Infected Tumor Cells Research Articles

Oncolytic herpes simplex virus treatment of metastatic breast cancer

The high prevalence and poor prognosis of breast cancer provides a strong rationale for developing new treatment strategies and preventive and therapeutic agents. Oncolytic replication-competent herpes simplex virus (HSV) can infect tumor cells, replicating and killing the cells by direct cytopathic effect and then spreading within the tumor. Replication of oncolytic HSV leads to the destruction of the infected tumor cell and release of new virions, which are able to infect adjacent cells until potentially all tumor cells are destroyed. In this study, the cytotoxicity of a third-generation oncolytic HSV vector, designated G47Δ, was examined in human breast cancer cell lines, as well as in immortalized and normal breast cells. A pulmonary metastatic model of breast cancer established in Balb/c nude mice was used to evaluate the efficacy of G47Δ treatment. Systemic treatment by intravenous administration of G47Δ for metastatic lung tumors was initiated 14 days after injection of tumor cells. On Day 56, the mice were sacrificed and tumor nodules on the surface of the lung were counted. G47Δ was highly cytotoxic to breast cancer and immortalized breast cells in vitro at low multiplicities of infection (MOI), while normal breast cells remained viable 5 days after infection. In the pulmonary metastatic model, the average number of surface lung tumor nodules in the G47Δ-treated group was approximately 9‑fold less than in the control-treated group. X-gal staining illustrated viral replication and spread in the tumor cells in vitro and in vivo. In conclusion, G47Δ effectively killed human breast cancer cells and immortalized breast cells but not normal breast cells. Systemic administration of G47Δ by tail vein injection was effective in inhibiting the growth of established breast cancer lung metastases.

Prognostic Value of Epstein-Barr Virus Infected Tumor Cell Size in Patients with Extranodal Natural Killer T-Cell Lymphoma, Nasal Type

Abstract 5191 Backgrounds:Extranodal natural killer T-cell lymphoma, nasal type (nasal ENKTL) is a distinct clinicopathologic entity of lymphoid tumors with variable size and differentiation of tumor cells. Nasal ENKTL is related to infection of the tumor cells with Epstein-Barr virus (EBV) and virtually all cases contain monoclonal episomal EBV DNA and detectable EBV encoded small nuclear RNAs (EBERs). Several clinical factors were known for their relation to the prognosis, but histopathologic prognostic factors of nasal ENKTL have not yet been well established. We evaluated prognostic value of the size and density of EBER-positive tumor cells along with immunohistochemistry (IHC) expression of CD30 and CD56. Patients and Methods:Patients with newly diagnosed nasal ENKTL treated in a single institution (Gachon University Gil Hospital) were evaluated with respect to clinical characteristics, survival, and histopathologic characteristics. IHC were performed using anti-CD56 (DAKO), CD30 (DAKO). EBV RNA was detected by an ISH (in situ hybridization) technique. Paraffin sections were pretreated with xylene followed by treatment with proteinase K and hybridized with FITC-conjugated EBV oligonucleotides (Novocastra) complementary to the nRNA portion of the EBER-1 and EBER-2 genes. The numbers of EBER-positive tumor cells were counted and the nuclear length of tumor cells were measured using a computerized image analysis system (IMT i-Solution Inc., Vancouver, British Columbia, Canada) that included a DP70 Digital camera (Olympus, Tokyo, Japan) installed on an Olympus light microscope (Olympus BX51) and attached to a personal computer. Five independent microscopic fields (at x400 magnification), representing the densest tumor infiltration, were selected for each patient sample to ensure representativeness and homogeneity. Each independent microscopic field contained an approximately 80% tumor ratio. The results were expressed as the mean number of cells (± standard error) in 1 computerized x400 microscopic field (9941.38mm2/field). More than 50 tumor cells were selected for the measurement of nuclear length of tumor cells. The results were expressed as the mean diameter of cells (± standard error). The mean density and mean diameter were used for statistical analysis. Results:Twenty-two patients were analyzed. Median age were 48.5 (range 15–81) and 20 of them were male (91%). Seven (32%) patients were Ann Arbor stage I, and 6 (27%), 3 (14%), and 6 (27%) were stage II, III, IV, respectively. Sixteen patients (73%) received combined chemotherapy and radiotherapy whereas 5 patients (23%) received chemotherapy alone and 1 patient received only radiotherapy. Median of the mean EBER-positive tumor cell diameters of the patients were 7.32 mm (range 5.15–11.27). Median of the mean tumor cell counts of the patients were 71.7/field.Patients with larger mean tumor cell diameters (≥ 7.32 mm) had a poorer event-free survival (EFS), which was defined as survival free from failure or death from any cause, than those with smaller (<7.32 mm) mean cell diameters (table 1 and figure 1). Density of tumor cells (≥71.7 vs. < 71.7/field) and IHC to CD56 or CD30 (≥50% vs. < 50% of expression) had no impact on EFS, whereas previously known clinical factors - local tumor invasion, type of therapy, performance status, and splenomegaly - maintained power enough to predict EFS (table 1).Table 1univariate Cox analysisFactorsPatient No.Hazard ratio (95% CI)pAge (>60)82.72 (0.76–9.79)0.125Local tumor invasion (+)64.26 (1.10–16.51)0.036Cervical nodes (+)91.36 (0.41–4.53)0.616Combined therapy (not doing)63.94 (1.1519–13.47)0.029ECOG PS (2,3,4)514.82 (2.57-85.36)0.003Serum LDH (elevated)91.72 (0.5219–5.70)0.378Ann Arbor stage (III and IV)91.74 (0.53–5.80)0.364Splenomegaly (present)94.52 (1.15-17.73)0.031Mean cell diameter (≥ 7.32 mm)124.73 (1.01–22.16)0.049Mean cell count (≥ 71.7/field)121.91 (0.51–7.23)0.340CD56 (≥ 50% expression)140.57 (0.17–1.91)0.572CD30 (≥ 50% expression)82.51 (0.67–9.38)0.173 [Display omitted] Conclusion:Larger EBER-positive tumor cell size had an adverse impact on EFS in patients with nasal ENKTL. Further larger scale study to define the prognostic value of EBV infected tumor cell size is warranted. Disclosures:No relevant conflicts of interest to declare.

Oncolytic plasmid: A novel strategy for tumor immuno-gene therapy

The oncolytic virus is expected to proliferate in and destroy tumor cells. The virus is also thought to generate antitumor immunity. Virally infected tumor cells express viral antigens on their surfaces. Such tumor cells or their fragments would be taken up by antigen-presenting cells (APCs) together with tumor-associated antigens (TAAs), and facilitated cross-priming of tumor-specific T cells. Virus-specific protein presented on the infected cells therefore played a crucial role in the enhancement of the adaptive antitumor immunity. In this study, a plasmid encoding adenovirus protein, the adenovirus death protein (ADP), was constructed, and a very fine complex of the plasmid with polyethylenimine (PEI) and chondroitin sulfate (CS) was injected into tumor-bearing mice. Transfection of the ADP gene was shown to suppress tumor growth as effectively as granulocyte-macrophage colony-stimulating factor (GM-CSF) transfection. When mice were administered plasmid coding ADP (pDNA-ADP) to generate an immune response to ADP prior to therapy, transfection of the ADP gene induced a much higher level of tumor growth suppression than that found in the non-immunized mice. An evident synergistic effect of ADP and GM-CSF genes was also observed, and at a pDNA-ADP/pDNA-GM-CSF ratio of 4:1, significant suppression of tumor growth was achieved even in the non-immunized mice.

Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies

BackgroundOncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies.MethodsHere, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system.ResultsUsing fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) renal excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay.ConclusionGusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells.

Systemic administration of 3-bromopyruvate in treating disseminated aggressive lymphoma

The Warburg hypothesis states that aggressive cancers obtain much of their adenosine triphosphate (ATP) by metabolizing glucose directly to lactic acid. As a result of its high tumor selectivity, 3-bromopyruvic acid (3-BrPA), a well-known inhibitor of energy metabolism, has been proposed as a specific anticancer agent. We investigated the effect of 3-BrPA in a mouse model of aggressive metastatic lymphoma. Epstein-Barr-virus-infected human Raji lymphoma cells with lentivirally transfected green fluorescent protein and luciferase were incubated with RPMI/fetal bovine serum, and various concentrations of 3-BrPA were used to determine the LD50 in vitro. In total, 18 severely combined immunodeficient mice were injected with 1 million human Raji lymphoma cells via the tail vein. Using bioluminescent imaging, tumor growth was measured daily for 12 days to determine the tumor burden. At day 0 (start of treatment), the mice were randomized. Six mice received 10 mg/kg 3-BrPA i.p. daily for 7 days, 6 mice received 1 treatment at day 0, and 6 mice received the control buffer. Tumor growth was assessed daily from day 0 until day 7 using bioluminescent imaging. All data were normalized to acquisition time (luminescence/second; L/s). Body weight was measured daily to determine the toxicity of 3-BrPA. The LD50 for Raji lymphoma cells exposed to 3-BrPA in vitro was 11 μM with an extremely steep dose response curve. At day 0, tumor activity medians in the group with daily treatment was 2131 L/s (244-12,725), with a 1-day dose of 3095 L/s (523-9650) and in the nontreated control group, 2997 L/s (1521-6911). In mice treated with a daily dose of 10 mg/kg 3-BrPa for 7 days, a significant reduction in tumor activity was found during the whole treatment period compared with the control mice (P = 0.0043 at day 7). In mice with a single treatment at day 0, growth delay was only evident at day 2 (P = 0.0152 at day 2) but not for the rest of the observation period. The only manifestation of toxicity of the daily administration of 10 mg/kg 3-BrPA was a reduction in body weight. Body weight at day 0 was 17.22 g ± 0.84 g in the treatment group and 17.58 g ± 0.86 g in the control group. Body weight at day +6 was 15.02 g ± 2.04 g in the treated group and 19.4 g ± 0.63 g in the control group. 3-BrPA demonstrated a significant positive tumor response both in vitro and in vivo. This, to our knowledge, is the first report of the use of 3-BrPA in a systemic tumor model. Based on these data, 3-BrPA holds promise for treatment of systemic metastatic cancers.

Virotherapy induces massive infiltration of neutrophils in a subset of tumors defined by a strong endogenous interferon response activity

Oncolytic virotherapy has shown substantial promises as an alternative therapeutic modality for solid tumors in both preclinical studies and clinical trials. The main therapeutic activity of virotherapy derives from the direct lytic effect associated with virus replication and the induction of host immune responses to the infected tumor cells. Here we show that some human and murine tumor cell lines are highly resistant to the lytic effect of a type II herpes simplex virus-derived oncolytic virus, FusOn-H2, which was constructed by deleting the N-terminal region of the ICP10 gene. However, these tumor cells still respond exceptionally well to FusOn-H2 virotherapy in vivo. Histological examination of the treated tumors revealed that, in contrast to tumors supporting FusOn-H2 replication, implants of these highly resistant lines showed massive infiltration of neutrophils after virotherapy. Further analysis showed that there is a correlation between an intrinsically strong interferon response activity and the recruitment of neutrophils in these tumors. These results suggest that an innate immune response mainly represented by neutrophils may be part of the virotherapy-mediated antitumor mechanism in these tumors.

Newly emerging therapies targeting viral-related lymphomas.

Gamma-(γ)-herpes virus lymphomas comprise a heterogenous group of B-cell and T-cell neoplasms most commonly associated with Epstein-Barr virus and rarely human herpes virus-8 infection. Adult T-cell leukemia/lymphoma (ATLL) is a unique disease entity caused by the human T-cell lymphotrophic virus, type 1 (HTLV-I), the only retrovirus known to cause cancer in humans. Viral lymphomas behave aggressively and disproportionally affect immunocompromised individuals and those living in underdeveloped regions. These diseases are often difficult to treat with conventional approaches. Despite recent advancements using cytotoxic, lymphoma-specific, and adoptive therapies, the long-term outcome of patients with γ-herpesvirus lymphomas occurring in severely immunocompromised patients and ATLL continues to be poor. Lytic-inducing therapies targeting NF-κB, and viral and tumor cell epigenetic mechanisms afford the advantage of exploiting the intrinsic presence of oncogenic viruses to eradicate infected tumor cells. In this review, viral-related lymphomas and newly emerging clinical approaches targeting viral latency are discussed.

Effect of a caspase inhibitor, zVADfmk, on the inhibition of breast cancer cells by herpes simplex virus type 1

The oncolytic effects of herpes simplex virus-1 (HSV-1) are limited, possibly because of premature death of infected cells by apoptosis, which limits the amount of progeny virus that is produced. It has been proposed that inhibition of apoptosis in infected tumor cells would allow increased viral persistence, replication and therapeutic effect. To test this hypothesis, we infected monocyte chemoattractant factor-7 (MCF-7) and MDA-MB-231 breast cancer cells with HSV-1 strain 17(+) and 17Δγ34.5 in the presence or absence of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVADfmk), a pan-caspase inhibitor. At low doses of HSV-1 strain 17(+) and 17Δγ34.5, the growth of MCF-7 cells was reduced to 37% or 42%, respectively, of uninfected cells. However, when cells were infected in the presence of zVADfmk, cell growth was further reduced to 24 and 33%. Similar results were seen in MDA-MB-231 cells. Cells treated with zVADfmk contained roughly 10 times more infectious viral particles than cells infected without zVADfmk, as shown by both plaque-forming and quantitative polymerase chain reaction assays. To model the situation within an infected tumor, supernatant fluids were collected from infected and non-infected cell cultures and then passed to non-infected cells. In the presence of zVADfmk, the cell growth inhibitory effect became stronger with repeated passages and was attributed to viral replication, because it could be prevented by anti-HSV antibody. These results suggest that caspases represent a novel target for drugs that increase the therapeutic efficacy of oncolytic herpes viruses against breast cancer.

AMC075: A sequential phase I/randomized phase II trial of vorinostat and risk-adapted chemotherapy with rituximab in HIV-related B-cell non-Hodgkin’s lymphoma.

e13002 Background: HIV infected individuals are at an increased risk of developing NHL and they usually present with more aggressive disease. Diffuse large B-cell lymphoma (DLBCL) is the most commonly encountered HIV-NHL. Unlike non-HIV, one-third of these cases are EBV+. Recent phase II trials suggest a superior outcome using infusional EPOCH over conventional CHOP, especially in high-risk groups. This warrants the investigation of risk-adapted chemotherapy and new drugs which can target the biology of HIV-DLBCL. At the AIDS Malignancy Consortium we have developed a phase I/randomized phase II trial using risk-adapted chemotherapy with the histone deacetylase inhibitor (HDAC-I) vorinostat (V) for untreated HIV-DLBCL. HDAC-Is have anti-tumor activity in lymphomas. These drugs alter the functional state of chromatin, facilitating the expression of abnormally silenced genes in neoplastic disease and latent viruses. We hypothesize that V will enhance the anti-tumor effect of chemotherapy in HIV-DLBCL and transiently activate HIV and latent oncogenic γ-herpesviruses thus eliminating infected reservoirs under antiviral treatment and inducing lysis of EBV infected tumor cells. Methods: Eligibility: HIV+ subjects with untreated CD20+ DLBCL and CD4 count ≥50 cell/mm3 Phase I (27-40 subjects): Will determine the recommended phase II dose (RPTD) of V in combination with R-CHOP in low risk disease (aa-IPI 0-1, Ki-67<80%, GCB subtype) and R-DA-EPOCH in high risk groups (aa-IPI 2-3, Ki-67≥80% or ABC subtype). The starting dose of V will be 300 mg on Days 1-5 on each 21-day cycle, and will be escalated to a maximum of 500 mg on a 3 + 3 design. Randomized phase II (90 subjects): Will determine the overall toxicity and efficacy of R-CHOP (in low-risk disease) or R-DA-EPOCH (in high-risk disease) with and without V. Correlative studies will measure the effect of V and chemotherapy on latent HIV infected reservoirs, and EBV and HHV-8. Results: This study is currently open to accrual nationwide. Conclusions: We have designed a risk-adapted chemotherapy approach for the treatment of HIV-DLBCL incorporating the novel use of an agent which can target the biology of these tumors.

Disparate Antiviral Responses in Molluscum contagiosum Virus–Induced Skin Lesions

Molluscum contagiosum virus (MCV) is a poxvirus that causes tumor-like skin lesions. New evidence indicates that plasmacytoid dendritic cells, type I interferon production, and interferon-induced dendritic cells have prominent roles in anti-MCV responses, and these features characterize the inflammatory response in lesions that will likely undergo spontaneous regression.

Adaptive Antiviral Immunity Is a Determinant of the Therapeutic Success of Oncolytic Virotherapy

Oncolytic virotherapy, the selective killing of tumor cells by oncolytic viruses (OVs), has emerged as a promising avenue of anticancer research. We have previously shown that KM100, a Herpes simplex virus type-1 (HSV) deficient for infected cell protein 0 (ICP0), possesses substantial oncolytic properties in vitro and has antitumor efficacy in vivo, in part by inducing antitumor immunity. Here, we illustrate through T-cell immunodepletion studies in nontolerized tumor-associated antigen models of breast cancer that KM100 treatment promotes antiviral and antitumor CD8(+) cytotoxic T-cell responses necessary for complete tumor regression. In tolerized tumor-associated antigen models of breast cancer, antiviral CD8(+) cytotoxic T-cell responses against infected tumor cells correlated with the induction of significant tumoristasis in the absence of tumor-associated antigen-specific CD8(+) cytotoxic T-cells. To enhance oncolysis, we tested a more cytopathic ICP0-null HSV and a vesicular stomatitis virus M protein mutant and found that despite improved in vitro replication, oncolysis in vivo did not improve. These studies illustrate that the in vitro cytolytic properties of OVs are poor prognostic indicators of in vivo antitumor activity, and underscore the importance of adaptive antiviral CD8(+) cytotoxic T-cells in effective cancer virotherapy.

Mathematical Modeling and Analysis of Tumor Therapy with Oncolytic Virus

In this paper, we have proposed and analyzed a nonlinear mathematical model for the study of interaction between tumor cells and oncolytic viruses. The model is analyzed using stability theory of differential equa- tions. Positive equilibrium points of the system are investigated and their stability analysis is carried out. Moreover, the numerical simulation of the proposed model is also performed by using fourth order Runge- Kutta method which supports the theoretical findings. It is found that both infected and uninfected tumor cells and hence tumor load can be eliminated with time, and complete recovery is possible because of virus therapy, if certain conditions are satisfied. It is further found that the system appears to exhibit periodic limit cycles and chaotic attractors for some ranges of the system parameters.

The replicability of oncolytic virus: Defining conditions in tumor virotherapy

The replicability of an oncolytic virus is measured by its burst size. The burst size is the number of new viruses coming out from a lysis of an infected tumor cell. Some clinical evidences show that the burst size of an oncolytic virus is a defining parameter for the success of virotherapy. This article analyzes a basic mathematical model that includes burst size for oncolytic virotherapy. The analysis of the model shows that there are two threshold values of the burst size: below the first threshold, the tumor always grows to its maximum (carrying capacity) size; while passing this threshold, there is a locally stable positive equilibrium solution appearing through transcritical bifurcation; while at or above the second threshold, there exits one or three families of periodic solutions arising from Hopf bifurcations. The study suggests that the tumor load can drop to a undetectable level either during the oscillation or when the burst size is large enough.

402 JX-594, a targeted multi-mechanistic oncolytic poxvirus, infects tumor vasculature and causes acute tumor vascular disruption and necrosis in advanced cancer patients

Successful cancer control relies on overcoming resistance to cell death and on activation of host antitumor immunity. Oncolytic viruses are particularly attractive in this regard, as they lyse infected tumor cells and trigger robust immune responses during the infection. However, repeated injections of the same virus promote antiviral rather than antitumor immunity and tumors may mount innate antiviral defenses to restrict oncolytic virus replication. In this article, we have explored if alternating the therapy virus could circumvent these problems. We demonstrate in two virus-resistant animal models a substantial delay in antiviral immune- and innate cellular response induction by alternating injections of two immunologically distinct oncolytic viruses, adenovirus, and vaccinia virus. Our results are in support of clinical development of heterologous adeno-/vaccinia virus therapy of cancer.

Identification of the BCL2/adenovirus E1B-19K protein-interacting protein 2 (BNIP-2) as a granzyme B target during human natural killer cell-mediated killing

Cytotoxic lymphocytes eliminate infected cells and tumours via the perforin-mediated delivery of pro-apoptotic serine proteases known as granzymes. Granzyme B triggers apoptosis via the cleavage of a repertoire of cellular proteins, leading to caspase activation and mitochondrial depolarization. A simple bioinformatics strategy identified a candidate granzyme B cleavage site in the widely expressed BNIP-2 (BCL2/adenovirus E1B-19K protein-interacting protein 2). Granzyme B cleaved recombinant BNIP-2 in vitro and endogenous BNIP-2 was cleaved during the NK (natural killer) cell-mediated killing of tumour cells. Cleavage required the site identified in the bioinformatics screen and was caspase-independent. Expression of either full-length BNIP-2 or a truncated molecule mimicking the granzyme B cleaved form was pro-apoptotic and led to the caspase-dependent cleavage of BNIP-2 at a site distinct from granzyme B cleavage. Inhibition of BNIP-2 expression did not affect the susceptibility to NK cell-mediated killing. Furthermore, target cells in which BID (BH3-interacting domain death agonist) expression was inhibited also remained highly susceptible to NK cell-mediated killing, revealing redundancy in the pro-apoptotic response to human cytotoxic lymphocytes. Such redundancy reduces the opportunity for escape from apoptosis induction and maximizes the chances of immune-mediated clearance of infected cells or tumour cells.

Dual E1A oncolytic adenovirus: targeting tumor heterogeneity with two independent cancer-specific promoter elements, DF3/MUC1 and hTERT

The therapeutic utility of oncolytic adenoviruses controlled by a single, tumor-specific regulatory element may be limited by the intra- and inter-tumoral heterogeneity that characterizes many cancers. To address this issue, we constructed an oncolytic adenovirus that uses two distinct tumor-specific promoters (DF3/Muc1 and hTERT) to drive separate E1A expression cassettes, in combination with deletion of the viral E1B region, which confers additional tumor selectivity and increased oncolytic activity. The resultant virus, Adeno-DF3-E1A/hTERT-E1A, induced higher levels of E1A oncoprotein, enhanced oncolysis, and an earlier and higher apoptotic index in infected tumor cells than following infection with Adeno-hTERT-E1A, which harbors a single hTERT promoter-driven E1A cassette. In isolated U251 human gliosarcoma cell holoclones (putative cancer stem cells), where DF3/Muc1 expression is substantially enriched and hTERT expression is decreased compared to the parental U251 cell population, E1A production and oncolysis were strongly decreased following infection with Adeno-hTERT-E1A but not Adeno-DF3-E1A/hTERT-E1A. The strong oncolytic activity of Adeno-DF3-E1A/hTERT-E1A translated into superior anti-tumor activity over Adeno-hTERT-E1A in vivo in a U251 solid tumor xenograft model, where hTERT levels were >90% suppressed and the DF3/Muc1 to hTERT expression ratio was substantially increased compared to cultured U251 cells. The enhanced anti-tumor activity of the dual-targeted Adeno-DF3-E1A/hTERT-E1A was achieved despite premature viral host cell death and decreased production of functional viral progeny, which limited tumor cell spread of the viral infection. These findings highlight the therapeutic benefit of targeting oncolytic viruses to heterogeneous tumor cell populations.

The detection of CMV pp65 and IE1 in glioblastoma multiforme

Glioblastoma multiforme (GBM) is a highly lethal brain tumor affecting children and adults, with the majority of affected individuals dying from their disease by 2 years following diagnosis. Other groups have reported the association of cytomegalovirus (CMV) with GBM, and we sought to confirm these findings in a large series of patients with primary GBM from our institution. Immunohistochemical analysis of paraffin embedded tissue sections was performed on 49 newly diagnosed GBM tumors, the largest series reported to date. We confirmed the presence of CMV pp65 on 25/49 (51%) and of IE1 on 8/49 (16%) of these tumors. While pp65 and IE1 are generally found in the nucleus of cells that are permissibly infected by CMV, GBM in this series had mostly cytoplasmic staining, with only 16% having nuclear staining for one or both of these antigens. We infected GBM cell lines with a laboratory strain of CMV, and found that most of the staining was cytoplasmic, with some perinuclear localization of IE1. To test the potential for CMV infected GBM cells to be recognized by CMV pp65 and IE1 specific cytotoxic T lymphocytes (CTL), we used CMV infected GBM cell lines in cytotoxicity assays with human leukocyte antigen partially matched CMV CTL. Lysis of CMV infected GBM tumor cells was accentuated by pre-treating these cell lines with either the demethylating agent decitabine or interferon-γ, both of which were shown to increase MHC Class I and II expression on tumor cells in vitro. These studies confirm the presence of CMV pp65 or IE1 on approximately half of GBM, with the possibility that CMV positive tumor cells can be recognized by CMV pp65/IE1 specific T cells.

Increasing the Efficacy of Oncolytic Adenovirus Vectors

Oncolytic adenovirus (Ad) vectors present a new modality to treat cancer. These vectors attack tumors via replicating in and killing cancer cells. Upon completion of the vector replication cycle, the infected tumor cell lyses and releases progeny virions that are capable of infecting neighboring tumor cells. Repeated cycles of vector replication and cell lysis can destroy the tumor. Numerous Ad vectors have been generated and tested, some of them reaching human clinical trials. In 2005, the first oncolytic Ad was approved for the treatment of head-and-neck cancer by the Chinese FDA. Oncolytic Ads have been proven to be safe, with no serious adverse effects reported even when high doses of the vector were injected intravenously. The vectors demonstrated modest anti-tumor effect when applied as a single agent; their efficacy improved when they were combined with another modality. The efficacy of oncolytic Ads can be improved using various approaches, including vector design, delivery techniques, and ancillary treatment, which will be discussed in this review.

Potentiating Cancer Immunotherapy Using an Oncolytic Virus

Oncolytic viruses (OVs) are highly immunogenic and this limits their use in immune-competent hosts. Although immunosuppression may improve viral oncolysis, this gain is likely achieved at the cost of antitumoral immunity. We have developed a strategy wherein the immune response against the OV leads to enhanced therapeutic outcomes. We demonstrate that immunization with an adenoviral (Ad) vaccine before treatment with an oncolytic vesicular stomatitis virus (VSV) expressing the same tumor antigen (Ag) leads to significantly enhanced antitumoral immunity. Intratumoral replication of VSV was minimally attenuated in Ad-immunized hosts but extending the interval between treatments reduced the attenuating effect and further increased antitumoral immunity. More importantly, our combination approach shifted the immune response from viral Ags to tumor Ags and further reduced OV replication in normal tissues, leading to enhancements in both efficacy and safety. These studies also highlight the benefits of using a replicating, OV to boost a pre-existing antitumoral immune response as this approach generated larger responses versus tumor Ag in tumor-bearing hosts than could be achieved in tumor-free hosts. This strategy should be applicable to other vector combinations, tumor Ags, and tumor targets.

Low dosage 5-fluorouracil increases the transfection efficiency of Ad/VEGF-A in mouse lung carcinoma cell line LA795 and inhibits tumor growth

Adenovirus vectors were widely used in gene therapy for tumors. We used adenovirus vector to transfer small interfering RNA (siRNA) against vascular epithelium growth factor A (VEGF-A) molecules to mouse lung adenoma LA795 cells and used low dose of chemotherapeutic drugs to further elevate the infection efficiency of adenovirus vector in and therapeutic effect of RNAi on tumor cells. LA795 cells were infected by Ad/EGFP and treated with different dosages of gemcitabin, epirubicin, cisplatin, or 5-fluorouracil (5-FU). Cells were observed under fluorescence microscope continuously using green fluorescent protein (GFP) as the reporter gene. The percentage of GFP-positive cells and fluorescent intensity were tested by flow cytometry to determine optimum concentrations of drugs. Ad/siVEGF-A containing VEGF-A siRNA was constructed. Real-time PCR and ELISA were applied to measure the expression level of VEGF-A after LA795 cells were infected by Ad/siVEGF-A and treated with 5-FU. The combination of Ad/siVEGF-A and 5-FU was also applied in treating subcutaneous tumor in mice. Low dose of 5-FU elevated the Ad/EGFP infection in LA795 cells significantly, and also enhanced the effect of Ad/siVEGF-A in down-regulating VEGF-A mRNA and protein levels in tumor cells. When used in tumor in vivo, the combination strategy repressed tumor growth effectively. Low dose of 5-FU can enhance the capability of adenovirus infecting tumor cells and promote the efficiency of gene therapy by adenovirus.