Neutrophil recruitment and apoptosis.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153975#pone.0153975.s001" target="_blank">S1 File</a>).</p

Relapse and survival following ingenol mebutate treatment of B16 tumours grown in MyD88^-/- and C57BL/6 mice.

<p>(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.</p

IL-1α and IL-1β protein levels after ingenol mebutate treatment.

<p>(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.</p

Relapse and survival following ingenol mebutate treatment of B16 tumours grown in C57BL/6 mice treated with anakinra.

<p>(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.</p

Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models

<div><p>Intra-lesional chemotherapy for treatment of cutaneous malignancies has been used for many decades, allowing higher local drug concentrations and less toxicity than systemic agents. Here we describe a novel diterpene ester, EBC-46, and provide preclinical data supporting its use as an intra-lesional treatment. A single injection of EBC-46 caused rapid inflammation and influx of blood, followed by eschar formation and rapid tumor ablation in a range of syngeneic and xenograft models. EBC-46 induced oxidative burst from purified human polymorphonuclear cells, which was prevented by the Protein Kinase C inhibitor bisindolylmaleimide-1. EBC-46 activated a more specific subset of PKC isoforms (PKC-βI, -βII, -α and -γ) compared to the structurally related phorbol 12-myristate 13-acetate (PMA). Although EBC-46 showed threefold less potency for inhibiting cell growth than PMA <i>in vitro</i>, it was more effective for cure of tumors <i>in vivo</i>. No viable tumor cells were evident four hours after injection by <i>ex vivo</i> culture. Pharmacokinetic profiles from treated mice indicated that EBC-46 was retained preferentially within the tumor, and resulted in significantly greater local responses (erythema, oedema) following intra-lesional injection compared with injection into normal skin. The efficacy of EBC-46 was reduced by co-injection with bisindolylmaleimide-1. Loss of vascular integrity following treatment was demonstrated by an increased permeability of endothelial cell monolayers <i>in vitro</i> and by CD31 immunostaining of treated tumors <i>in vivo</i>. Our results demonstrate that a single intra-lesional injection of EBC-46 causes PKC-dependent hemorrhagic necrosis, rapid tumor cell death and ultimate cure of solid tumors in pre-clinical models of cancer.</p></div

EBC-46 causes disruption and permeability of endothelial cells within tumor.

<p><b>A</b>. Immunohistochemical analysis of CD31 staining of FaDu head and neck cancer tumors treated with EBC-46. FaDu tumors were allowed to reach 100 mm³ before they were treated with either vehicle (20% propylene glycol in water) or 50 nmol (30 µg) EBC-46, and harvested at the indicated times. Representative photomicrographs are shown of the tumor site. Black arrows – examples of vessels with compromised or disrupted structural integrity. Scale bars  = 100 µm. <b>B</b>. Monolayers of HUVEC cells were treated with 350 µM (200 µg/ml) EBC-46 for 30 mins, before being assessed for permeability to FITC labeled Dextran. (***, p = 0.0013; t-test). <b>C</b>. HUVEC cells were treated with 350 µM (200 µg/ml) EBC-46 for 30 mins with or without 5 µM bisindolylmaleimide-1, before being assessed by propidium iodide exclusion. Error bars – SD, n = 3.</p

EBC-46 efficacy <i>in vivo</i> is independent of tumor cell sensitivity <i>in vitro</i>.

<p><b>A</b>. Dose response for cell killing by EBC-46 compared to PMA. B16-F0 (circles) or SK-MEL-28 (squares) melanoma cells were treated with the indicated doses of either EBC-46 (blue) or PMA (red) for 4 days, before assay for cell survival using the sulforhodamine B assay. Data shown are mean ± SD from triplicate readings from three independent experiments, n = 3. <b>B</b>. Kaplan Meier analysis of survival of C57BL/6J mice with B16-F0 tumors. Mice with two tumors were treated with single bolus doses of vehicle alone (20% propylene glycol in water; light grey), 50 nmol (30 µg) PMA (mid grey) or 50 nmol (30 µg) EBC-46 (black). Mice were euthanized once the total tumor volume reached 1,000 mm³ per animal. Squares – censored data. Difference between survival following treatment with EBC-46 or PMA was significant (*** p = 0.0004; Log-rank (Mantel-Cox) Test). Data was obtained from 6 mice per group, 2 tumors per mouse; n = 12. <b>C</b>. Kaplan Meier analysis of SK-MEL-28 melanoma tumors reaching >100 mm³ in BALB/c <i>Foxn1^nu</i> mice following single treatment with vehicle alone (grey) or 30 µg EBC-46 (black) (***, p<0.0001; Log-rank (Mantel-Cox) Test). Data was obtained from 5 mice per group, 2 tumors per mouse; n = 10. <b>D</b>. Kaplan Meier analysis of MM649 melanoma tumors reaching >100 mm³ in BALB/c <i>Foxn1^nu</i> mice following single treatment with vehicle alone (grey) or 50 nmol (30 µg) EBC-46 (black) (***, p<0.0001; Log-rank (Mantel-Cox) Test). Data was obtained from 5 mice per group, 2 tumors per mouse; n = 10.</p

EBC-46 anti-cancer efficacy is PKC-dependent.

<p><b>A</b>. The effect of neutrophil depletion on EBC-46 treatment of SK-MEL-28 tumors grown on BALB/c <i>Foxn1^nu</i> mice. Tumor growth in mice given anti-Ly-6G antibody, isotype control antibody or no antibody. SK-MEL-28 cells were injected sub-cutaneously (n = 10 tumors/group; 2 tumors/mouse, 5 mice/group) and allowed to reach approximately 100 mm³. Mice were injected with anti-Ly-6G antibody (clone 1A8; 100 µg i.p, on days −2, 0, and 2), with isotype control antibody (IgG2a, clone 2A3; 100 µg i.p, on days −2, 0, and 2) or with nothing. Tumors were treated by intra-lesional injection of vehicle (50 µl of 20% propylene glycol in water) on day 0. The tumor volumes represent the mean volume of individual tumors. • – no antibody, ▪ – control IgG2a antibody, ▴ – anti-mLy-6G antibody. <b>B</b>. Tumor growth in mice given anti-Ly-6G antibody, isotype control antibody or no antibody after EBC-46 treatment. As for A, SK-MEL-28 tumors (approximately 100 mm³) were treated by intra-lesional injection with 25 nmol (15 µg) EBC-46 (in 50 µl of 20% propylene glycol in water) on day 0. • – no antibody, ▪ – control IgG2a antibody, ▴ – anti-mLy-6G antibody. Error bars – SD. <b>C</b>. 500,000 B16-F0 cells were injected into BALB/c <i>Foxn1^nu</i> mice, and allowed to reach >50 mm³. Tumors were then treated with either 50 µl vehicle (20% propylene glycol in water), 16.7 nmol (10 µg) EBC-46 in vehicle or 16.7 nmol (10 µg) EBC-46 after pre-treatment with 5 µM bisindolylmaleimide-1 (BIS-1) in vehicle. Tumor size was measured 8 days after treatment. Error bars  =  SD, n = 12. (****, p<0.0001; **, p = 0.0024; t-test). <b>D</b>. Number of tumors (as percentage) present 8 days after treatment. Total tumor number n = 12 for each group.</p

EBC-46 treatment induces greater effects when injected into tumors compared to normal skin.

<p>EBC-46 was injected into male BALB/c <i>Foxn1^nu</i> mice either bearing (n = 9) or not bearing (n = 9) tumors, as a single intra-lesional or sub-cutaneous injection, respectively. <b>A</b>. Erythema at injection site 24 hours following treatment with 50 nmol (30 µg) EBC-46 in tumor- or non-tumor-bearing mice. <b>B</b>. Oedema at injection site 24 hours following treatment with 50 nmol (30 µg) EBC-46 in tumor- or non-tumor-bearing mice. <b>C</b>. Percentage weight change 24 hours following treatment with 50 nmol (30 µg) EBC-46 in tumor- or non-tumor-bearing mice. <b>D</b>. Concentration in serum following treatment with 50 nmol (30 µg) EBC-46 in tumor- or non-tumor-bearing mice. Data is from serum from three animals unless otherwise indicated. Error bars – SEM. (**, p<0.01; ***, p<0.005; t-test). a - single data point from EBC-46-treated animals. b - below lower limit of detection of the assay, set at 0.01 ng/ml.</p

Clinical scoring for Drug Treated and Tumor Bearing Mice.

<p>Clinical scoring for Drug Treated and Tumor Bearing Mice.</p