The purpose of this original work is to examine the ability of an oncolytic vaccinia virus expressing the human sodium iodine transporter (hNIS) to provide real time monitoring of viral therapy and effective treatment of malignant pleural mesothelioma (MPM).
Infectivity and cytotoxic effect of GLV-1h153 on mesothelioma cell lines of all histologic subtypes was assayed in vitro. Viral replication was examined by standard viral plaque assay. Orthotopic MPM xenografts were generated in athymic nude mice and treated with intrapleural GLV-1h153 and assessed for effect on tumor burden and survival. Orthotopic tumors were also imaged on SPECT/CT after 131I administration.
GLV-1h153 infected and killed all cell lines in a time and concentration dependent manner. Viral replication demonstrated over a 2.5 log increase in titer over 4 days. Intrapleural treatment of orthotopic MPM xenografts resulted in a significant reduction in tumor burden one week after treatment and an improvement in survival. Infection of orthotopic xenografts was both therapeutic and facilitated monitoring by 131I-SPECT/CT via expression of hNIS in infected tissue.
Our results suggest GLV-1h153 is a promising therapeutic agent for MPM and warrants further investigation.
Virotherapy on the basis of oncolytic vaccinia virus (VACV) infection is a promising approach for cancer therapy. In this study we describe the establishment of a new preclinical model of feline mammary carcinoma (FMC) using a recently established cancer cell line, DT09/06. In addition, we evaluated a recombinant vaccinia virus strain, GLV-5b451, expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as an oncolytic agent against FMC. Cell culture data demonstrate that GLV-5b451 virus efficiently infected, replicated in and destroyed DT09/06 cancer cells. In the selected xenografts of FMC, a single systemic administration of GLV-5b451 led to significant inhibition of tumor growth in comparison to untreated tumor-bearing mice. Furthermore, tumor-specific virus infection led to overproduction of functional scAb GLAF-2, which caused drastic reduction of intratumoral VEGF levels and inhibition of angiogenesis.
In summary, here we have shown, for the first time, that the vaccinia virus strains and especially GLV-5b451 have great potential for effective treatment of FMC in animal model.
The capacity of the recombinant Vaccinia virus GLV-1h68 as a single agent to efficiently treat different human or canine cancers has been shown in several preclinical studies. Currently, its human safety and efficacy are investigated in phase I/II clinical trials. In this study we set out to evaluate the oncolytic activity of GLV-1h68 in the human lung adenocarcinoma cell line PC14PE6-RFP in cell cultures and analyzed the antitumor potency of a combined treatment strategy consisting of GLV-1h68 and cyclophosphamide (CPA) in a mouse model of PC14PE6-RFP lung adenocarcinoma.
PC14PE6-RFP cells were treated in cell culture with GLV-1h68. Viral replication and cell survival were determined by plaque assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Subcutaneously implanted PC14PE6-RFP xenografts were treated by systemic injection of GLV-1h68, CPA or a combination of both. Tumor growth and viral biodistribution were monitored and immune-related antigen profiling of tumor lysates was performed.
GLV-1h68 efficiently infected, replicated in and lysed human PC14PE6-RFP cells in cell cultures. PC14PE6-RFP tumors were efficiently colonized by GLV-1h68 leading to much delayed tumor growth in PC14PE6-RFP tumor-bearing nude mice. Combination treatment with GLV-1h68 and CPA significantly improved the antitumor efficacy of GLV-1h68 and led to an increased viral distribution within the tumors. Pro-inflammatory cytokines and chemokines were distinctly elevated in tumors of GLV-1h68-treated mice. Factors expressed by endothelial cells or present in the blood were decreased after combination treatment. A complete loss in the hemorrhagic phenotype of the PC14PE6-RFP tumors and a decrease in the number of blood vessels after combination treatment could be observed.
CPA and GLV-1h68 have synergistic antitumor effects on PC14PE6-RFP xenografts. We strongly suppose that in the PC14PE6-RFP model the enhanced tumor growth inhibition achieved by combining GLV-1h68 with CPA is due to an effect on the vasculature rather than an immunosuppressive action of CPA. These results provide evidence to support further preclinical studies of combining GLV-1h68 and CPA in other highly angiogenic tumor models. Moreover, data presented here demonstrate that CPA can be combined successfully with GLV-1h68 based oncolytic virus therapy and therefore might be promising as combination therapy in human clinical trials.
Vaccinia virus; Chemotherapy; Combination therapy; Cyclophosphamide; Lung cancer
More than 90% of cancer mortalities are due to cancer that has metastasized. Therefore, it is crucial to intensify research on metastasis formation and therapy. Here, we describe for the first time the metastasizing ability of the human cervical cancer cell line C33A in athymic nude mice after subcutaneous implantation of tumor cells. In this model, we demonstrated a steady progression of lumbar and renal lymph node metastases during tumor development. Besides predominantly occurring lymphatic metastases, we visualized the formation of hematogenous metastases utilizing red fluorescent protein (RFP) expressing C33A-RFP cells. RFP positive cancer cells were found migrating in blood vessels and forming micrometastases in lungs of tumor-bearing mice. Next, we set out to analyze the influence of oncolytic virotherapy in the C33A-RFP model and demonstrated an efficient virus-mediated reduction of tumor size and metastatic burden. These results suggest the C33A-RFP cervical cancer model as a new platform to analyze cancer metastases as well as to test novel treatment options to combat metastases.
Cancer is the leading cause of disease-related death in companion animals such as dogs and cats. Despite recent progress in the diagnosis and treatment of advanced canine and feline cancer, overall patient treatment outcome has not been substantially improved. Virotherapy using oncolytic viruses is one promising new strategy for cancer therapy. Oncolytic viruses (OVs) preferentially infect and lyse cancer cells, without causing excessive damage to surrounding healthy tissue, and initiate tumor-specific immunity. The current review describes the use of different oncolytic viruses for cancer therapy and their application to canine and feline cancer.
cancer; canine and feline cancer therapy; oncolytic virus; oncolysis
Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is one novel approach for canine cancer therapy. In this study we described for the first time the characterization and the use of new VACV strain LIVP6.1.1 as an oncolytic agent against canine cancer in a panel of four canine cancer cell lines including: soft tissue sarcoma (STSA-1), melanoma (CHAS), osteosarcoma (D-17) and prostate carcinoma (DT08/40). Cell culture data demonstrated that LIVP6.1.1 efficiently infected and destroyed all four tested canine cancer cell lines. In two different xenograft models on the basis of the canine soft tissue sarcoma STSA-1 and the prostate carcinoma DT08/40 cell lines, a systemic administration of the LIVP6.1.1 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice.
In summary, the pre-clinical evaluation has demonstrated the efficacy of LIVP6.1.1 for canine cancer therapy. Furthermore, a clinical trial with canine cancer patients has already been started.
cancer; canine cancer therapy; oncolytic virus; oncolysis; canine xenografts
Gastric cancers have poor overall survival despite recent advancements in early detection methods, endoscopic resection techniques, and chemotherapy treatments. Vaccinia viral therapy has had promising therapeutic potential for various cancers and has a great safety profile. We investigated the therapeutic efficacy of a novel genetically-engineered vaccinia virus carrying the human sodium iodide symporter (hNIS) gene, GLV-1 h153, on gastric cancers and its potential utility for imaging with 99mTc pertechnetate scintigraphy and 124I positron emission tomography (PET).
GLV-1 h153 was tested against five human gastric cancer cell lines using cytotoxicity and standard viral plaque assays. In vivo, subcutaneous flank tumors were generated in nude mice with human gastric cancer cells, MKN-74. Tumors were subsequently injected with either GLV-1 h153 or PBS and followed for tumor growth. 99mTc pertechnetate scintigraphy and 124I microPET imaging were performed.
GFP expression, a surrogate for viral infectivity, confirmed viral infection by 24 hours. At a multiplicity of infection (MOI) of 1, GLV-1 h153 achieved > 90% cytotoxicity in MNK-74, OCUM-2MD3, and AGS over 9 days, and >70% cytotoxicity in MNK- 45 and TMK-1. In vivo, GLV-1 h153 was effective in treating xenografts (p < 0.001) after 2 weeks of treatment. GLV-1 h153-infected tumors were readily imaged by 99mTc pertechnetate scintigraphy and 124I microPET imaging 2 days after treatment.
GLV-1 h153 is an effective oncolytic virus expressing the hNIS protein that can efficiently regress gastric tumors and allow deep-tissue imaging. These data encourages its continued investigation in clinical settings.
Oncolytic viral therapy; GLV-1 h153; Gastric cancer; Human sodium iodide symporter (hNIS)
Vaccinia virus (VACV) has emerged as an attractive tool in oncolytic virotherapy. VACV replication efficiency plays a crucial role in the therapeutic outcome. However, little is known about the influence of host factors on viral replication efficiency and permissiveness of a host cell line to infection and oncolysis. In this study, replication of the attenuated VACV GLV-1h68 strain and three wild-type VACV isolates was determined in two autologous human melanoma cell lines (888-MEL and 1936-MEL). Host gene expression and viral gene expression in infected cells were evaluated via respective expression array platforms.
Microarray analyses followed by sequential statistical approaches characterized human genes that change specifically due to virus infection. Viral gene transcription correlated with viral replication in a time-dependent manner. A set of human genes revealed strong correlations with the respective viral gene expression. Finally we identified a set of human genes with possible predictive value for viral replication in an independent dataset.
The results demonstrate a probable correlation between viral replication, early gene expression, and the respective host response, and thus a possible involvement of human host factors in viral early replication. The characterization of human target genes that influence viral replication could help answer the question of host cell permissiveness to oncolytic virotherapy and provide important information for the development of novel recombinant vaccinia viruses with improved features to enhance replication rate and hence trigger therapeutic outcome.
Reinboth and colleagues use a whole genome human array and a custom-made vaccinia virus (VACV) array to simultaneously analyze viral and host gene expression. Using this approach they identify a set of human genes that may influence viral replication and help to answer questions pertaining to host cell permissiveness during oncolytic virotherapy.
Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.
Glioblastoma multiforme (GBM) is one of the most aggressive forms of cancer with a high rate of recurrence. We propose a novel oncolytic vaccinia virus (VACV)-based therapy using expression of the bone morphogenetic protein (BMP)-4 for treating GBM and preventing recurrence.
We have utilized clinically relevant, orthotopic xenograft models of GBM based on tumor-biopsy derived, primary cancer stem cell (CSC) lines. One of the cell lines, after being transduced with a cDNA encoding firefly luciferase, could be used for real time tumor imaging. A VACV that expresses BMP-4 was constructed and utilized for infecting several primary glioma cultures besides conventional serum-grown glioma cell lines. This virus was also delivered intracranially upon implantation of the GBM CSCs in mice to determine effects on tumor growth.
We found that the VACV that overexpresses BMP-4 demonstrated heightened replication and cytotoxic activity in GBM CSC cultures with a broad spectrum of activity across several different patient-biopsy cultures. Intracranial inoculation of mice with this virus resulted in a tumor size equal to or below that at the time of injection. This resulted in survival of 100% of the treated mice up to 84 days post inoculation, significantly superior to that of a VACV lacking BMP-4 expression. When mice with a higher tumor burden were injected with the VACV lacking BMP-4, 80% of the mice showed tumor recurrence. In contrast, no recurrence was seen when mice were injected with the VACV expressing BMP-4, possibly due to induction of differentiation in the CSC population and subsequently serving as a better host for VACV infection and oncolysis. This lack of recurrence resulted in superior survival in the BMP-4 VACV treated group.
Based on these findings we propose a novel VACV therapy for treating GBM, which would allow tumor specific production of drugs in the future in combination with BMPs which would simultaneously control tumor maintenance and facilitate CSC differentiation, respectively, thereby causing sustained tumor regression without recurrence.
Vaccinia virus (VACV); Glioblastoma multiforme (GBM); Bone morphogenetic protein (BMP); Cancer stem cells (CSCs) and differentiation
Approximately one fourth of bone and soft-tissue sarcomas recur after prior treatment. GLV-1h68 is a recombinant, replication-competent vaccinia virus that has been shown to have oncolytic effects against many human cancer types. We sought to determine whether GLV-1h68 could selectively target and lyse a panel of human bone and soft-tissue sarcoma cell lines in vitro and in vivo.
GLV-1h68 was tested in a panel of four cell lines including: fibrosarcoma HT-1080, osteosarcoma U-2OS, fibrohistiocytoma M-805 and rhabdomyosarcoma HTB-82. Gene expression, infectivity, viral proliferation, and cytotoxicity were characterized in vitro. HT-1080 xenograft flank tumors grown in vivo were injected intratumorally with a single dose of GLV-1h68.
All four cell lines supported robust viral transgene expression in vitro. At a multiplicity of infection (MOI) of 5, GLV-1h68 was cytotoxic to three cell lines, resulting in >80% cytotoxicity over 7 days. In vivo, a single injection of GLV-1h68 into HT-1080 xenografts exhibited localized intratumoral luciferase activity peaking at days 2–4, with gradual resolution over 8 days and no evidence of spread to normal tissues. Treated animals exhibited near-complete tumor regression over a 28-day period without observed toxicity.
GLV-1h68 has potent direct oncolytic effects against human sarcoma in vitro and in vivo. Recombinant vaccinia oncolytic virotherapy could provide a new platform for the treatment of patients with bone and soft tissue sarcomas. Future clinical trials investigating oncolytic vaccinia as a therapy for sarcomas are warranted.
oncolysis; replication-competent; recombinant; virus
The 27th annual meeting of the Society for Immunotherapy of Cancer (SITC) was held on October 26–28, 2012 in North Bethesda, Maryland and the highlights of the meeting are summarized. The topics covered at this meeting included advances in cancer treatment using adoptive cell therapy (ACT), oncolytic viruses, dendritic cells (DCs), immune check point modulators and combination therapies. Advances in immune editing of cancer, immune modulation by cancer and the tumor microenvironment were also discussed as were advances in single cell analysis and the manufacture and potency testing of tumor infiltrating lymphocytes (TIL).
Immunotherapy; Cancer; Adoptive cellular therapy
Despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers.
Oncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models.
In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke’s type A-stage HCT-116 and Duke’s type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls.
The anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression.
Cancer; Vaccinia virus; Colorectal; Oncolytic virotherapy; Metastasis
Surgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model.
GLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 μCi of 124I-iodide.
Viral infectivity, measured by green fluorescent protein (GFP) expression, was time- and concentration-dependent. All cell lines showed less than 10% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( <10,000-fold increase from the initial viral dose ) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm3 versus 168 mm3 in untreated controls (P < 0.05).
This is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors.
Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate 19F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy.
The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by 1H/19F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the 19F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the 19F signal hot spots and CD68+-macrophages. Thereby, the CD68+-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the 19F signal correlated with the extent of viral spreading within tumors.
These results suggest 19F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, 19F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response.
Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor) single-chain antibody (scAb) GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.
Recently, we showed that the oncolytic vaccinia virus GLV-1h68 has a significant therapeutic potential in treating lymph node metastases of human PC-3 prostate carcinoma in tumor xenografts. In this study, underlying mechanisms of the virus-mediated metastases reduction were analyzed. Immunohistochemistry demonstrated that virus-treatment resulted in a drastically decrease of blood and lymph vessels, representing essential routes for PC-3 cell migration, in both tumors and metastases. Thus, GLV-1h68 drastically reduced essential routes for the metastatic spread of PC-3 cells. Furthermore, analysis of viral distribution in GLV-1h68-injected tumor-bearing mice by plaque assays, revealed significantly higher virus titers in metastases compared to solid tumors. To elucidate conditions potentially mediating the preferential viral colonization and eradication of metastases, microenvironmental components of uninfected tumors and metastases were compared by microscopic studies. These analyses revealed that PC-3 lymph node metastases showed increased vascular permeability, higher proliferation status of tumor cells as determined by BrdU- and Ki-67 assays and lesser necrosis of PC-3 cells than solid tumors. Moreover, an increased number of immune cells (MHCII+/CD68+ macrophages, MHCII+/CD19+ B lymphocytes) combined with an up-regulated expression of pro-inflammatory cytokines was observed in metastases in comparison to primary PC-3 tumors. We propose that these microenvironmental components mediated the metastatic tropism of GLV-1h68. Therefore, vaccinia virus-based oncolytic virotherapy might offer a novel treatment of metastatic prostate carcinomas in humans.
Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors.
For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation.
GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV.
Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome.
Oncolytic viruses show promise for treating cancer. However, to assess therapy and potential toxicity, a noninvasive imaging modality is needed. This study aims to determine the in vivo biodistribution, and imaging and timing characteristics of a vaccinia virus, GLV-1h153, encoding the human sodium iodide symporter (hNIS.
GLV-1h153 was modified from GLV-1h68 to encode the hNIS gene. Timing of cellular uptake of radioiodide 131I in human pancreatic carcinoma cells PANC-1 was assessed using radiouptake assays. Viral biodistribution was determined in nude mice bearing PANC-1 xenografts, and infection in tumors confirmed histologically and optically via Green Fluorescent Protein (GFP) and bioluminescence. Timing characteristics of enhanced radiouptake in xenografts were assessed via 124I-positron emission tomography (PET). Detection of systemic administration of virus was investigated with both 124I-PET and 99m-technecium gamma-scintigraphy.
GLV-1h153 successfully facilitated time-dependent intracellular uptake of 131I in PANC-1 cells with a maximum uptake at 24 hours postinfection (P<0.05). In vivo, biodistribution profiles revealed persistence of virus in tumors 5 weeks postinjection at 109 plaque-forming unit (PFU)/gm tissue, with the virus mainly cleared from all other major organs. Tumor infection by GLV-1h153 was confirmed via optical imaging and histology. GLV-1h153 facilitated imaging virus replication in tumors via PET even at 8 hours post radiotracer injection, with a mean %ID/gm of 3.82±0.46 (P<0.05) 2 days after intratumoral administration of virus, confirmed via tissue radiouptake assays. One week post systemic administration, GLV-1h153-infected tumors were detected via 124I-PET and 99m-technecium-scintigraphy.
GLV-1h153 is a promising oncolytic agent against pancreatic cancer with a promising biosafety profile. GLV-1h153 facilitated time-dependent hNIS-specific radiouptake in pancreatic cancer cells, facilitating detection by PET with both intratumoral and systemic administration. Therefore, GLV-1h153 is a promising candidate for the noninvasive imaging of virotherapy and warrants further study into longterm monitoring of virotherapy and potential radiocombination therapies with this treatment and imaging modality.
Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy.
Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44+CD24+ESA+ cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models.
We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44+CD24+ESA+ cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44+CD24-ESA+ cells.
Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors.
Aldehyde dehydrogenase 1; Cancer stem cells; Oncolytic virotherapy; Vaccinia virus; EMT
Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS.
Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic.
Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects.
We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia.
Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects.
vaccinia virus; cancer; cytokine; hyper-IL-6; oncolysis; chemotherapy