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Cpt1a, PPARs and FAO-related gene expression

Glycolytic rate assay and ROS staining

Memory CD8+ T cells (Tmem) are superior mediators of adoptive cell therapy (ACT) compared with effector CD8+ T cells (Teff) due to increased persistence in vivo. Underpinning Tmem survival is a shift in cellular metabolism away from aerobic glycolysis towards fatty acid oxidation (FAO). Here we investigated the impact of the peroxisome proliferator-activated receptor (PPAR) agonist GW501516 (GW), an agent known to boost FAO in other tissues, on CD8+ T-cell metabolism, function, and efficacy in a murine ACT model. Via activation of both PPARα and PPARδ/β, GW treatment increased expression of carnitine palmitoyl transferase 1a, the rate-limiting enzyme of FAO, in activated CD8+ T cells. Using a metabolomics approach, we demonstrated that GW increased the abundance of multiple different acylcarnitines, consistent with enhanced FAO. T cells activated in the presence of GW and inflammatory signals, either mature dendritic cells or IL12, also demonstrated enhanced production of IFNγ and expression of T-bet. Despite high expression of T-bet, a characteristic of short-lived effector cells, GW-treated cells demonstrated enhanced persistence in vivo and superior efficacy in a model of ACT. Collectively, these data identify combined PPARα and PPARδ/β agonists as attractive candidates for further studies and rapid translation into clinical trials of ACT.Significance:Dual activation of peroxisome proliferator-activated receptors α and δ improves the efficacy of adoptive cell therapy by reprogramming T-cell metabolism and cytokine expression.

By conferring systemic protection and durable benefits, cancer immunotherapies are emerging as long-term solutions for cancer treatment. One such approach that is currently undergoing clinical testing is a therapeutic anti-cancer vaccine that uses two different viruses expressing the same tumor antigen to prime and boost anti-tumor immunity. By providing the additional advantage of directly killing cancer cells, oncolytic viruses (OVs) constitute ideal platforms for such treatment strategy. However, given that the targeted tumor antigen is encoded into the viral genomes, its production requires robust infection and therefore, the vaccination efficiency partially depends on the unpredictable and highly variable intrinsic sensitivity of each tumor to OV infection. In this study, we demonstrate that anti-cancer vaccination using OVs (Adenovirus (Ad), Maraba virus (MRB), Vesicular stomatitis virus (VSV) and Vaccinia virus (VV)) co-administered with antigenic peptides is as efficient as antigen-engineered OVs and does not depend on viral replication. Our strategy is particularly attractive for personalized anti-cancer vaccines targeting patient-specific mutations. We suggest that the use of OVs as adjuvant platforms for therapeutic anti-cancer vaccination warrants testing for cancer treatment. Viruses expressing tumour antigens can prime and boost anti-tumour immunity but the efficiency of this approach depends on the capacity of the virus to infect the host. Here, the authors show that vaccination with oncolytic viruses co-administered with tumour antigenic peptides is as efficient as antigen-engineered oncolytic viruses.

Advances in immunotherapy have underscored the importance of antitumor immune responses in controlling cancer. However, the tumor microenvironment (TME) imposes several obstacles to the proper function of immune cells, including a metabolically challenging and immunosuppressive microenvironment. The increased metabolic activity of tumor cells can lead to the depletion of key nutrients required by immune cells and the accumulation of byproducts that hamper antitumor immunity. Furthermore, the presence of suppressive immune cells, such as regulatory T cells and myeloid-derived suppressor cells, and the expression of immune inhibitory receptors can negatively impact immune cell metabolism and function. This review summarizes the metabolic reprogramming that is characteristic of various immune cell subsets, discusses how the metabolism and function of immune cells are shaped by the TME, and highlights how therapeutic interventions aimed at improving the metabolic fitness of immune cells and alleviating the metabolic constraints in the TME can boost antitumor immunity.

Recent advances in cancer therapeutics clearly demonstrate the need for innovative multiplex therapies that attack the tumour on multiple fronts. Oncolytic or “cancer-killing” viruses (OVs) represent up-and-coming multi-mechanistic immunotherapeutic drugs for the treatment of cancer. In this study, we perform an in-vitro screen based on virus-encoded artificial microRNAs (amiRNAs) and find that a unique amiRNA, herein termed amiR-4, confers a replicative advantage to the VSVΔ51 OV platform. Target validation of amiR-4 reveals ARID1A, a protein involved in chromatin remodelling, as an important player in resistance to OV replication. Virus-directed targeting of ARID1A coupled with small-molecule inhibition of the methyltransferase EZH2 leads to the synthetic lethal killing of both infected and uninfected tumour cells. The bystander killing of uninfected cells is mediated by intercellular transfer of extracellular vesicles carrying amiR-4 cargo. Altogether, our findings establish that OVs can serve as replicating vehicles for amiRNA therapeutics with the potential for combination with small molecule and immune checkpoint inhibitor therapy.

The factors that promote T cell expansion are not fully known. Creatine is an abundant circulating metabolite that has recently been implicated in T cell function; however, its cell-autonomous role in immune-cell function is unknown. Here, we show that creatine supports cell-intrinsic CD8

Memory CD8+ T cells (Tmem) are superior mediators of adoptive cell therapy (ACT) compared with effector CD8+ T cells (Teff) due to increased persistence in vivo. Underpinning Tmem survival is a shift in cellular metabolism away from aerobic glycolysis towards fatty acid oxidation (FAO). Here we investigated the impact of the peroxisome proliferator-activated receptor (PPAR) agonist GW501516 (GW), an agent known to boost FAO in other tissues, on CD8+ T-cell metabolism, function, and efficacy in a murine ACT model. Via activation of both PPARα and PPARδ/β, GW treatment increased expression of carnitine palmitoyl transferase 1a, the rate-limiting enzyme of FAO, in activated CD8+ T cells. Using a metabolomics approach, we demonstrated that GW increased the abundance of multiple different acylcarnitines, consistent with enhanced FAO. T cells activated in the presence of GW and inflammatory signals, either mature dendritic cells or IL12, also demonstrated enhanced production of IFNγ and expression of T-bet. Despite high expression of T-bet, a characteristic of short-lived effector cells, GW-treated cells demonstrated enhanced persistence in vivo and superior efficacy in a model of ACT. Collectively, these data identify combined PPARα and PPARδ/β agonists as attractive candidates for further studies and rapid translation into clinical trials of ACT. Significance: Dual activation of peroxisome proliferator-activated receptors α and δ improves the efficacy of adoptive cell therapy by reprogramming T-cell metabolism and cytokine expression.

One-carbon metabolism fuels the high demand of cancer cells for nucleotides and other building blocks needed for increased proliferation. Although inhibitors of this pathway are widely used to treat many cancers, their global impact on anabolic and catabolic processes remains unclear. Using a combination of real-time bioenergetics assays and metabolomics approaches, we investigated the global effects of methotrexate on cellular metabolism. We show that methotrexate treatment increases the intracellular concentration of the metabolite AICAR, resulting in AMPK activation. Methotrexate-induced AMPK activation leads to decreased one-carbon metabolism gene expression and cellular proliferation as well as increased global bioenergetic capacity. The anti-proliferative and pro-respiratory effects of methotrexate are AMPK-dependent, as cells with reduced AMPK activity are less affected by methotrexate treatment. Conversely, the combination of methotrexate with the AMPK activator, phenformin, potentiates its anti-proliferative activity in cancer cells. These data highlight a reciprocal effect of methotrexate on anabolic and catabolic processes and implicate AMPK activation as a metabolic determinant of methotrexate response.

Oncolytic viruses (OVs) are a class of biotherapeutics that are currently being explored for the treatment of cancer. While showing promise in several pre-clinical and clinical studies, systemic delivery of these anti-cancer agents is hampered by inefficient tumor targeting and a host immune system that is highly evolved to detect and neutralize pathogens. To shield the virus from immune recognition and destruction, the use of cells as delivery vehicles has been explored for the systemic delivery of OVs. Though several types of cell carriers are able to protect OVs during intravenous delivery, many still lack the ability to specifically home to or accumulate within the tumor microenvironment. Overall, OV-based therapeutics could benefit from tumor targeting strategies to maximize tumor-specific delivery and minimize infection of off-target tissues. In the current study, we examine magnetic targeting as a strategy to improve OV infection of tumor cells in vitro. We found that magnetic targeting of magnetically-labeled VSV particles or VSV-infected cell carriers resulted in increased infection and killing of tumor cells. Furthermore, this enhanced infection of target tumor cells was observed even in the presence of virus-specific neutralizing antibodies. Overall, our findings suggest that magnetic targeting strategies can improve the infection of tumor cells and may be a viable strategy to improve the tumor-targeted delivery of oncolytic VSV-based therapeutics.

Upon immune recognition of viruses, the mammalian innate immune response activates a complex signal transduction network to combat infection. This activation requires phosphorylation of key transcription factors regulating IFN production and signaling, including IFN regulatory factor 3 (IRF3) and STAT1. The mechanisms regulating these STAT1 and IRF3 phosphorylation events remain unclear. Here, using human and mouse cell lines along with gene microarrays, quantitative RT-PCR, viral infection and plaque assays, and reporter gene assays, we demonstrate that a microRNA cluster conserved among bilaterian animals, encoding miR-96, miR-182, and miR-183, regulates IFN signaling. In particular, we observed that the miR-183 cluster promotes IFN production and signaling, mediated by enhancing IRF3 and STAT1 phosphorylation. We also found that the miR-183 cluster activates the IFN pathway and inhibits vesicular stomatitis virus infection by directly targeting several negative regulators of IRF3 and STAT1 activities, including protein phosphatase 2A (PPP2CA) and tripartite motif–containing 27 (TRIM27). Overall, our work reveals an important role of the evolutionarily conserved miR-183 cluster in the regulation of mammalian innate immunity.

The use of oncolytic viruses (OVs) for cancer treatment is emerging as a successful strategy that combines the direct, targeted killing of the cancer with the induction of a long-lasting anti-tumor immune response. Using multiple aggressive murine models of triple-negative breast cancer, we have recently demonstrated that the early administration of oncolytic Maraba virus (MRB) prior to surgical resection of the primary tumor is sufficient to minimize the metastatic burden, protect against tumor rechallenge, cure a fraction of the mice and sensitize refractory tumors to immune checkpoint blockade without the need for further treatment. Here, we apply our surgical model to other OVs: Vesicular stomatitis virus (VSV), Adenovirus (Ad), Reovirus (Reo) and Herpes simplex virus (HSV) and show that all of the tested OVs could positively change the outcome of the treated animals. The growth of the primary and secondary tumors was differently affected by the various OVs and most of the viruses conferred survival benefits in this neoadjuvant setting despite the absence of direct treatment following rechallenge. This study establishes that OV-therapy confers long-term protection when administered in the pre-operative window of opportunity.

Metabolic reprogramming is an essential part of the T cell activation program. Upon activation, T cells undergo dramatic rewiring of their metabolic pathways to promote ATP production and biosynthesis sufficient to support the rapid exponential growth of antigen-specific T cells. The metabolic profile of T cells is shaped by both cell-intrinsic factors, such as genetics and receptor-mediated signaling, and environmental conditions, such as nutrient availability. However, our understanding of T cell metabolism has largely been shaped by studies in vitro, where oxygen and nutrients are in excess. Here, we combined bioenergetic profiling and 13C-glutamine infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. Similar to in vitro-activated T cells, glutamine is a prominent source of fuel for the TCA cycle during early expansion (3 dpi). However, at later timepoints of infection (6 dpi), the use of glutamine is substantially decreased by Teff cells. Similar to the observation of decreased glucose usage by T cells over the course of infection (Ma et al Immunity 2019). Our data suggests a dynamic flexibly in nutrient usage by T cells in vivo to fuel the different phases of T cell functions, from the early T cell response (3 dpi) to the late (6 dpi). Our work highlights the differences in T cell metabolism in vivo compared to in vitro cultures and a new method to study T cell nutrient utilization in vivo.

MicroRNAs (miRNA) are emerging as important regulators of immune function due to their fast action and ability to regulate programs of gene expression. Dendritic cell (DC) responses to stimuli involves a rapid transition from steady-state an activated state, leading to numerous phenotypic changes underpinned by changes in gene expression. We have found that microRNA-9 (miR-9) expression is rapidly increased upon LPS stimulation. Pathway analysis on predicted miR-9 targets show a significant enrichment of negative regulators of gene expression. We investigated whether miR-9 promotes DC activation through targeting negative regulators. We found that DCs overexpressing miR-9 showed an increased activation phenotype whereas DCs sequestering miR-9 showed blunted activation. Co-culture of miR-9 sequestering DCs with T cells led to decreased T-cell activation, whereas miR-9 overexpressing DCs promoted T cell activation. Mice immunized with miR-9 overexpressing DCs following injection with B16-OVA melanoma cells displayed decreased tumour volume compared to controls. This work demonstrates that miR-9 promotes the activation and function of DCs, adding to the growing evidence that miRNAs are involved in the regulation of immune responses.

Summary Dendritic cells, cells of the innate immune system, are found in a steady state poised to respond to activating stimuli. Once stimulated, they rapidly undergo dynamic changes in gene expression to adopt an activated phenotype capable of stimulating immune responses. We find that the microRNA miR-9 is upregulated in both bone marrow-derived DCs and conventional DC1s but not in conventional DC2s following stimulation. miR-9 expression in BMDCs and conventional DC1s promotes enhanced DC activation and function, including the ability to stimulate T cell activation and control tumor growth. We find that miR-9 regulated the expression of several negative regulators of transcription, including the transcriptional repressor Polycomb group factor 6 (Pcgf6). These findings demonstrate that miR-9 facilitates the transition of DCs from steady state to mature state by regulating the expression of several negative regulators of DC function in a cell-type-specific manner.

Mycobacterium tuberculosis ( Mtb ) is one of the most ancient human pathogens, yet the exact mechanism(s) of host defense against Mtb remains unclear. Although one-third of the world’s population is chronically infected with Mtb , only 5 to 10% develop active disease. This indicates that, in addition to resistance mechanisms that control bacterial burden, the host has also evolved strategies to tolerate the presence of Mtb to limit disease severity. We identify mitochondrial cyclophilin D (CypD) as a critical checkpoint of T cell metabolism that controls the expansion of activated T cells. Although loss of CypD function in T cells led to enhanced Mtb antigen–specific T cell responses, this increased T cell response had no impact on bacterial burden. Rather, mice containing CypD-deficient T cells exhibited substantially compromised disease tolerance and succumbed to Mtb infection. This study establishes a mechanistic link between T cell–mediated immunity and disease tolerance during Mtb infection.

Due to cancer's genetic complexity, significant advances in the treatment of metastatic disease will require sophisticated, multi-pronged therapeutic approaches. Here we demonstrate the utility of a Drosophila melanogaster cell platform for the production and in vivo delivery of multi-gene biotherapeutic systems. We show that cultured Drosophila S2 cell carriers can stably propagate oncolytic viral therapeutics that are highly cytotoxic for mammalian cancer cells without adverse effects on insect cell viability or gene expression. Drosophila cell carriers administered systemically to immunocompetent animals trafficked to tumors to deliver multiple biotherapeutics with little apparent off-target tissue homing or toxicity, resulting in a therapeutic effect. Cells of this Dipteran invertebrate provide a genetically tractable platform supporting the integration of complex, multi-gene biotherapies while avoiding many of the barriers to systemic administration of mammalian cell carriers. These transporters have immense therapeutic potential as they can be modified to express large banks of biotherapeutics with complementary activities that enhance anti-tumor activity.

Supplemental Digital Content is available in the text., Anticancer vaccination is becoming a popular therapeutic approach for patients with cancers expressing common tumor antigens. One variation on this strategy is a heterologous virus vaccine where 2 viruses encoding the same tumor antigen are administered sequentially to prime and boost antitumor immunity. This approach is currently undergoing clinical investigation using an adenovirus (Ad) and the oncolytic virus Maraba (MRB). In this study, we show that Listeria monocytogenes can be used in place of the Ad to obtain comparable immune priming efficiency before MRB boosting. Importantly, the therapeutic benefits provided by our heterologous L. monocytogenes-MRB prime-boost strategy are superior to those conferred by the Ad-MRB combination. Our study provides proof of concept for the heterologous oncolytic bacteria-virus prime-boost approach for anticancer vaccination and merits its consideration for clinical testing.

Triple-negative breast cancer (TNBC) is an aggressive disease for which treatment options are limited and associated with severe toxicities. Immunotherapeutic approaches like immune checkpoint inhibitors (ICIs) are a potential strategy, but clinical trials have demonstrated limited success in this patient cohort. Clinical studies using ICIs have revealed that patients with preexisting anticancer immunity are the most responsive. Given that oncolytic viruses (OVs) induce antitumor immunity, we investigated their use as an ICI-sensitizing approach. Using a therapeutic model that mimics the course of treatment for women with newly diagnosed TNBC, we demonstrate that early OV treatment coupled with surgical resection provides long-term benefits. OV therapy sensitizes otherwise refractory TNBC to immune checkpoint blockade, preventing relapse in most of the treated animals. We suggest that OV therapy in combination with immune checkpoint blockade warrants testing as a neoadjuvant treatment option in the window of opportunity between TNBC diagnosis and surgical resection.

Oncolytic viruses (OVs) and bacteria share the property of tumor-selective replication following systemic administration. In the case of nonpathogenic bacteria, tumor selectivity relates to their ability to grow extracellularly within tumor stroma and is therefore ideally suited to restricting the production of bacterially produced therapeutic agents to tumors. We have previously shown the ability of the type 1 interferon antagonist B18R to enhance the replication and spread of vesicular stomatitis virus (VSV) by overcoming related cellular innate immunity. In this study, we utilized nonpathogenic bacteria (E. coli) expressing B18R to facilitate tumor-specific production of B18R, resulting in a microenvironment depleted of bioactive antiviral cytokine, thus "preconditioning" the tumor to enhance subsequent tumor destruction by the OV. Both in vitro and in vivo infection by VSVΔ51 was greatly enhanced by B18R produced from E. coli. Moreover, a significant increase in therapeutic efficacy resulted from intravenous (i.v.) injection of bacteria to tumor-bearing mice 5 days prior to i.v. VSVΔ51 administration, as evidenced by a significant reduction in tumor growth and increased survival in mice. Our strategy is the first example where two such diverse microorganisms are rationally combined and demonstrates the feasibility of combining complementary microorganisms to improve therapeutic outcome.

Summary Naive CD8+ T cells differentiating into effector T cells increase glucose uptake and shift from quiescent to anabolic metabolism. Although much is known about the metabolism of cultured T cells, how T cells use nutrients during immune responses in vivo is less well defined. Here, we combined bioenergetic profiling and 13C-glucose infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. In contrast to in vitro-activated T cells, which display hallmarks of Warburg metabolism, physiologically activated CD8+ T cells displayed greater rates of oxidative metabolism, higher bioenergetic capacity, differential use of pyruvate, and prominent flow of 13C-glucose carbon to anabolic pathways, including nucleotide and serine biosynthesis. Glucose-dependent serine biosynthesis mediated by the enzyme Phgdh was essential for CD8+ T cell expansion in vivo. Our data highlight fundamental differences in glucose use by pathogen-specific T cells in vivo, illustrating the impact of environment on T cell metabolic phenotypes.

T cell subsets including effector (T(eff)), regulatory (T(reg)) and memory (T(mem)) cells are characterized by distinct metabolic profiles that influence their differentiation and function. Previous research suggests that engagement of long-chain fatty acid oxidation (LC-FAO) supports Foxp3(+) T(reg) cell and T(mem) cell survival. However, evidence for this is mostly based on inhibition of Cpt1a, the rate limiting enzyme for LC-FAO, with the drug etomoxir. Using genetic models to target Cpt1a specifically in T cells, we dissected the role of LC-FAO in primary, memory and regulatory T cell responses. Here we show that the ACC2/Cpt1a axis is largely dispensable for T(eff), T(mem) or T(reg) cell formation, and that the effects of etomoxir on T cell differentiation and function are independent of Cpt1a expression. Together our data argue that metabolic pathways other than LC-FAO fuel T(mem) or T(reg) differentiation and suggest alternative mechanisms for the effects of etomoxir that involve mitochondrial respiration.

Oncolytic viruses are known to stimulate the antitumor immune response by specifically replicating in tumor cells. This is believed to be an important aspect of the durable responses observed in some patients and the field is rapidly moving toward immunotherapy. As a further means to engage the immune system, we engineered a virus, vesicular stomatitis virus (VSV), to encode the proinflammatory cytokine interferon-γ. We used the 4T1 mammary adenocarcinoma as well as other murine tumor models to characterize immune responses in tumor-bearing animals generated by treatment with our viruses. The interferon-γ-encoding virus demonstrated greater activation of dendritic cells and drove a more profound secretion of proinflammatory cytokines compared to the parental virus. From a therapeutic point of view, the interferon-γ virus slowed tumor growth, minimized lung tumors, and prolonged survival in several murine tumor models. The improved efficacy was lost in immunocompromized animals; hence the mechanism appears to be T-cell-mediated. Taken together, these results demonstrate the ability of oncolytic viruses to act as immune stimulators to drive antitumor immunity as well as their potential for targeted gene therapy.

Immune regulation of cellular metabolism can be responsible for successful responses to invading pathogens. Viruses alter their hosts' cellular metabolism to facilitate infection. Conversely, the innate antiviral responses of mammalian cells target these metabolic pathways to restrict viral propagation. We identified miR-130b and miR-185 as hepatic microRNAs (miRNAs) whose expression is stimulated by 25-hydroxycholesterol (25-HC), an antiviral oxysterol secreted by interferon-stimulated macrophages and dendritic cells, during hepatitis C virus (HCV) infection. However, 25-HC only directly stimulated miR-185 expression, whereas HCV regulated miR-130b expression. Independently, miR-130b and miR-185 inhibited HCV infection. In particular, miR-185 significantly restricted host metabolic pathways crucial to the HCV life cycle. Interestingly, HCV infection decreased miR-185 and miR-130b levels to promote lipid accumulation and counteract 25-HC's antiviral effect. Furthermore, miR-185 can inhibit other viruses through the regulation of immunometabolic pathways. These data establish these microRNAs as a key link between innate defenses and metabolism in the liver.

Vaccines have classically been used for disease prevention. Modern clinical vaccines are continuously being developed for both traditional use as well as for new applications. Typically thought of in terms of infectious disease control, vaccination approaches can alternatively be adapted as a cancer therapy. Vaccines targeting cancer antigens can be used to induce anti-tumour immunity and have demonstrated therapeutic efficacy both pre-clinically and clinically. Various approaches now exist and further establish the tremendous potential and adaptability of anti-cancer vaccination. Classical strategies include ex vivo-loaded immune cells, RNA- or DNA-based vaccines and tumour cell lysates. Recent oncolytic virus development has resulted in a surge of novel viruses engineered to induce powerful tumour-specific immune responses. In addition to their use as cancer vaccines, oncolytic viruses have the added benefit of being directly cytolytic to cancer cells and thus promote antigen recognition within a highly immune-stimulating tumour microenvironment. While oncolytic viruses are perfectly equipped for efficient immunization, this complicates their use upon previous exposure. Indeed, the host’s anti-viral counter-attacks often impair multiple-dosing regimens. In this review we will focus on the use of oncolytic viruses for anti-tumour vaccination. We will explore different strategies as well as ways to circumvent some of their limitations.

Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses. Also, vaccinia virus appears to be sensitive to SFV-induced antiviral interference.

Oncolytic viruses are complex biological agents that interact at multiple levels with both tumour and normal tissues. Antiviral pathways induced by interferon are known to have a critical role in determining tumour cell sensitivity and normal cell resistance to infection with oncolytic viruses. Here we pursue a synthetic biology approach to identify methods that enhance antitumour activity of oncolytic viruses through suppression of interferon signalling. On the basis of the mathematical analysis of multiple strategies, we hypothesize that a positive feedback loop, established by virus-mediated expression of a soluble interferon-binding decoy receptor, increases tumour cytotoxicity without compromising normal cells. Oncolytic rhabdoviruses engineered to express a secreted interferon antagonist have improved oncolytic potential in cellular cancer models, and display improved therapeutic potential in tumour-bearing mice. Our results demonstrate the potential of this methodology in evaluating potential caveats of viral immune-evasion strategies and improving the design oncolytic viruses.

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Oncolytic viruses (OVs) have been engineered or selected for cancer cell-specific infection however, we have found that following intravenous administration of vesicular stomatitis virus (VSV), tumor cell killing rapidly extends far beyond the initial sites of infection. We show here for the first time that VSV directly infects and destroys tumor vasculature in vivo but leaves normal vasculature intact. Three-dimensional (3D) reconstruction of infected tumors revealed that the majority of the tumor mass lacks significant blood flow in contrast to uninfected tumors, which exhibit relatively uniform perfusion. VSV replication in tumor neovasculature and spread within the tumor mass, initiates an inflammatory reaction including a neutrophil-dependent initiation of microclots within tumor blood vessels. Within 6 hours of intravenous administration of VSV and continuing for at least 24 hours, we observed the initiation of blood clots within the tumor vasculature whereas normal vasculature remained clot free. Blocking blood clot formation with thrombin inhibitors prevented tumor vascular collapse. Our results demonstrate that the therapeutic activity of an OV can go far beyond simple infection and lysis of malignant cells.

Oncolytic viruses constitute a promising therapy against malignant gliomas (MGs). However, virus-induced type I IFN greatly limits its clinical application. The kinase mammalian target of rapamycin (mTOR) stimulates type I IFN production via phosphorylation of its effector proteins, 4E-BPs and S6Ks. Here we show that mouse embryonic fibroblasts and mice lacking S6K1 and S6K2 are more susceptible to vesicular stomatitis virus (VSV) infection than their WT counterparts as a result of an impaired type I IFN response. We used this knowledge to employ a pharmacoviral approach to treat MGs. The highly specific inhibitor of mTOR rapamycin, in combination with an IFN-sensitive VSV-mutant strain (VSV ΔM51 ), dramatically increased the survival of immunocompetent rats bearing MGs. More importantly, VSV ΔM51 selectively killed tumor, but not normal cells, in MG-bearing rats treated with rapamycin. These results demonstrate that reducing type I IFNs through inhibition of mTORC1 is an effective strategy to augment the therapeutic activity of VSV ΔM51 .

The optimal route for clinical delivery of oncolytic viruses is thought to be systemic intravenous injection; however, the immune system is armed with several highly efficient mechanisms to remove pathogens from the circulatory system. To overcome the challenges faced in trying to delivery oncolytic viruses specifically to tumors via the bloodstream, carrier cells have been investigated to determine their suitability as delivery vehicles for systemic administration of oncolytic viruses. Cell carriers protect viruses from neutralization, one of the most limiting aspects of oncolytic virus interaction with the immune system. Cell carriers can also possess inherent tumor tropism, thus directing the delivery of the virus more specifically to a tumor. With preclinical studies already demonstrating the success and feasibility of this approach with multiple oncolytic viruses, clinical evaluation of cell-mediated delivery of viruses is on the horizon. Meanwhile, ongoing preclinical studies are aimed at identifying new cellular vehicles for oncolytic viruses and improving current promising cell carrier platforms.

Oncolytic viruses constitute a promising therapy against malignant gliomas (MGs). However, virus-induced type-I interferon (IFN) greatly limits its clinical application. The kinase mTOR (mammalian target of rapamycin) stimulates type-I IFN production via phosphorylation of its effector proteins, 4E-BPs and S6Ks. Here we show that both mouse embryonic fibroblasts (MEFs) and mice lacking S6K1 and S6K2, are susceptible to Vesicular Stomatitis Virus (VSV) infection due to an impaired type-I interferon response. We used this knowledge to employ a pharmacoviral approach to treat MGs. The highly specific inhibitor of mTOR, rapamycin, in combination with an IFN-sensitive VSV-mutant strain (VSVΔM51), dramatically increased the survival of immunocompetent rats bearing MGs. More importantly, VSVΔM51 selectively killed tumor, but not normal cells, in MG-bearing rats, which were treated with rapamycin. These results demonstrate that reducing type-I IFNs through inhibition of mTOR is an effective strategy to augment the therapeutic activity of VSVΔM51. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5349.

Background Breast cancer is the most common malignant disease amongst Western women. The lack of treatment options for patients with chemotherapy-resistant or recurrent cancers is pushing the field toward the rapid development of novel therapies. The use of oncolytic viruses is a promising approach for the treatment of disseminated diseases like breast cancer, with the first candidate recently approved by the Food and Drug Administration for use in patients. In this report, we demonstrate the compatibility of oncolytic virotherapy and chemotherapy using various murine breast cancer models. This one-two punch has been explored in the past by several groups with different viruses and drugs and was shown to be a successful approach. Our strategy is to combine Paclitaxel, one of the most common drugs used to treat patients with breast cancer, and the oncolytic Rhabdovirus Maraba-MG1, a clinical trial candidate in a study currently recruiting patients with late-stage metastatic cancer. Methods We used the EMT6, 4 T1 and E0771 murine breast cancer models to evaluate in vitro and in vivo the effects of co-treatment with MG1 and Paclitaxel. Treatment-induced cytotoxicity was assessed and plaque assays, flow cytometry, microscopy and immunocytochemistry analysis were performed to quantify virus production and transgene expression. Orthotopically implanted tumors were measured during and after treatment to evaluate efficacy and Kaplan-Meier survival curves were generated. Results Our data demonstrate not only the compatibility of the treatments, but also their synergistic cytopathic activity. With Paclitaxel, EMT6 and 4 T1 tumors demonstrated increased virus production both in vitro and in vivo. Our results also show that Paclitaxel does not impair the safety profile of the virus treatment. Importantly, when combined, MG1 and the drug controlled tumor growth and prolonged survival. Conclusions The combination of MG1 and Paclitaxel improved efficacy in all of the breast cancer models we tested and thus is a promising alternative approach for the treatment of patients with refractory breast cancer. Our strategy has potential for rapid translation to the clinic, given the current clinical status of both agents. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0744-y) contains supplementary material, which is available to authorized users.