Temozolomide renders murine cancer cells susceptible to oncolytic adenovirus replication and oncolysis

The preclinical evaluation of oncolytic adenoviruses (OAds) has been limited to cancer xenograft mouse models because OAds replicate poorly in murine cancer cells. The alkylating agent temozolomide (TMZ) has been shown to enhance oncolytic virotherapy in human cancer cells; therefore, we investigated whether TMZ could increase OAd replication and oncolysis in murine cancer cells. To test our hypothesis, three murine cancer cells were infected with OAd (E1b-deleted) alone or in combination with TMZ. TMZ increased OAd-mediated oncolysis in all three murine cancer cells tested. This increased oncolysis was, at least in part, due to productive virus replication, apoptosis, and autophagy induction. Most importantly, murine lung non-cancerous cells were not affected by OAd+TMZ. Moreover, TMZ increased Ad transduction efficiency. However, TMZ did not increase coxsackievirus and adenovirus receptor; therefore, other mechanism could be implicated on the transduction efficiency. These results showed, for the first time, that TMZ could render murine tumor cells more susceptible to oncolytic virotherapy. The proposed combination of OAds with TMZ presents an attractive approach towards the evaluation of OAd potency and safety in syngeneic mouse models using these murine cancer cell-lines in vivo.     

Potent systemic antitumor activity from an oncolytic herpes simplex virus of syncytial phenotype

Conditionally replicating (oncolytic) viruses, which selectively replicate in tumor cells but not in normal cells, show great promise as antitumor agents for cancer therapy. The principal antitumor activity of these viruses derives from their replication within tumor cells, which results in cell destruction and the production of progeny virions that can spread to adjacent tumor cells. However, one potential limitation of this approach is that viral gene deletions conferring tumor selectivity also result frequently in reduced potency of the virus in tumors. Therefore, strategies designed to enhance the potency of current oncolytic viruses will likely increase their chance of clinical success. Here we report the construction of an oncolytic herpes simplex virus (HSV) of which the infection also causes strong cell membrane fusion (syncytial formation). In vitro characterization on a variety of human tumor cells of different tissue origins showed that the plaques from this virus (Fu-10) are phenotypically unique and are significantly larger than those from the parental G207 virus, a well-characterized oncolytic HSV lacking fusogenic function. Furthermore, the syncytial formation caused by this virus depended on HSV replication, indicating that cell membrane fusion will only occur in dividing cells (such as tumor cells) where the virus can undergo a full infection cycle but not in normal cells where the viral replication is restricted. Systemic administration of Fu-10 into mice with established lung metastatic breast cancer resulted in a dramatic therapeutic effect. These studies demonstrate that incorporation of fusogenic function into an oncolytic virus can significantly increase the potency of viral oncolysis; this may lead to an enhanced clinical performance, especially in late-stage cancer patients.     

The potential of oncolytic virus therapy for pancreatic cancer

The objective of this paper was to review a new category of gene therapy using oncolytic viruses for the treatment of pancreatic cancer. The eligibility and feasibility of oncolytic virus therapy as a novel therapeutic agent against pancreatic cancer are discussed as well as basic research for clinical trials, including a historical perspective and the current status of these novel agents. Even combination therapy, such as surgery with radiation and chemotherapy, has not significantly improved the survival rate of pancreatic cancer. Recently, a clinical trial (phase I and II) using an oncolytic adenovirus, ONYX-015, was completed in patients with pancreatic cancer. The phase II trial yielded beneficial results (tumor reduction or stabilization) in about 50% of the patients. A phase I study of the efficacy of oncolytic herpes viruses, G207, OncoVEX GM-CSF, and 1716 against a variety of tumors has been completed, and G207 is in phase II trials for use against brain tumors. In addition, a phase I trial using the herpesvirus showed good tolerance at all dosages. We discuss the basic scientific principles and current results of the above clinical trials with respect to these oncolytic viruses, and then compare the relative advantages and disadvantages of adenoviruses and herpesviruses as oncolytic agents. We also review the published literature on newly developed oncolytic viruses. The concept of oncolytic therapy has been studied for a century. Recent technological developments have made these oncolytic viruses more tumor-specific by exploiting the tumor cell environments. In addition, these viruses have been reported to increase the immunosusceptibility of the tumor cells, and have been designed to express other genes to increase the susceptibility of tumor cells to other therapeutic agents. Oncolytic virus therapy certainly appears to be a feasible treatment for pancreatic cancer.     

Mesenchymal stem cells regulate epithelial–mesenchymal transition and tumor progression of pancreatic cancer cells

Cancer‐associated fibroblasts contribute to cancer progression that is caused by epithelial–mesenchymal transition (EMT). Recently, mesenchymal stem cells (MSCs) were found to be the major candidate involved in the development of tumor‐promoting cancer stroma. Here we report that α‐smooth muscle actin‐positive myofibroblast‐like cells originating from MSCs contribute to inducing EMT in side population cells of pancreatic cancer. More importantly, MSC‐derived myofibroblasts function to maintain tumor‐initiating stem cell‐like characteristics, including augmenting expression levels of various stemness‐associated genes, enhancing sphere‐ forming activity, promoting tumor formation in a mouse xenograft model, and showing resistance to anticancer drugs. Furthermore, both γ‐secretase inhibitor and siRNA directed against Jagged‐1 attenuated MSC‐associated E‐cadherin suppression and sphere formation in pancreatic cancer side population cells. Thus, our results suggest that MSC‐derived myofibroblasts play important roles in regulating EMT and tumor‐initiating stem cell‐like properties of pancreatic cancer cells through an intermediating Notch signal.     

Enhanced killing of therapy‐induced senescent tumor cells by oncolytic measles vaccine viruses

Therapy‐induced senescence (TIS) as a permanent growth arrest can be induced by various stimuli, including anticancer compounds. TIS emerged as a promising strategy to overcome resistance phenomena. However, senescent cancer cells might regain proliferation activity in vivo or even secrete tumor‐promoting cytokines. Therefore, successful exploitation of TIS in cancer treatment simultaneously requires the development of effective strategies to eliminate senescent cancer cells. Virotherapy aims to selectively hit tumor cells, thus a combination with senescence‐inducing drugs was explored. As a model, we chose measles vaccine virus (MeV), which does not interfere with cellular senescence by itself. In different tumor cell types, such as hepatoma, pancreatic and mammary gland carcinoma, we demonstrate efficient viral replication and lysis after TIS by gemcitabine, doxorubicin or taxol. Applying real time imaging, we even found an accelerated lysis of senescent cancer cells, supporting an enhanced viral replication with an increase in cell‐associated and released infectious MeV particles. In summary, we show as a proof‐of‐concept that senescent tumor cells can be efficiently exploited as virus host cells by oncolytic MeV. These observations open up a new field for preclinical and clinical research to further investigate TIS and oncolytic viruses as an attractive combinatorial future treatment approach.     

Use of replicating oncolytic adenoviruses in combination therapy for cancer.

Oncolytic virotherapy is the use of genetically engineered viruses that specifically target and destroy tumor cells via their cytolytic replication cycle. Viral-mediated tumor destruction is propagated through infection of nearby tumor cells by the newly released progeny. Each cycle should amplify the number of oncolytic viruses available for infection. Our understanding of the life cycles of cytolytic viruses has allowed manipulation of their genome to selectively kill tumor cells over normal tissue. Because the mechanism of tumor destruction is different, oncolytic virotherapy should work synergistically with current modes of treatment such as chemotherapy and radiation therapy. This article focuses on oncolytic adenoviruses that have been created and tested in preclinical and clinical trials in combination with chemotherapy, radiation therapy, and gene therapy.     

Lymphokine‐activated killer and dendritic cell carriage enhances oncolytic reovirus therapy for ovarian cancer by overcoming antibody neutralization in ascites

Reovirus is an oncolytic virus (OV), which acts by both direct tumor cell killing and priming of antitumor immunity. A major obstacle for effective oncolytic virotherapy is effective delivery of OV to tumor cells. Ovarian cancer is often confined to the peritoneal cavity and therefore i.p. delivery of reovirus may provide the ideal locoregional delivery, avoiding systemic dissemination. However, ovarian cancer is associated with an accumulation of ascitic fluid, which may interfere with oncolytic viral therapy. Here, we investigated the effect of ascites on reovirus‐induced oncolysis against primary ovarian cancer cells and ovarian cancer cell lines. In the absence of ascites, reovirus was cytotoxic against ovarian cancer cells; however, cytotoxicity was abrogated in the presence of ascitic fluid. Neutralizing antibodies (NAb) were identified as the cause of this inhibition. Loading OV onto cell carriers may facilitate virus delivery in the presence of NAb and immune cells which have their own antitumor effector activity are particularly appealing. Immature dendritic cells (iDC), Lymphokine‐activated killer (LAK) cells and LAKDC cocultures were tested as potential carriers for reovirus for tumor cell killing and immune cell priming. Reovirus‐loaded LAKDC, and to a lesser degree iDC, were able to: (i) protect from NAb and hand‐off reovirus for tumor cell killing; (ii) induce a proinflammatory cytokine milieu (IFN?, IL‐12, IFNα and TNFα) and (iii) generate an innate and specific antitumor adaptive immune response. Hence, LAKDC pulsed with reovirus represent a novel, clinically practical treatment for ovarian cancer to maximise both direct and innate/adaptive immune‐mediated tumor cell killing.     

Future directions: oncolytic viruses

Oncolytic viruses offer a promising new modality for cancer treatment. The strategy of this therapy is to develop viruses capable of selectively infecting and replicating in malignant tumor cells. Oncolytic viruses can spread and destroy malignant tumors without deleterious effects in normal tissues. These viruses are genetically engineered based on both the biology of replicating viruses and the major genetic defects in human cancer cells, so that they can replicate in cancer cells but not in normal cells. The key to the development of such viruses is the identification of viral genes, the deletion or modification of which enables tumor-specific cell destruction. Several clinical trials have demonstrated the safety of oncolytic viruses as cancer therapy and have also shown some encouraging results. Much evidence suggests that oncolytic viral therapy works in synergy with standard cancer therapies. In this review, we will focus on the oncolytic viruses that may be beneficial to patients with lung cancer in the near future.     

Effects of capsid-modified oncolytic adenoviruses and their combinations with gemcitabine or silica gel on pancreatic cancer

Conventional cancer treatments often have little impact on the course of advanced pancreatic cancer. Although cancer gene therapy with adenoviruses is a promising developmental approach, the primary receptor is poorly expressed in pancreatic cancers which might compromise efficacy and thus targeting to other receptors could be beneficial. Extended stealth delivery, combination with standard chemotherapy or circumvention of host antiadenoviral immune response might improve efficacy further. In this work, capsid-modified adenoviruses were studied for transduction of cell lines and clinical normal and tumor tissue samples. The respective oncolytic viruses were tested for oncolytic activity in vitro and in vivo. Survival was studied in a peritoneally disseminated pancreas cancer model, with or without concurrent gemcitabine while silica implants were utilized for extended intraperitoneal virus delivery. Immunocompetent mice and Syrian hamsters were used to study the effect of silica mediated delivery on antiviral immune responses and subsequent in vivo gene delivery. Capsid modifications selectively enhanced gene transfer to malignant pancreatic cancer cell lines and clinical samples. The respective oncolytic viruses resulted in increased cell killing in vitro, which translated into a survival benefit in mice. Early proinfammatory cytokine responses and formation of antiviral neutralizing antibodies was partially avoided with silica implants. The implant also shielded the virus from pre-existing neutralizing antibodies, while increasing the pancreas/liver gene delivery ratio six-fold. In conclusion, capsid modified adenoviruses would be useful for testing in pancreatic cancer trials. Silica implants might increase the safety and efficacy of the approach.     

Genetic delivery of an immunoRNase by an oncolytic adenovirus enhances anticancer activity

Antibody therapy of solid cancers is well established, but suffers from unsatisfactory tumor penetration of large immunoglobulins or from low serum retention of antibody fragments. Oncolytic viruses are in advanced clinical development showing excellent safety, but suboptimal potency due to limited virus spread within tumors. Here, by developing an immunoRNase‐encoding oncolytic adenovirus, we combine viral oncolysis with intratumoral genetic delivery of a small antibody‐fusion protein for targeted bystander killing of tumor cells (viro‐antibody therapy). Specifically, we explore genetic delivery of a small immunoRNase consisting of an EGFR‐binding scFv antibody fragment fused to the RNase Onconase (ONC_EGFR) that induces tumor cell death by RNA degradation after cellular internalization. Onconase is a frog RNase that combines lack of immunogenicity and excellent safety in patients with high tumor killing potency due to its resistance to the human cytosolic RNase inhibitor. We show that ONC_EGFR expression by oncolytic adenoviruses is feasible with an optimized, replication‐dependent gene expression strategy. Virus‐encoded ONC_EGFR induces potent and EGFR‐dependent bystander killing of tumor cells. Importantly, the ONC_EGFR‐encoding oncolytic adenovirus showed dramatically increased cytotoxicity specifically to EGFR‐positive tumor cells in vitro and significantly enhanced therapeutic activity in a mouse xenograft tumor model. The latter demonstrates that ONC_EGFR is expressed at levels sufficient to trigger tumor cell killing in vivo. The established ONC_EGFR‐encoding oncolytic adenovirus represents a novel agent for treatment of EGFR‐positive tumors. This viro‐antibody therapy platform can be further developed for targeted/personalized cancer therapy by exploiting antibody diversity to target further established or emerging tumor markers or combinations thereof.     

Mre11 inhibition by oncolytic adenovirus associates with autophagy and underlies synergy with ionizing radiation

New treatment approaches are needed for hormone refractory prostate cancer. Oncolytic adenoviruses are promising anti‐cancer agents, and their efficacy can be improved by combining with conventional therapies such as ionizing radiation. The aim of this study was to determine the timing of oncolytic adenovirus treatment with regard to radiation and study the mechanisms of synergy in combination treatment. Prostate cancer cells were infected with oncolytic adenoviruses, irradiated and synergy mechanisms were assessed. In vivo models of combination treatment were tested. Radiation and oncolytic viruses were synergistic when viral infection was scheduled 24 hr after irradiation. Combination of oncolytic adenovirus with radiotherapy significantly increased antitumor efficacy in vivo compared to either agent alone. Microarray analysis showed dysregulated pathways including cell cycle, mTOR and antigen processing pathways. Functional analysis showed that adenoviral infection was accompanied with degradation of proteins involved in DNA break repair. Mre11 was degraded for subsequent inactivation of Chk2‐Thr68 in combination treated cells, while γH2AX‐Ser139 was elevated implicating the persistence of DNA double strand breaks. Increased autophagocytosis was seen in combination treated cells. Combination treatment did not increase apoptosis or virus replication. The results provide evidence of the antitumor efficacy of combining oncolytic adenoviruses with irradiation as a therapeutic strategy for the treatment of prostate cancer. Further, these findings propose a molecular mechanism that may be important in radiation induced cell death, autophagy and viral cytopathic effect. © 2009 UICC     

Oncolytic virus therapy: A new era of cancer treatment at dawn

Oncolytic virus therapy is perhaps the next major breakthrough in cancer treatment following the success in immunotherapy using immune checkpoint inhibitors. Oncolytic viruses are defined as genetically engineered or naturally occurring viruses that selectively replicate in and kill cancer cells without harming the normal tissues. T‐Vec (talimogene laherparepvec), a second‐generation oncolytic herpes simplex virus type 1 (HSV‐1) armed with GM‐CSF, was recently approved as the first oncolytic virus drug in the USA and Europe. The phase III trial proved that local intralesional injections with T‐Vec in advanced malignant melanoma patients can not only suppress the growth of injected tumors but also act systemically and prolong overall survival. Other oncolytic viruses that are closing in on drug approval in North America and Europe include vaccinia virus JX‐594 (pexastimogene devacirepvec) for hepatocellular carcinoma, GM‐CSF‐expressing adenovirus CG0070 for bladder cancer, and Reolysin (pelareorep), a wild‐type variant of reovirus, for head and neck cancer. In Japan, a phase II clinical trial of G47?, a third‐generation oncolytic HSV‐1, is ongoing in glioblastoma patients. G47? was recently designated as a “Sakigake” breakthrough therapy drug in Japan. This new system by the Japanese government should provide G47? with priority reviews and a fast‐track drug approval by the regulatory authorities. Whereas numerous oncolytic viruses have been subjected to clinical trials, the common feature that is expected to play a major role in prolonging the survival of cancer patients is an induction of specific antitumor immunity in the course of tumor‐specific viral replication. It appears that it will not be long before oncolytic virus therapy becomes a standard therapeutic option for all cancer patients.     

Stroma‐directed imatinib therapy impairs the tumor‐promoting effect of bone marrow‐derived mesenchymal stem cells in an orthotopic transplantation model of colon cancer

Bone marrow‐derived mesenchymal stem cells (MSCs) are reported to contribute to formation of tumor‐promoting stromal cells. We reported recently that, in an orthotopic nude mice model of colon cancer, MSCs traveled to tumor stroma, where they differentiated into carcinoma‐associated fibroblast (CAF)‐like cells. We also found that CAFs express platelet‐derived growth factor receptor (PDGFR) at a high level and that imatinib therapy targeting PDGFR in CAFs inhibits growth and metastasis of human colon cancer. These findings led us to examine whether the tumor‐promoting effect of MSCs is impaired by blockade of PDGFR signaling achieved with imatinib. Orthotopic transplantation and splenic injection of human MSCs along with KM12SM human colon cancer cells, in comparison with transplantation of KM12SM cells alone, resulted in significantly greater promotion of tumor growth and liver metastasis. The KM12SM + MSC xenograft enhanced cell proliferation and angiogenesis and inhibited tumor cell apoptosis. When tumor‐bearing animals were treated with imatinib, there was no significant increase in primary tumor volume or total volume of liver metastases, despite the KM12SM+MSC xenograft, and survival in the mixed‐cell group was prolonged by imatinib treatment. Moreover, the ability of MSCs to migrate to tumor stroma was impaired, and the number of MSCs surviving in the tumor microenvironment was significantly decreased. In in vitro experiments, treatment with imatinib inhibited migration of MSCs. Our data suggest that blockade of PDGF signaling pathways influences the interaction between bone marrow‐derived MSCs and tumor cells in the tumor microenvironment and, hence, inhibits the progressive growth of colon cancer.     

Systemic delivery of HER2-retargeted oncolytic-HSV by mesenchymal stromal cells protects from lung and brain metastases

Fully retargeted oncolytic herpes simplex viruses (o-HSVs) gain cancer-specificity from redirection of tropism to cancer-specific receptors, and are non-attenuated. To overcome the hurdles of systemic delivery, and enable oncolytic viruses (o-viruses) to reach metastatic sites, carrier cells are being exploited. Mesenchymal stromal cells (MSCs) were never tested as carriers of retargeted o-viruses, given their scarse-null expression of the cancer-specific receptors. We report that MSCs from different sources can be forcedly infected with a HER2-retargeted oncolytic HSV. Progeny virus spread from MSCs to cancer cells in vitro and in vivo. We evaluated the organ distribution and therapeutic efficacy in two murine models of metastatic cancers, following a single i.v. injection of infected MSCs. As expected, the highest concentration of carrier-cells and of viral genomes was in the lungs. Viral genomes persisted throughout the body for at least two days. The growth of ovarian cancer lung metastases in nude mice was strongly inhibited, and the majority of treated mice appeared metastasis-free. The treatment significantly inhibited also breast cancer metastases to the brain in NSG mice, and reduced by more than one-half the metastatic burden in the brain.     

Effects of tumor selective replication-competent herpes viruses in combination with gemcitabine on pancreatic cancer

Purpose Pancreatic cancer still has a poor prognosis, even if aggressive therapy is pursued. Currently, new modalities of oncolytic virus therapy are being tested against this cancer. The combination of one of two representative mutant herpes simplex viruses (R3616: γ₁34.5 inactivated, hrR3: UL39 inactivated) with a standard anti-pancreatic cancer chemotherapy drug (gemcitabine), was investigated in this study. Experimental design The intracellular concentration of ribonucleotide reductase was estimated by Western blotting. The effect of gemcitabine on viral replication and the total cytotoxic effect of the combination therapy were investigated on pancreatic cancer cell lines. We compared the results of two oncolytic viruses, R3616 and hrR3. A mouse model of pancreatic cancer with peritoneal dissemination was used to evaluate the in vivo effect of the combination therapy. Results Although the replication of both viruses was inhibited by gemcitabine, the combination caused more tumor cell cytotoxicity than did virus alone in vitro. The results with R3616 were more striking. Although the difference was not statistically significant, R3616 with gemcitabine had a greater effect than did R3616 alone, while hrR3 with gemcitabine had a weaker effect than did hrR3 alone in vivo experiments. Conclusion The combination of oncolytic virus with gemcitabine is a promising new strategy against advanced pancreatic cancer. Each virus has different functional characteristics, and can affect the results of the combination of viruses and chemotherapy drugs. The results indicate that there is a complicated interaction among viruses, cells, and chemotherapy drugs and that the best combination of oncolytic virus and chemotherapeutic agents should be studied more extensively before embarking on a clinical trial.     

Future prospects for oncolytic therapy

Viruses have been engineered to replicate selectively in cancer cells, based on a number of innovative principles. Several of these viruses have entered clinical trials and have proven relatively safe, and have shown evidence of efficacy. However, further research is required to enable these agents to function systemically. This might involve attempts to suppress immune responses to virus antigens, and re-targeting of viruses to favor tumor infection and increased potency. When these barriers are overcome, oncolytic viruses could enter the mainstream of clinical oncology.     

Intracellular prodrug gene therapy for cancer mediated by tumor cell suicide gene exosomes

Recently, we reported about exosomes possessing messenger RNA (mRNA) of suicide gene secreted from mesenchymal stem/stromal cells (MSCs) engineered to express the suicide gene—fused yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT). The yCD::UPRT‐MSC exosomes are internalized by tumor cells and intracellularly convert prodrug 5‐fluorocytosine (5‐FC) to cytotoxic drug 5‐fluorouracil (5‐FU). Human tumor cells with the potential to metastasize release exosomes involved in the creation of a premetastatic niche at the predicted organs. We found that cancer cells stably transduced with yCD::UPRT gene by retrovirus infection released exosomes acting similarly like yCD::UPRT‐MSC exosomes. Different types of tumor cells were transduced with the yCD::UPRT gene. The homogenous cell population of yCD::UPRT‐transduced tumor cells expressed the yCD::UPRT suicide gene and secreted continuously exosomes with suicide gene mRNA in their cargo. All tumor cell suicide gene exosomes upon internalization into the recipient tumor cells induced the cell death by intracellular conversion of 5‐FC to 5‐FU and to 5‐FUMP in a dose‐dependent manner. Most of tumor cell‐derived suicide gene exosomes were tumor tropic, in 5‐FC presence they killed tumor cells but did not inhibit the growth of human skin fibroblast as well as DP‐MSCs. Tumor cell‐derived suicide gene exosomes home to their cells of origin and hold an exciting potential to become innovative specific therapy for tumors and potentially for metastases.     

An armed oncolytic herpes simplex virus expressing thrombospondin‐1 has an enhanced in vivo antitumor effect against human gastric cancer

Advanced gastric cancer is a common disease, but the conventional treatments are unsatisfactory because of the high recurrence rate. One of the promising new therapies is oncolytic virotherapy, using oncolytic herpes simplex viruses (HSVs). Thrombospondin‐1 (TSP‐1) suppresses tumor progression via multiple mechanisms including antiangiogenesis. Our approach to enhance the effects of oncolytic HSVs is to generate an armed oncolytic HSV that combines the direct viral oncolysis with TSP‐1‐mediated function for gastric cancer treatment. Using the bacterial artificial chromosome (BAC) system, a 3rd generation oncolytic HSV (T‐TSP‐1) expressing human TSP‐1 was constructed for human gastric cancer treatment. The enhanced efficacy of T‐TSP‐1 was determined in both human gastric cancer cell lines in vitro and subcutaneous tumor xenografts of human gastric cancer cells in vivo. In addition, we examined the apoptotic effect of T‐TSP‐1 in vitro, and the antiangiogenic effect of T‐TSP‐1 in vivo compared with a non‐armed 3rd generation oncolytic HSV, T‐01. No apparent apoptotic induction by T‐TSP‐1 was observed for human gastric cancer cell lines TMK‐1 cells but for MKN1 cells in vitro. Arming the viruses with TSP‐1 slightly inhibited their replication in some gastric cancer cell lines, but the viral cytotoxicity was not attenuated. In addition, T‐TSP‐1 exhibited enhanced therapeutic efficacy and inhibition of angiogenesis compared with T‐01 in vivo. In this study, we established a novel armed oncolytic HSV, T‐TSP‐1, which enhanced the antitumor efficacy by providing a combination of direct viral oncolysis with antiangiogenesis. Arming oncolytic HSVs may be a useful therapeutic strategy for gastric cancer therapy.     

High CD46 receptor density determines preferential killing of tumor cells by oncolytic measles virus

Live attenuated Edmonston B strain of measles virus (MV-Edm) is a potent and specific oncolytic agent, but the mechanism underlying its tumor selectivity is unknown. The virus causes cytopathic effects (CPEs) of extensive syncytial formation in tumor cells but minimal damage or cell killing in normal cells. The CPE is dependent on expression of viral proteins and the presence of CD46, the major cellular receptor of MV-Edm. Using a virally encoded soluble marker peptide to provide a quantitative readout of the level of viral gene expression, we determined that tumor cells and normal cells expressed comparable levels of viral proteins. CD46 mediates virus attachment, entry, and virus-induced cell-to-cell fusion. Using engineered cells expressing a range of CD46 densities, we determined that whereas virus entry increased progressively with CD46 density, cell fusion was minimal at low receptor densities but increased dramatically above a threshold density of CD46 receptors. It is well established that tumor cells express abundant CD46 receptors on their surfaces compared with their normal counterparts. Thus, at low CD46 densities typical of normal cells, infection occurs, but intercellular fusion is negligible. At higher densities typical of tumor cells, infection leads to extensive cell fusion. Intercellular fusion also results in enhancement of viral gene expression through recruitment of neighboring uninfected cells into the syncytium, further amplifying the CPE. Discrimination between high and low CD46 receptor density provides a compelling basis for the oncolytic specificity of MV-Edm and establishes MV-Edm as a promising CD46-targeted cancer therapeutic agent.     

Replication of an integrin targeted conditionally replicating adenovirus on primary ovarian cancer spheroids

Replication competent viruses hold promise for treatment of advanced cancers resistant to available therapeutic modalities. Although preliminary clinical results have substantiated their efficacy, preclinical development of these novel approaches is limited by assay substrates. The evaluation of candidate agents could be confounded by differences between primary tumor cells and tumor cell lines, as discordance in the levels of surface receptors relevant for viral entry has been reported. Since primary tumor cells are difficult to analyze ex vivo for longitudinal observation of virus replication, we developed three-dimensional aggregates or spheroids of unpassaged and purified ovarian cancer cells as a means for prolonging primary tumor cell viability and as a three-dimensional in vitro model for replicative viral infection. Ovarian cancer cells purified from ascites samples were sustained for 30 days while retaining the infection profile with tropism modified and unmodified adenoviruses (Ads). Cell line and primary cell spheroids were used to quantitate the replication and oncolytic potency of replicative Ads in preclinical testing for human ovarian cancer trials. Therefore, spheroids provide a method to sustain purified unpassaged primary ovarian cancer cells for extended periods and to allow evaluation of replicative viruses in a three-dimensional model.