Oncolytic alphavirus SFV-VA7 efficiently eradicates subcutaneous and orthotopic human prostate tumours in mice.

BACKGROUND: Despite recent therapeutic and diagnostic advances, prostate cancer remains the second leading cause of cancer-related deaths among men in the Western world. Oncolytic viruses that replicate selectively in tumour cells represent a novel treatment candidate for these malignancies. METHODS: We analysed infectivity of avirulent Semliki Firest virus SFV-VA7 in human prostate cancer cell lines VCaP, LNCaP and 22Rv1 and in nonmalignant prostate epithelial cell line RWPE-1. Therapeutic potency of SFV-VA7 was evaluated in subcutaneous and orthotopic mouse LNCaP xenograft models. RESULTS: SFV-VA7 infected and killed the tested human prostate cancer cell lines irrespective of their hormone response status, while the nonmalignant prostate epithelial cell line RWPE-1 proved highly virus resistant. Notably, a single peritoneal dose of SFV-VA7 was sufficient to eradicate all subcutaneous and orthotopic LNCaP tumours. CONCLUSIONS: Our results indicate that SFV-VA7 is a novel, promising therapeutic virus against prostate cancer warranting further testing in early clinical trials.

Interferon-ß sensitivity of tumor cells correlates with poor response to VA7 virotherapy in mouse glioma models.

In our recent study, replicative alphaviral vector VA7 was found to be effective against orthotopic human U87-glioma xenografts in an athymic mouse model eradicating the tumors with single intravenous (i.v.) injection. Here, we tested the efficacy of VA7 in immunocompetent orthotopic GL261 and CT-2A glioma models of C57BL/6 mouse in vivo. The cell lines were susceptible to VA7 infection in vitro, but GL261 infection was highly restricted in confluent cell cultures, and mouse interferon-ß (IFNß) pretreatment prevented the replication of VA7 in both cell lines. When mice bearing orthotopic GL261 or CT-2A tumors were administered neurotropic VA7, either i.v. or intracranially (i.c.), the vector was unable to infect the tumor and no survival benefit was achieved. Pretreatments with immunosuppressive cyclophosphamide (CPA) and rapamycin markedly lowered serum-neutralizing antibodies (NAbs) but had no effect on tumor infection or survival. Intracranial GL261 tumors were refractory also in athymic C57BL/6 mice, which have serious defects in their adaptive immunity. Implanted VA7-infected GL261 cells formed tumors with only slightly delayed kinetics and without improving survival thus excluding the participation of physical barriers and indicating robust host IFN action. Mouse and human IFNß do not seem be species cross-reactive, which might limit the translational relevance of xenograft models in oncolytic virotherapy.

Geographical variation in host use of a blood-feeding ectoparasitic fly: implications for population invasiveness.

Invasive generalist ectoparasites provide a tool to study factors affecting expansion rates. An increase in the number of host species may facilitate geographic range expansion by increasing the number of suitable habitats and by affecting local extinction and colonization rates. A geographic perspective on parasite host specificity and its implications on range expansion are, however, insufficiently understood. We conducted a field study to explore if divergent host specificity could explain the observed variation in expansion rates between Fennoscandian populations of the deer ked (Lipoptena cervi), which is a blood-feeding ectoparasitic fly of cervids. We found that the rapidly expanding eastern population in Finland appears to specialize on moose, whereas the slowly expanding western population in Norway breeds successfully on both moose and roe deer. The eastern population was also found to utilize the wild forest reindeer as an auxiliary host, but this species is apparently of low value for L. cervi in terms of adult maintenance, reproductive output and offspring quality. Abundant numbers of roe deer and white-tailed deer were observed to be apparently uninfected in Finland, suggesting that host use is not a plastic response to host availability, but rather a consequence of population-level evolutionary changes. Locally compatible hosts were found to be the ones sharing a long history with the deer ked in the area. Cervids that sustained adult deer keds also allowed successful reproduction. Thus, host use is probably determined by the ability of the adult to exploit particular host species. We conclude that a wide host range alone does not account for the high expansion rate or wide geographic distribution of the deer ked, although loose ecological requirements would increase habitat availability.

Human embryonic stem cell-derived mesenchymal stromal cell transplantation in a rat hind limb injury model.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have been used in a wide variety of pre-clinical experiments and in an increasing number of human clinical trials. Although many of these studies have shown different levels of engraftment, the exact fate of MSC after transplantation and the tissue response to their engraftment have not been investigated in detail. In the present work we studied the distribution of human MSC in a rat hind limb ischemic injury model immediately after transplantation and also analyzed the recipient tissue response to transplanted cells. METHODS: We tracked the in vivo fate of the transplanted MSC utilizing bioluminescence imaging, fluorescence microscopy and gene/protein expression analysis in a rat hind limb ischemia model. We also monitored the viability of transplanted cells by graft versus recipient expression analysis and determined the angiogenic and proliferative effect of transplantation by histologic staining. RESULTS: According to imaging analysis only a small portion of cells persisted for an extended period of time at the site of injury. Interestingly, recipient versus graft expression studies showed increased synthesis of rat-origin angiogenic factors and no human-origin mRNA or protein synthesis in transplanted tissues. More importantly, despite the lack of robust engraftment or growth factor secretion the transplantation procedure exerted a significant pro-angiogenic and pro-proliferative effect, which was mediated by angiogenic and mitogenic signaling pathways. CONCLUSIONS: Our results show an immediate temporal tissue effect in response to MSC transplantation that may represent a novel indirect paracrine mechanism for the beneficial effects of cell transplantation observed in injured tissues.

Oncolytic capacity of attenuated replicative semliki forest virus in human melanoma xenografts in severe combined immunodeficient mice.

Oncolytic viruses have gained attention as a novel form of cancer treatment. Many viral vectors in use today have been rendered safe by deletion of genes encoding viral structural proteins, thus making them unable to spread beyond the first infected cells. Hence, such replication-deficient constructs may lack efficacy. Here, we analyzed the oncolytic potential of the replication-competent vector VA7-EGFP, based on the avirulent Semliki Forest virus (SFV) strain A7(74), to kill cancer cells in culture as well as to target s.c. human melanoma xenografts in severe combined immunodeficient (SCID) mice. VA7-EGFP was able to infect most cancer cell lines studied, leading to complete lysis of the cells within 72 hours after infection. In SCID mice grafted with A2058 human melanoma, marked regression of the xenografts was observed following a single injection of 10(6) plaque-forming units of virus given either i.p., i.v., or intratumorally. Histologic analysis revealed the presence of virus not only in all treated tumors but also in the brains of the treated mice, causing progressing neuropathology beginning at day 16 after infection. Following initial oncolysis, clusters of viable tumor cells were observed embedded in connective tissue, and at later stages, encapsulated tumor nodules had formed. Infection of melanoma cells from explant cultures of these nodules revealed that a portion of the cells were resistant to virus. To be eligible for use in virotherapy, the ability of avirulent SFV to spread within tumor tissue may have to be improved and the biological safety of the virus may have to be addressed thoroughly in higher animals.

Oncolytic viruses in cancer therapy.

Oncolytic virotherapy is a promising form of gene therapy for cancer, employing nature's own agents to find and destroy malignant cells. The purpose of this review is to provide an introduction to this very topical field of research and to point out some of the current observations, insights and ideas circulating in the literature. We have strived to acknowledge as many different oncolytic viruses as possible to give a broader picture of targeting cancer using viruses. Some of the newest additions to the panel of oncolytic viruses include the avian adenovirus, foamy virus, myxoma virus, yaba-like disease virus, echovirus type 1, bovine herpesvirus 4, Saimiri virus, feline panleukopenia virus, Sendai virus and the non-human coronaviruses. Although promising, virotherapy still faces many obstacles that need to be addressed, including the emergence of virus-resistant tumor cells.

Targeted inhibition of human collagenase-3 (MMP-13) expression inhibits squamous cell carcinoma growth in vivo.

Squamous cell carcinomas (SCCs) of the head and neck are characterized by a high tendency for local invasion and metastasis to lymph nodes. Collagenase-3 (MMP-13) is specifically expressed by tumor cells in SCCs of the head and neck and its expression correlates with their invasion capacity. To specifically examine the role of MMP-13 in the growth and invasion of SCC, we constructed a hammerhead ribozyme targeted against human MMP-13 mRNA. The anti-MMP-13 ribozyme effectively cleaved MMP-13 transcripts in vitro. Adenoviral delivery of the anti-MMP-13 ribozyme to cutaneous metastatic SCC cells in culture resulted in potent and specific inhibition of the production of proMMP-13 and markedly suppressed invasion of SCC cells through Matrigel. In addition, adenoviral delivery of anti-MMP-13 ribozyme promoted apoptosis in SCC cells within 72 h. Intratumoral injection of anti-MMP-13 ribozyme coding adenovirus into human SCC xenografts established in SCID mice potently suppressed tumor growth, inhibited MMP-13 expression and gelatinolytic activity and reduced the number of proliferating cells within the tumors. These results provide evidence for an important role for MMP-13 in SCC growth and invasion and identify MMP-13 as a promising target for ribozyme-based therapy of SCC in vivo.

Removal of cholera toxin from aqueous solution by probiotic bacteria.

Cholera remains a serious health problem, especially in developing countries where basic hygiene standards are not met. The symptoms of cholera are caused by cholera toxin, an enterotoxin, which is produced by the bacterium Vibrio cholerae. We have recently shown that human probiotic bacteria are capable of removing cyanobacterial toxins from aqueous solutions. In the present study we investigate the ability of the human probiotic bacteria, Lactobacillus rhamnosus strain GG (ATCC 53103) and Bifidobacterium longum 46 (DSM 14583), to remove cholera toxin from solution in vitro. Lactobacillus rhamnosus strain GG and Bifidobacterium longum 46 were able to remove 68% and 59% of cholera toxin from aqueous solutions during 18 h of incubation at 37 °C, respectively. The effect was dependent on bacterial concentration and L. rhamnosus GG was more effective at lower bacterial concentrations. No significant effect on cholera toxin concentration was observed when nonviable bacteria or bacterial supernatant was used.

Differential roles of fibroblast growth factor receptors (FGFR) 1, 2 and 3 in the regulation of S115 breast cancer cell growth.

Fibroblast growth factors (FGFs) regulate the growth and progression of breast cancer. FGF signaling is transduced through FGF receptors 1-4, which have oncogenic or anti-oncogenic roles depending on the ligand and the cellular context. Our aim was to clarify the roles of FGFR1-3 in breast cancer cell growth in vitro and in vivo. Pools of S115 mouse breast cancer cells expressing shRNA against FGFR1, 2 and 3 were created by lentiviral gene transfer, resulting in cells with downregulated expression of FGFR1, FGFR2 or FGFR3 (shR1, shR2 and shR3 cells, respectively) and shLacZ controls. FGFR1-silenced shR1 cells formed small, poorly vascularized tumors in nude mice. Silencing of FGFR2 in shR2 cells was associated with strong upregulation of FGFR1 expression and the formation of large, highly vascularized tumors compared to the control tumors. Silencing FGFR3 did not affect cell survival or tumor growth. Overexpressing FGFR2 in control cells did not affect FGFR1 expression, suggesting that high FGFR1 expression in shR2 cells and tumors was associated with FGFR2 silencing by indirect mechanisms. The expression of FGFR1 was, however, increased by the addition of FGF-8 to starved shLacZ or MCF-7 cells and decreased by the FGFR inhibitor PD173074 in shR2 cells with an elevated FGFR1 level. In conclusion, our results demonstrate that FGFR1 is crucial for S115 breast cancer cell proliferation and tumor growth and angiogenesis, whereas FGFR2 and FGFR3 are less critical for the growth of these cells. The results also suggest that the expression of FGFR1 itself is regulated by FGF-8 and FGF signaling, which may be of importance in breast tumors expressing FGFs at a high level.

Intravenously administered alphavirus vector VA7 eradicates orthotopic human glioma xenografts in nude mice.

BACKGROUND: VA7 is a neurotropic alphavirus vector based on an attenuated strain of Semliki Forest virus. We have previously shown that VA7 exhibits oncolytic activity against human melanoma xenografts in immunodeficient mice. The purpose of this study was to determine if intravenously administered VA7 would be effective against human glioma. METHODOLOGY/PRINCIPAL FINDINGS: In vitro, U87, U251, and A172 human glioma cells were infected and killed by VA7-EGFP. In vivo, antiglioma activity of VA7 was tested in Balb/c nude mice using U87 cells stably expressing firefly luciferase in subcutaneous and orthotopic tumor models. Intravenously administered VA7-EGFP completely eradicated 100% of small and 50% of large subcutaneous U87Fluc tumors. A single intravenous injection of either VA7-EGFP or VA7 expressing Renilla luciferase (VA7-Rluc) into mice bearing orthotopic U87Fluc tumors caused a complete quenching of intracranial firefly bioluminescence and long-term survival in total 16 of 17 animals. In tumor-bearing mice injected with VA7-Rluc, transient intracranial and peripheral Renilla bioluminescence was observed. Virus was well tolerated and no damage to heart, liver, spleen, or brain was observed upon pathological assessment at three and ninety days post injection, despite detectable virus titers in these organs during the earlier time point. CONCLUSION: VA7 vector is apathogenic and can enter and destroy brain tumors in nude mice when administered systemically. This study warrants further elucidation of the mechanism of tumor destruction and attenuation of the VA7 virus.

Sphingosine kinase as a regulator of calcium entry through autocrine sphingosine 1-phosphate signaling in thyroid FRTL-5 cells.

Calcium entry is one of the main regulators of intracellular signaling. Here, we have described the importance of sphingosine, sphingosine kinase 1 (SK1), and sphingosine 1-phosphate (S1P) in regulating calcium entry in thyroid FRTL-5 cells. In cells incubated with the phosphatase inhibitor calyculin A, which evokes calcium entry without mobilizing sequestered intracellular calcium, sphingosine inhibited calcium entry in a concentration-dependent manner. Furthermore, inhibiting SK1 or the ATP-binding cassette ABCC1 multidrug transporter attenuated calcium entry. The addition of exogenous S1P restored calcium entry. Neither sphingosine nor inhibition of SK1 attenuated thapsigargin-evoked calcium entry. Blocking S1P receptor 2 or phospholipase C attenuated calcium entry, whereas blocking S1P receptor 3 did not. Overexpression of wild-type SK1, but not SK2, enhanced calyculin-evoked calcium entry compared with mock-transfected cells, whereas calcium entry was decreased in cells transfected with the dominant-negative G82D SK1 mutant. Exogenous S1P restored calcium entry in G82D cells. Our results suggest that the calcium entry pathway is blocked by sphingosine and that activation of SK1 and the production of S1P, through an autocrine mechanism, facilitate calcium entry through activation of S1P receptor 2. This is a novel mechanism by which the sphingosine-S1P rheostat regulates cellular calcium homeostasis.

Semliki Forest virus vectors expressing transforming growth factor beta inhibit experimental autoimmune encephalomyelitis in Balb/c mice.

Cytokine immunomodulation of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, has remained a formidable treatment option, but access into the CNS is hampered due to the impermeability of the blood-brain barrier. In this report, we describe the construction and characterization of CNS-homing gene delivery/therapy vectors based on avirulent Semliki Forest virus (SFV) expressing either native or mutant transforming growth factor beta 1 (TGF-beta1). Biological activity of the expressed inserts was demonstrated by PAI-1 promoter driven luciferase production in mink cells and TGF-beta1 mRNA was demonstrated in the CNS of virus treated mice by in situ hybridization and RT-PCR. Both vectors, when given intraperitoneally to EAE mice significantly reduced disease severity compared to untreated mice. Our results imply that immunomodulation by neurotropic viral vectors may offer a promising treatment strategy for autoimmune CNS disorders.