Variation in susceptibility of human malignant melanomas to oncolytic vesicular stomatitis virus

Abstract

Background Vesicular stomatitis virus (VSV) is a novel, anti-cancer therapy that selectively targets cancer cells with defective antiviral responses; however, not all malignant cells are sensitive to the oncolytic effects of VSV. Herein, we explore the mechanistic determinants of mutant M protein VSV (M51R-VSV) susceptibility in malignant melanoma cells. Methods Cell viability after VSV infection was measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) viability assay in a panel of melanoma cell lines. VSV infectability, viral protein synthesis and viral progeny production were quantified by flow cytometry, 35S-methionine electrophoresis, and viral plaque assays, respectively. Interferon (IFN) responsiveness was determined using MTS assay after β-IFN pre-treatment. Xenografts were established in athymic nude mice and treated with intratumoral M51R-VSV. Results Cell viability after M51R-VSV infection at a multiplicity of infection (MOI) of 10 pfu/mL, 48 hours post-infection) ranged between 0±1 and 59±9% (mean ± standard deviation). Sensitive cell lines supported VSV infection, viral protein synthesis, and viral progeny production. In addition, when pre-treated with β-IFN, sensitive cells became resistant to M51R-VSV, suggesting that IFN-mediated antiviral signaling is defective in these cells. In contrast, resistant melanoma cells do not support VSV infection, viral protein synthesis, or viral replication, indicating that anti-viral defenses remain intact. In a murine xenograft model, intratumoral M51R-VSV treatment decreased tumor growth relative to controls after 26 days in SK-Mel 5 (−21±19% vs. 2100±770%, p<0.0001) and SK-Mel 3 (2000±810% vs 7000±3000%, p=0.008) established tumors. Conclusions M51R-VSV is a viable, anti-cancer therapy, but susceptibility varies among melanomas. Future work will exploit specific mechanisms of resistance to expand the therapeutic efficacy of M51R-VSV.

Vesicular Stomatitis Virus Variants Selectively Infect and Kill Human Melanomas but Not Normal Melanocytes

“…In our work with primary human melanoma in vitro, only 5 of the 19 melanoma samples showed a low rate of infection after low-titer virus application; the remaining 14 melanoma cultures were very susceptible to VSV. This ratio (26% resistance) is consistent with a previous report of VSV infection resistance rates (22) of established cancer lines from the NCI 60 panel, which included some melanomas, and with a recent report on 6 established melanoma lines (56). In our hands, the observed cytopathic effect (CPE) rates and EthD-1-staining of dead cells across the panel correlated very highly with infection rates (R ϭ 0.94 and R ϭ 0.87, respectively), consistent with the rapid cell death-inducing consequences of VSV infection (57) and supportive of the potential efficacy of VSV as an oncolytic agent in melanoma.…”

“…This supports the notion that VSV-rp30 targets both established and developing tumors. VSV-M51 mutants have been described as safe in mice after intratumoral injection (56). Although VSV-rp30 infected melanoma tumors faster than the two M51 mutants (VSV-M51 and VSV-CT9/M51) that we tested, control immunocompetent mice given a high intravenous dose of VSVrp30 showed no adverse health consequences.…”

“…Our samples were directly derived from excised human melanoma samples with minimal cell passaging. Recent work on melanoma cell lines that have been long established shows a similar refractoriness to virus infection in a minority of lines (56). Established cancer cell lines often accumulate additional mutations that promote long-term propagation in vitro.…”

Re-engineering Vesicular Stomatitis Virus to Abrogate Neurotoxicity, Circumvent Humoral Immunity, and Enhance Oncolytic Potency

“…Similar to attenuated VSV recombinants, which are engineered to have an impaired ability to antagonize cellular antiviral responses, the IFNb transgene of the study virus restricts viral spread to IFN-unresponsive tumor cells (9-11, 21, 36). However, the extent of defective IFN responses in malignancies is variable and thus such vectors may require alternative strategies to improve their efficacy (37)(38)(39).…”

Interferon Beta and Interferon Alpha 2a Differentially Protect Head and Neck Cancer Cells from Vesicular Stomatitis Virus-Induced Oncolysis

“…Cancer cell resistance to VSV and other oncolytic viruses often is due to partial or full retention of type I IFN responsiveness and/or to constitutive expression of IFN-stimulated antiviral genes (6,13,(29)(30)(31)(32)(33)(34). The human type I IFN family includes 12 subtypes of IFN-␣ and one subtype of IFN-␤, all of which share significant amino acid homology (35).…”