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. 2013 Jan;20(1):7-15.
doi: 10.1038/gt.2011.205. Epub 2011 Dec 15.

Measles virus causes immunogenic cell death in human melanoma

O G Donnelly  1 , F Errington-Mais, L Steele, E Hadac, V Jennings, K Scott, H Peach, R M Phillips, J Bond, H Pandha, K Harrington, R Vile, S Russell, P Selby, A A Melcher
Affiliations

Measles virus causes immunogenic cell death in human melanoma

O G Donnelly et al. Gene Ther. 2013 Jan.

Abstract

Oncolytic viruses (OV) are promising treatments for cancer, with several currently undergoing testing in randomised clinical trials. Measles virus (MV) has not yet been tested in models of human melanoma. This study demonstrates the efficacy of MV against human melanoma. It is increasingly recognised that an essential component of therapy with OV is the recruitment of host antitumour immune responses, both innate and adaptive. MV-mediated melanoma cell death is an inflammatory process, causing the release of inflammatory cytokines including type-1 interferons and the potent danger signal HMGB1. Here, using human in vitro models, we demonstrate that MV enhances innate antitumour activity, and that MV-mediated melanoma cell death is capable of stimulating a melanoma-specific adaptive immune response.

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Figures

Figure 1
Figure 1
Oncolytic activity of MV against human melanoma cell lines. A; Levels of surface expression of CD46, the MV receptor, were determined by flow cytometry in four human melanoma cell lines (Mel888, Mel624, MeWo, SkMel28). Filled histograms are isotype controls. Data shown are representative of three separate experiments. B; Melanoma cells were infected with GFP-expressing MV at a range of MOI. Photographs were taken 48hr after infection by phase contrast (left) and fluorescence (right) microscopy. C; Cytotoxicity was further measured in each cell line using the Live/Dead assay. Data shown are mean results from five separate experiments; bars demonstrate SE. D; Viral replication. Melanoma Cells were infected at an MOI of 0.1 and after 48 hours virus quantification was determined using the TCID50 method. Data shown are representative of 3 separate experiments. E; Mel624 were grown in a three dimensional model using transwell inserts for 5 days, then infected from the inferior (as indicated) surface with MV-GFP. i) Transverse sections of the multilayer model stained with H&E. ii) Confocal Z-stack image showing a lateral projection of a GFP-expressing syncytium.
Figure 2
Figure 2
The inflammatory response associated with MV infection. A; Cytokine/chemokine release. Cell-free supernatants were collected 48 hours after infection with MV and cytokine levels determined by ELISA. Data shown are representative of three independent experiments. B; HMGB1 release. Melanoma cells were treated with MV at MOI from 0.01 to 5. 48hours after infection cell-free supernatant was collected then analysed by western blot for HMGB1. Lanes with protein markers (M) and untreated controls (C) are indicated. Data shown are representative of two separate experiments.
Figure 3
Figure 3
Effects of MV on primary melanoma cells. A; Primary cells from freshly explanted melanoma B; Characteristic CPE 48 hours following treatment with MV-GFP by phase contrast (left) and fluorescence (right) microscopy. C; Live/Dead assay 2, 5 and 9 days after treatment of primary cells with MV. Data shown are representative of primary cells from three donors. D; ELISA of supernatant from primary cells treated with MV 72 hours previously.
Figure 4
Figure 4
Activation of innate immunity by MV. PBMC were treated overnight with MV (MOI 1). A; Chromium release elicited from labelled Mel888 targets on 4 hour culture with MV-treated PBMC. B; Levels of CD69 on CD3-CD56+ NK cells with or without treatment with MV. C; CD107 upregulation on CD3-CD56+ NK cells within PBMC, following four hour co-culture with Mel888. All figures are representative of experiments in three healthy donors.
Figure 5
Figure 5
Effect of MV on dendritic cells. A; DC were directly treated with MV at a range of MOI, or LPS as an alternative maturation agent, and then analysed by Live/Dead. B; DC were cultured in filtered virus-free tumour conditioned media (TCM) from Mel888 infected with MV at MOI of 0, 0.1 or 2.5 for 48 hours, and expression of surface markers measured. B.i; shows levels of expression normalised to untreated DC, and are mean values from four donors. B.ii; is a representative histogram plot of CD86 expression from one donor; Black line is isotype control, blue line is untreated DC, red line is DC treated with TCM from untreated Mel888 and purple line is DC treated with TCM from Mel888 infected with MV at 2.5 MOI. C. DC were cultured with infected or uninfected Mel888 for 24 hours then levels of surface marker expression measured from non-adherent cells gated on class II positive populations. Data are mean values from four donors and bars indicate SEM throughout. Asterisks indicate statistically significant differences between MFI values for DC treated with Mel888 or Mel888 TCM, versus 2.5 Mel888 or 2.5 TCM
Figure 6
Figure 6
Priming an adaptive T cell immune response. CTL were primed from PBMC cocultured with autologous DC that had been loaded with either Mel888 (control) or Mel888 treated with Mv at an MOI of 0.1 (0.1). A; Degranulation of CTL following coculture with either Mel888, SkOV or an equal volume of media free of target cells. B; Intracellular IFNγ within CTL primed from PBMC following coculture with Mel888, SkOV or media control. C; Cr-51 release from Mel888 following coculture with CTL; no significant release of Cr51 was detected from irrelevant SkOV targets. Data shown are from one donor, representative of experiments in 12 donors, in 9 of which a specific anti-melanoma response was elicited.
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