represent a double-edged sword during therapy and that specific blockade of TNF might enhance the efficacy of these treatments.

The use of oncolytic virotherapy in the neoadjuvant setting.

Surgical removal of tumors remains a front-line therapy for many types of cancer. However, this treatment often fails to eradicate disease due to either recurrence of the original tumor or development of distant micrometastases. To address these challenges, patients are often given non-curative treatments presurgery with the intent of improving surgical outcomes. These treatments, collectively known as neoadjuvant therapies, have traditionally focused on the presurgical use of chemotherapeutics. Recently, however, a variety of immunotherapies have also been identified as potentially effective in the neoadjuvant setting. One of these immunotherapies is oncolytic virotherapy, whose clinical use has exploded with the Food and Drug Administration approval of Talimogene Laherparepvec. This review summarizes both the preclinical and clinical literature examining the use of oncolytic virotherapy in the neoadjuvant setting for different types of cancers and discusses some of the major questions that still need to be addressed in order for this unique use of immunotherapy to become clinically viable.

B cells imprint adoptively transferred CD8+ T cells with enhanced tumor immunity.

BACKGROUND: Adoptive T cell transfer (ACT) therapy improves outcomes in patients with advanced malignancies, yet many individuals relapse due to the infusion of T cells with poor function or persistence. Toll-like receptor (TLR) agonists can invigorate antitumor T cell responses when administered directly to patients, but these responses often coincide with toxicities. We posited that TLR agonists could be repurposed ex vivo to condition T cells with remarkable potency in vivo, circumventing TLR-related toxicity. METHODS: In this study we investigated how tumor-specific murine CD8+ T cells and human tumor infiltrating lymphocytes (TILs) are impacted when expanded ex vivo with the TLR9 agonist CpG. RESULTS: Herein we reveal a new way to reverse the tolerant state of adoptively transferred CD8+ T cells against tumors using TLR-activated B cells. We repurposed the TLR9 agonist, CpG, commonly used in the clinic, to bolster T cell-B cell interactions during expansion for ACT. T cells expanded ex vivo from a CpG-treated culture demonstrated potent antitumor efficacy and prolonged persistence in vivo. This antitumor efficacy was accomplished without in vivo administration of TLR agonists or other adjuvants of high-dose interleukin (IL)-2 or vaccination, which are classically required for effective ACT therapy. CpG-conditioned CD8+ T cells acquired a unique proteomic signature hallmarked by an IL-2RαhighICOShighCD39low phenotype and an altered metabolic profile, all reliant on B cells transiently present in the culture. Likewise, human TILs benefitted from expansion with CpG ex vivo, as they also possessed the IL-2RαhighICOShighCD39low phenotype. CpG fostered the expansion of potent CD8+ T cells with the signature phenotype and antitumor ability via empowering a direct B-T cell interaction. Isolated B cells also imparted T cells with the CpG-associated phenotype and improved tumor immunity without the aid of additional antigen-presenting cells or other immune cells in the culture. CONCLUSIONS: Our results demonstrate a novel way to use TLR agonists to improve immunotherapy and reveal a vital role for B cells in the generation of potent CD8+ T cell-based therapies. Our findings have immediate implications in the clinical treatment of advanced solid tumors.

Inhibition of Polyamine Biosynthesis Using Difluoromethylornithine Acts as a Potent Immune Modulator and Displays Therapeutic Synergy With PD-1-blockade.

Polyamines are known to play a significant role in cancer progression and treatment using difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, has shown some clinical promise. It is interesting to note that, while DFMO is directly cytostatic in vitro, recent work has suggested that it achieves its antitumor efficacy in vivo by enhancing adaptive antitumor immune responses. On the basis of these data, we hypothesized that DFMO might act as an immune sensitizer to increase tumor responsiveness to checkpoint blockade. To test this hypothesis, we treated tumors with DFMO, in either the presence or absence of additional PD-1 blockade, and subsequently analyzed their immunological and therapeutic responses. Our data demonstrates that treatment with DFMO significantly enhances both the viability and activation status of intratumoral CD8+ T cells, most likely through an indirect mechanism. When combined with PD-1 blockade, this increased viability resulted in unique proinflammatory cytokine profiles and transcriptomes within the tumor microenvironment and improved therapeutic outcomes. Taken together, these data suggest that DFMO might represent a potential immunomodulatory agent that can enhance current PD-1-based checkpoint therapies.

Interleukin-23 receptor signaling by interleukin-39 potentiates T cell pathogenicity in acute graft-versus-host disease.

IL-12 (p35/p40) and IL-23 (p19/p40) signal through IL-12R (IL-12Rß2/ß1) and IL-23R (IL-23Rα/IL-12Rß1), respectively, which can promote pathogenic T lymphocyte activation, differentiation, and function in graft-versus-host disease (GVHD). With the use of murine models of allogeneic hematopoietic cell transplantation (HCT), we found that IL-12Rß1 on donor T cells was dispensable to induce acute GVHD development in certain circumstances, while IL-23Rα was commonly required. This observation challenges the current paradigm regarding IL-12Rß1 as a prerequisite to transmit IL-23 signaling. We hypothesized that p19/EBI3 (IL-39) may have an important role during acute GVHD. With the use of gene transfection and immunoprecipitation approaches, we verified that p19 and EBI3 can form biological heterodimers. We found that IL-39 levels in recipient serum positively correlated with development of acute GVHD in experimental models and in clinical settings, thereby implicating IL-39 in the pathogenesis of acute GVHD. Furthermore, we observed that human T cells can signal in response to IL-39. In chronic GVHD, IL-23Rα and IL-12Rß1 were similarly required for donor T cell pathogenicity, and IL-39 levels were not significantly different from controls without GVHD. Collectively, we identify a novel cytokine, IL-39, as a pathogenic factor in acute GVHD, which represents a novel potential therapeutic target to control GVHD and other inflammatory disorders.

Decreasing the Susceptibility of Malignant Cells to Infection Does Not Impact the Overall Efficacy of Myxoma Virus-Based Oncolytic Virotherapy.

Oncolytic virotherapy relies on the induction of anti-tumor immune responses to achieve therapeutic efficacy. The factors that influence the induction of these responses, however, are not well understood. To begin to address this lack of knowledge, we asked how decreasing the susceptibility of malignant cells to direct viral infection would impact the induction of immune responses and therapeutic efficacy caused by oncolytic myxoma virus treatment. To accomplish this, we used CRISPR-Cas9 genome editing to remove the essential sulfation enzyme N-deacetylase/N-sulfotransferase-1 from B16/F10 murine melanoma cells. This eliminates the negative cell surface charges associated with glycosaminoglycan sulfation, which reduces a cell's susceptibility to infection with the myxoma virus by ∼3- to 10-fold. With the use of these cells as a model of reduced susceptibility to oncolytic infection, our data demonstrate that 3- to 10-fold reductions in in vivo infection do not hinder the ability of the oncolytic myxoma virus to induce anti-tumor immunity and do not lower the overall efficacy of localized treatment. Additionally, our data show that in mice bearing multiple distinct tumor masses, the choice to treat a less-susceptible tumor mass does not reduce the overall therapeutic impact against either the injected or noninjected lesion. Taken together, these data suggest that minor changes in the susceptibility of malignant cells to direct oncolytic infection do not necessarily influence the overall outcomes of treatment.

Initial dose of oncolytic myxoma virus programs durable antitumor immunity independent of in vivo viral replication.

BACKGROUND: Oncolytic therapy uses live-replicating viruses to improve the immunological status of treated tumors. Critically, while these viruses are known to self-amplify in vivo, clinical oncolytic therapies still appear to display a strong dose dependence and the mechanisms mediating this dose dependence are not well understood. METHODS: To explore this apparent contradiction, we investigated how the initial dose of oncolytic myxoma virus affected the subsequent ability of treatment to alter the immunological status of tumors as well as synergize with programmed cell death protein 1 (PD1) blockade. RESULTS: Our results indicate that, due to viral self-amplification in vivo, the overall load of myxoma virus rapidly normalizes within treated tumors despite up to 3-log differences in inoculating dose. Because of this, therapeutic efficacy in the absence of checkpoint blockade is largely dose independent. Despite this rapid normalization, however, treatment with high or low doses of myxoma virus induces distinct immunological changes within treated tumors. Critically, these changes appear to be durably programmed based on the initial oncolytic dose with low-dose treatment failing to induce immunological improvements despite rapidly achieving equivalent viral burdens. Finally, due to the distinct immunological profiles induced by high and low myxoma virus doses, oncolytic efficacy resulting from combination with PD1 blockade therapy displays a strong dose dependence. CONCLUSIONS: Taken together, these data suggest that the ability of oncolytic myxoma virus to immunologically reprogram treated tumors is dependent on initial viral dose. Additionally, this work could provide a possible mechanistic explanation for clinical results observed with other oncolytic viruses.

Reduced cellular binding affinity has profoundly different impacts on the spread of distinct poxviruses.

Poxviruses are large enveloped viruses that replicate exclusively in the cytoplasm. Like all viruses, their replication cycle begins with virion adsorption to the cell surface. Unlike most other viral families, however, no unique poxviral receptor has ever been identified. In the absence of a unique receptor, poxviruses are instead thought to adhere to the cell surface primarily through electrostatic interactions between the positively charged viral envelope proteins and the negatively charged sulfate groups on cellular glycosaminoglycans (GAGs). While these negatively charged GAGs are an integral part of all eukaryotic membranes, their specific expression and sulfation patterns differ between cell types. Critically, while poxviral binding has been extensively studied using virally centered genetic strategies, the impact of cell-intrinsic changes to GAG charge has never been examined. Here we show that loss of heparin sulfation, accomplished by deleting the enzyme N-Deacetylase and N-Sulfotransferase-1 (NDST1) which is essential for GAG sulfation, significantly reduces the binding affinity of both vaccinia and myxoma viruses to the cell surface. Strikingly, however, while this lowered binding affinity inhibits the subsequent spread of myxoma virus, it actually enhances the overall spread of vaccinia by generating more diffuse regions of infection. These data indicate that cell-intrinsic GAG sulfation plays a major role in poxviral infection, however, this role varies significantly between different members of the poxviridae.

Impact of Induced Syncytia Formation on the Oncolytic Potential of Myxoma Virus.

INTRODUCTION: Cancer has become one of the most critical health issues of modern times. To overcome the ineffectiveness of current treatment options, research is being done to explore new therapeutic modalities. One such novel treatment is oncolytic virotherapy (OV) which uses tumor tropic viruses to specifically target and kill malignant cells. While OV has shown significant promise in recent clinical trials, the therapeutic use of viruses poses a number of unique challenges. In particular, obtaining effective viral spread throughout the tumor microenvironment remains problematic. Previous work has suggested this can be overcome by forcing oncolytic viruses to induce syncytia formation. METHODS: In the current work, we generated a series of recombinant myxoma viruses expressing exogenous fusion proteins from other viral genomes and examined their therapeutic potential in vitro and in vivo. RESULTS: Similar to previous studies, we observed that the expression of these fusion proteins during myxoma infection induced the formation of multinucleated syncytia which increased viral spread and lytic potential compared to non-fusogenic controls. Contrary to expectations, however, the treatment of established tumors with these viruses resulted in decreased therapeutic efficacy which corresponded with reduced viral persistence. DISCUSSION: These findings indicate that enhanced viral spread caused by syncytia formation can actually reduce the efficacy of OV and supports a number of previous works suggesting that the in vitro properties of viruses frequently fail to predict their in vivo efficacy.

Fueling Cancer Immunotherapy With Common Gamma Chain Cytokines.

Adoptive T cell transfer therapy (ACT) using tumor infiltrating lymphocytes or lymphocytes redirected with antigen receptors (CAR or TCR) has revolutionized the field of cancer immunotherapy. Although CAR T cell therapy mediates robust responses in patients with hematological malignancies, this approach has been less effective for treating patients with solid tumors. Additionally, toxicities post T cell infusion highlight the need for safer ACT protocols. Current protocols traditionally expand T lymphocytes isolated from patient tumors or from peripheral blood to large magnitudes in the presence of high dose IL-2 prior to infusion. Unfortunately, this expansion protocol differentiates T cells to a full effector or terminal phenotype in vitro, consequently reducing their long-term survival and antitumor effectiveness in vivo. Post-infusion, T cells face further obstacles limiting their persistence and function within the suppressive tumor microenvironment. Therapeutic manipulation of T cells with common γ chain cytokines, which are critical growth factors for T cells, may be the key to bypass such immunological hurdles. Herein, we discuss the primary functions of the common γ chain cytokines impacting T cell survival and memory and then elaborate on how these distinct cytokines have been used to augment T cell-based cancer immunotherapy.

Chimeric tumor modeling reveals role of partial PDL1 expression in resistance to virally induced immunotherapy.

Expression of PDL1 on the surface of tumor cells can blunt the efficacy of many cancer immunotherapies. For example, our lab has previously shown that tumors derived from malignant cells incapable of expressing PDL1 are highly susceptible to immunotherapy induced by oncolytic virus treatment while tumors derived from PDL1 capable cells are highly resistant. In patient biopsies, however, expression of PDL1 on malignant cells is often not uniform with some cells expressing PDL1 while others do not. Importantly, how this partial PDL1 positivity influences the outcomes of immunotherapy remains largely unknown. In the current work, we expand on our previous findings by generating partially PDL1 positive tumors in immune competent animals and asking what percentage of tumor cells must express PDL1 for a tumor to become functionally resistant to oncolytic treatment. Our results indicate that the responsiveness of partially PDL1+ tumors correlates linearly with the percentage of PDL1 capable cells present at the initiation of treatment. Additionally, we observe that tumors which relapse after treatment display a significant increase in the numbers of PDL1 capable cells present suggesting that specific editing of mixed tumors might play a role in disease relapse. These data indicate that varying levels of PDL1 expression can play a significant role in the outcomes of oncolytic immunotherapy and challenges the concept that tumors should be viewed as simply PDL1+ or PDL1-.

Syncytia Formation in Oncolytic Virotherapy.

Oncolytic virotherapy uses replication-competent virus as a means of treating cancer. Whereas this field has shown great promise as a viable treatment method, the limited spread of these viruses throughout the tumor microenvironment remains a major challenge. To overcome this issue, researchers have begun looking at syncytia formation as a novel method of increasing viral spread. Several naturally occurring fusogenic viruses have been shown to possess strong oncolytic potential and have since been studied to gain insight into how this process benefits oncolytic virotherapy. Whereas these naturally fusogenic viruses have been beneficial, there are still challenges associated with their regular use. Because of this, engineered/recombinant fusogenic viruses have also been created that enhance nonfusogenic oncolytic viruses with the beneficial property of syncytia formation. The purpose of this review is to examine the existing body of literature on syncytia formation in oncolytics and offer direction for potential future studies.

Oncolytic myxoma virus synergizes with standard of care for treatment of glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is an aggressive form of brain cancer which is associated with poor prognosis. A variety of oncolytic viruses have previously shown positive efficacy against GBM, potentially offering new treatment options for patients. One such oncolytic virus is Myxoma virus (MYXV), a rabbit-specific poxvirus that has been shown to be efficacious against a variety of tumor models including GBM. PURPOSE: The purpose of this study was to test the efficacy of MYXV combined with current treatment regimens for GBM in both established cell lines as well as patient biopsy samples. MATERIALS AND METHODS: U118 gliobastoma cell lines were treated under various standard of care combinations (untreated, radiation and chemotherapeutic) prior to infection with MYXV. Infection was then monitored for differences in rate of infection, titer and rate of spread. Cellular death was measured by MTT assay and Caspase-3 colorimetric assay. Patient biopsies were harvested and treated under similar treatment conditions. RESULTS: The addition of GBM standard of care to MYXV infection resulted in an increased rate of spread compared to single treatment with either radiation or chemotherapeutic alone. SOC did not alter viral replication or infection rates. Similar effects were seen in ex vivo patient biopsies. Cellular viability was significantly decreased with the combination therapy of SOC and MYXV infection compared to any other treatment outcome. Caspase-3 activity was also significantly increased in samples treated with combination therapy when compared to any other treatment combination. CONCLUSION: Our results show that the combination of MYXV with current SOC results in both increased killing of GBM cells compared to either treatment regime alone as well as increased spread of MYXV infection. These findings lay the foundation for future in vivo studies on combining MYXV with GBM SOC.

Refinement and Successful Implementation of a Scoring System for Myxomatosis in a Susceptible Rabbit (Oryctolagus cuniculus) Model.

Myxoma virus is a member of Leporipoxviridae whose tropism is tightly restricted to lagomorphs. In susceptible Oryctolagus rabbits, the virus causes a highly lethal disease known as myxomatosis, which begins as a localized infection but rapidly disseminates throughout the animal, leading to immune compromise, mucosal infections, multiorgan failure, and death. In a research setting, myxoma infection of susceptible Oryctolagus cuniculus rabbits is used as a model of poxviral disease progression and represents one of only a few means to study the pathogenesis of this viral family in a native host species. However, the rapid progression of myxomatosis makes accurate prediction of humane endpoints critical to limiting animal pain and distress and preventing death as an endpoint. Here we present case studies of myxomatosis at 2 institutions and offer a refined scoring system to reliably track the course of disease in susceptible rabbits infected with myxoma virus.