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The α v β 3 integrin is highly expressed in prostate cancer (PCa), in which it is a key player in tumour invasion, angiogenesis and metastasis formation. Therefore, α v β 3 integrin is considered a very promising target for molecular imaging of PCa. This study tested the potential of the novel α v β 3 integrin affine agent [ 68 Ga]NOTA-RGD in comparison with the established [ 18 F]fluoroethylcholine (FEC) and [ 18 F]fluorodeoxyglucose (FDG) for assessing PCa using positron emission tomography (PET). [ 68 Ga]NOTA-RGD showed a lower uptake in PC-3 and DU-145 cells compared with FEC and FDG. µPET imaging studies showed a good delineation of the PCa xenografts in mice. The means tumor-to-muscleand tumor-to-bone-ratio amounted 5.1 ± 1.4 and 5.2 ± 1.2 for [ 68 Ga]NOTA-RGD compared with 2.6 ± 0.9 and 2.9 ± 1.6 for FDG, and 2.4 ± 0.7 and 0.8 ± 0.2 for FEC, respectively. The uptake of [ 68 Ga]NOTA-RGD into tumor was fully inhibited by c(RGDfV), known to bind specifically to α v β 3 integrin, confirming the specificity of the tumor uptake in vivo. These results suggest that [ 68 Ga]NOTA-RGD is a promising candidate for PET imaging of α v β 3 integrin expression in PCa and warrant further in vivo validations to ascertain its potential as an imaging agent for clinical use. The simple and fast preparation of [ 68 Ga]NOTA-RGD may greatly facilitate its translation to a clinical setting.

Breast cancer is the most common cause of cancer-related death worldwide, thus remaining a crucial health problem among women despite advances in conventional therapy. Therefore, new alternative strategies are needed for effective diagnosis and treatment. One approach is the use of oncolytic viruses for gene-directed enzyme prodrug therapy. Here, the lacZ-carrying vaccinia virus (VACV) strain GLV-1h68 was used in combination with a β-galactosidase-activatable prodrug derived from a seco-analog of the natural antibiotic duocarmycin SA. Tumor cell infection with the VACV strain GLV-1h68 led to production of β-galactosidase, essential for the conversion of the prodrug to the toxic compound. Furthermore, drug-dependent cell kill and induction of the intrinsic apoptosis pathway in tumor cells was also observed on combination therapy using the prodrug and the GLV-1h68 strain, despite the fact that VACV strains encode antiapoptotic proteins. Moreover, GI-101A breast cancer xenografts were effectively treated by the combination therapy. In conclusion, the combination of a β-galactosidase-activatable prodrug with a tumor-specific vaccinica virus strain encoding this enzyme, induced apoptosis in cultures of the human GI-101A breast cancer cells, in which a synergistic oncolytic effect was observed. Moreover, in vivo, additional prodrug treatment had beneficial effects on tumor regression in GLV-1h68-treated GI-101A-xenografted mice.

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In the current study, we analyzed the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 against two human prostate cancer cell lines DU-145 and PC-3 in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 was able to infect, replicate in, and lyse these prostate cancer cells in culture. In DU-145 and PC-3 tumor xenograft models, a single intravenous injection with GLV-1h68 resulted in a significant reduction of primary tumor size. In addition, the GLV-1h68-infection led to strong inflammatory and oncolytic effects resulting in drastic reduction of regional lymph nodes with PC-3 metastases. Our data documented that the GLV-1h68 virus has a great potential for treatment of human prostate carcinoma.

Colorectal cancer (CRC) detected at an early stage has a good prognosis, however, recurrence of the disease is prevalent and late stage CRC (stage IIIC and IV) has observed 5-year survival rates of 28% and 6%, respectively. Oncolytic virotherapy has been explored as a potential treatment modality in cancer therapy for its safety and tumor specificity. We recently reported that oncolytic vaccinia virus GLV-1h68 efficiently infected, replicated in, and lysed different colorectal cancer cell lines derived from patients at different stages of the disease in culture and mouse xenograft models. However, the cytotoxicity of GLV-1h68 was cell line- dependent and, in particular, xenograft tumors of the colorectal cancer cell line HT-29 did not respond favorably to oncolytic treatment with GLV-1h68. Here we report on the generation of recombinant vaccinia viruses expressing the colorectal tumor suppressor Klf4. We show that a single injection of vaccinia virus expressing Klf4 led to significant tumor growth inhibition of HT-29 human colon cancer xenografts in comparison to treatment with GLV-1h68. Furthermore, virus-mediated expression of a membrane-permeable Klf4-TAT fusion protein, further improved the anti-tumoral effects. This is the first report demonstrating that arming oncolytic viruses with the colorectal tumor suppressor Klf4 led to improved therapy in a human colon cancer xenograft model.

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.

The α(v)β₃ integrin is highly expressed in prostate cancer (PCa), in which it is a key player in tumour invasion, angiogenesis and metastasis formation. Therefore, α(v)β₃ integrin is considered a very promising target for molecular imaging of PCa. This study tested the potential of the novel α(v)β₃ integrin affine agent [⁶⁸Ga]NOTA-RGD in comparison with the established [¹⁸F]fluoroethylcholine (FEC) and [¹⁸F]fluorodeoxyglucose (FDG) for assessing PCa using positron emission tomography (PET). [⁶⁸Ga]NOTA-RGD showed a lower uptake in PC-3 and DU-145 cells compared with FEC and FDG. µPET imaging studies showed a good delineation of the PCa xenografts in mice. The means tumor-to-muscle and tumor-to-bone-ratio amounted 5.1 ± 1.4 and 5.2 ± 1.2 for [⁶⁸Ga]NOTA-RGD compared with 2.6 ± 0.9 and 2.9 ± 1.6 for FDG, and 2.4 ± 0.7 and 0.8 ± 0.2 for FEC, respectively. The uptake of [⁶⁸Ga]NOTA-RGD into tumor was fully inhibited by c(RGDfV), known to bind specifically to α(v)β₃ integrin, confirming the specificity of the tumor uptake in vivo. These results suggest that [⁶⁸Ga]NOTA-RGD is a promising candidate for PET imaging of α(v)β₃ integrin expression in PCa and warrant further in vivo validations to ascertain its potential as an imaging agent for clinical use. The simple and fast preparation of [⁶⁸Ga]NOTA-RGD may greatly facilitate its translation to a clinical setting.

We reported earlier the delivery of antiangiogenic single chain antibodies by using oncolytic vaccinia virus strains to enhance their therapeutic efficacy. Here, we provide evidence that gene-evoked production of melanin can be used as a therapeutic and diagnostic mediator, as exemplified by insertion of only one or two genes into the genome of an oncolytic vaccinia virus strain. We found that produced melanin is an excellent reporter for optical imaging without addition of substrate. Melanin production also facilitated deep tissue optoacoustic imaging as well as MRI. In addition, melanin was shown to be a suitable target for laser-induced thermotherapy and enhanced oncolytic viral therapy. In conclusion, melanin as a mediator for thermotherapy and reporter for different imaging modalities may soon become a versatile alternative to replace fluorescent proteins also in other biological systems. After ongoing extensive preclinical studies, melanin overproducing oncolytic virus strains might be used in clinical trials in patients with cancer.

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.

Background Malignant pleural effusion (MPE) is associated with advanced stages of lung cancer and is mainly dependent on invasion of the pleura and expression of vascular endothelial growth factor (VEGF) by cancer cells. As MPE indicates an incurable disease with limited palliative treatment options and poor outcome, there is an urgent need for new and efficient treatment options. Methods In this study, we used subcutaneously generated PC14PE6 lung adenocarcinoma xenografts in athymic mice that developed subcutaneous malignant effusions (ME) which mimic pleural effusions of the orthotopic model. Using this approach monitoring of therapeutic intervention was facilitated by direct observation of subcutaneous ME formation without the need of sacrificing mice or special imaging equipment as in case of MPE. Further, we tested oncolytic virotherapy using Vaccinia virus as a novel treatment modality against ME in this subcutaneous PC14PE6 xenograft model of advanced lung adenocarcinoma. Results We demonstrated significant therapeutic efficacy of Vaccinia virus treatment of both advanced lung adenocarcinoma and tumor-associated ME. We attribute the efficacy to the virus-mediated reduction of tumor cell-derived VEGF levels in tumors, decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of the blood vessel-invading tumor cells. Moreover, we showed that the use of oncolytic Vaccinia virus encoding for a single-chain antibody (scAb) against VEGF (GLAF-1) significantly enhanced mono-therapy of oncolytic treatment. Conclusions Here, we demonstrate for the first time that oncolytic virotherapy using tumor-specific Vaccinia virus represents a novel and promising treatment modality for therapy of ME associated with advanced lung cancer.

Background 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. Methods 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. Results 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. Conclusions 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.

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.

Canine mammary carcinoma is a highly metastatic tumor that is poorly responsive to available treatment. Therefore, there is an urgent need to identify novel agents for therapy of this disease. Recently, we reported that the oncolytic vaccinia virus GLV-1h68 could be a useful tool for therapy of canine mammary adenomain vivo. In this study we analyzed the therapeutic effect of GLV-1h68 against canine mammary carcinoma. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the mammary carcinoma cell line MTH52c. Furthermore, after systemic administration, this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. Finally, infection with GLV-1h68 led to strong inflammatory and oncolytic effects resulting in significant growth inhibition of the tumors. In summary, the data showed that the GLV-1h68 virus strain has promising potential for effective treatment of canine mammary carcinoma.

The type 5 phosphodiesterase inhibitor vardenafil (VAR) reduces myocardial infarct size following ischemia/ reperfusion injury when applied prior to ischemia or at reperfusion in models of in situ hearts. Little is known about the underlying protective signaling. Here, we test whether VAR is protective in a cell model of calcium stress as well as in isolated rat hearts. HL-1 cardiomyocytes were loaded with tetramethylrhodamine ethyl ester (TMRE, 100 nM) which causes cells to fluoresce proportional to their mitochondrial membrane potential (Ψm). A reduction of fluorescence serves as an indicator of collapse of Ψm, and, presumably to permeability transition pore (mPTP) formation. Cells were subjected to the calcium ionophore calcimycin (CAL) (100 μM) which causes mPTP formation due to calcium overload. Fluorescence intensity was measured after 80 minutes of calcium stress with FACS technique. Treatment with CAL reduced the mean cell fluorescence (903 ± 28 arbitrary units (a.u.), vs. 548 ± 29 a.u. in untreated cells, p < 0.001) while VAR (1 nM) could protect the cells (698 ± 40 a.u., p < 0.001). This protection could be blocked by either the guanylyl cyclase (GC) inhibitor ODQ (20 μM) (519 ± 41 a.u.) or the protein kinase G blocker KT5823 (1 μM) (513 ± 22 a.u.), while inhibition of NO-synthase with L-NAME had no effect. To further support the results, isolated rat hearts were subjected to 30 min regional ischemia followed by 120 min reperfusion. As expected, VAR (10 nM) at 5 min prior to reperfusion reduced infarct size as percentage of the ischemic zone from 46.6 ± 2.1% in control hearts to 26.2 ± 2.7% (p < 0.001). This protective effect could be blocked with co-administration of ODQ (43.9 ± 2.2%), while LNAME was not effective, confirming a role for GC but not for NOS in VAR's protection. Taken together, these results further support the hypothesis that PDE-5 inhibitors induce protective effects in the ischemic heart in addition to their well known clinical effects in the treatment of erectile dysfunction in men. from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007 Published: 25 July 2007