Fig 1.
Relapse and survival following ingenol mebutate treatment of B16 tumours grown in MyD88-/- and C57BL/6 mice.
(A) Relapse rates following (i) ingenol mebutate treatment of B16 tumours grown in MyD88-/- mice (n = 25), (ii) ingenol mebutate treatment of B16 tumours grown in C57BL/6 mice (n = 21), (iii) placebo treatment of B16 tumours grown in MyD88-/- mice (n = 18) and (iv) placebo treatment of B16 tumours grown in C57BL/6 mice (n = 19). Mice were scored positive when a tumour was clearly visible (≥1–2 mm in diameter). Data from two independent experiments. Ingenol mebutate treatment groups were significantly different p = 0.021, log-rank (Mantel-Cox) test. (B) Survival rates of the same mice described in A; mice were euthanized when tumours reached 100 mm2. Ingenol mebutate treatment groups were significantly different p = 0.018, log-rank (Mantel-Cox) test.
Fig 2.
Relapse and survival following ingenol mebutate treatment of B16 tumours grown in C57BL/6 mice treated with anakinra.
(A) Relapse rates after C57BL/6 mice bearing B16 tumours were treated with ingenol mebuate or placebo, and received daily injections of PBS or anakinra, days 1–7. (n = 9–12 mice per group). Mice were scored positive when a tumour was clearly visible (≥1–2 mm in diameter). Statistics compared + anakinra with + PBS in ingenol mebutate treated groups using the log-rank (Mantel-Cox) test. (B) Survival of the mice described in A; mice were euthanized when tumours reached 100 mm2. Statistics as in A.
Fig 3.
Neutrophil recruitment and apoptosis.
(A) Neutrophil recruitment to treatments sites (left bar chart) and to within ≈200 μm of tumour (right bar chart). Treatment sites; day 2 post initiation of ingenol mebutate treatment, treatment sites were excised and processed for immunohistochemistry and stained with the neutrophil marker, anti-Ly6G. Slides were scanned and analysed by Aperio Pixel count software for brown staining (default settings) excluding areas containing tumour (as melanosomes provide a false positive signal). Two sections per mouse, 6 mice per group. Statistics by Kolmogorov-Smirnov tests (differences in variance between groups was >4). Within ≈200 μm of tumour; in sections where the tumour mass could be readily identified (by the presence of black melanosomes), brown staining surrounding the tumour (within ≈200 μm) was quantitated as above. One section per mouse, 4–5 mice per group. Statistics by Mann Whitney U test (non-parametric data distribution and differences in variance <4). (B) Images illustrating the reduced density of anti-Ly6G staining neutrophils (brown stain) within ≈200 μm of the tumour mass in anakinra versus PBS treated mice. The tumours are delineated by white lines and identified by the presence of black melanosomes. Sections are oriented with the skin (not shown) at the top, with the tumours located in the dermis. (C) ApoTag staining of the sections described in A. Six mice per group, 2/3 sections per mouse, statistics by 2 way ANOVA (parametric data distribution and differences in variance <4, drug and mouse as fixed factors, 2/3 sections per mouse as dependent variables). (Examples of the staining are shown in Figure F in S1 File).
Fig 4.
In vitro killing of B16 tumour cells in the presence of ingenol mebutate, anakinra and/or IL-1β.
(A) Bone marrow cells (≈40% neutrophils) were incubated with B16 cells in 96 well plates (6 replicates) at the indicated effector to target ratios and cell killing calculated relative to B16 cells with no drug or effectors. Statistics by Kolmogorov-Smirnov (difference in variance >4) p = 0.005, and Mann Whitney U test (non-parametric distribution and difference in variance <4) p = 0.002. Ingenol mebutate (40 ng/ml final) and/or anakinra (100 μg/ml final) was present during the assay. (B) As for A except a large range of effector to target ratios (i.e. 15:1 to 250:1) were used and IL-1β (40 ng/ml final) was added. Statistics by Kolmogorov-Smirnov (for data sets where difference in variance was >4) and t tests (for data sets with parametric distribution and differences in variance <4).
Fig 5.
IL-1α and IL-1β protein levels after ingenol mebutate treatment.
(A, B) C57BL/6 and MyD88-/- mice with B16 tumours were treated topically with ingenol mebutate day 0 and 1, treatment sites were excised and IL-1α and IL-1β levels were measured in extracts using BD BD™ Cytometric Bead Array (n = 6 mice per group and time point). Statistics by Kolmogorov-Smirnov tests (differences in variance >4). (C) Cultured adult human keratinocytes were treated with the indicated concentration of ingenol mebutate for 16 hours and the supernatants analysed by Western using an anti-IL-1α antibody. Arrow indicates the position of the 18 kDa bioactive form of IL-1α.