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The wound inflammatory response exacerbates growth of pre-neoplastic cells and progression to cancer.

Antonio N, Bønnelykke-Behrndtz ML, Ward LC, Collin J, Christensen IJ, Steiniche T, Schmidt H, Feng Y, Martin P - EMBO J. (2015)

Bottom Line: There is a long-standing association between wound healing and cancer, with cancer often described as a "wound that does not heal".In an adult model of chronic wounding in zebrafish, we show that repeated wounding with subsequent inflammation leads to a greater incidence of local melanoma formation.We find a strong correlation between neutrophil presence at sites of melanoma ulceration and cell proliferation at these sites, which is associated with poor prognostic outcome.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, University of Bristol, Bristol, UK.

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Related in: MedlinePlus

Wounding leads to increased proliferation of pre-neoplastic cellsA–D’ RasG12VeGFP larvae were left unwounded (A–D) or laser-wounded (yellow dotted line) at 2 dpf just dorsal to the cloaca (A’–D’). Larvae were left to grow for 3 days before being fixed and analysed for pre-neoplastic cell number (n = 30 larvae in each group). EdU accumulation (purple) is shown at low magnification (C and C’) and a representative pre-neoplastic cell clone is shown (D and D’).E Graph illustrating pre-neoplastic cell numbers in unwounded versus wounded larvae (n = 27 of each group). Data from three independent experiments showed the same level of significance. ***P ≤ 0.001.F–G’ Images of larvae injected with control morpholino at the one-cell stage, and either left unwounded (F, high magnification: G) or laser-wounded at 3 dpf (F’, high magnification: G’) and fixed at 5 dpf.H–J’ Larvae injected with a combination of PU-1 and GCSF morpholinos at the one-cell stage, left unwounded (H, high magnification: I) or laser-wounded at 3 dpf (H’, high magnification: I’) and fixed at 5 dpf. Images of larvae injected with irf8 morpholino, and either unwounded (J) or laser-wounded at 3 dpf (J’) before subsequent fixation at 5 dpf.K, K’ Larvae injected with GCSF morpholino, and either left unwounded (K) or laser-wounded at 3 dpf (K’) and fixed at 5 dpf.L, M Graphs to show the total number of Ras+ cells and clones (respectively) at 5 dpf after PU-1 and GCSF morpholinos.N, O Graphs showing the number of Ras+ cells at 5 dpf after irf8 morpholino (N) or after GCSF morpholino injection (O).Data information: All scale bars represent 50 μm except (D) and (D’) which represent 15 μm. n = 15–20 larvae in each group (L–O). All graphs display mean ± SEM.
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fig03: Wounding leads to increased proliferation of pre-neoplastic cellsA–D’ RasG12VeGFP larvae were left unwounded (A–D) or laser-wounded (yellow dotted line) at 2 dpf just dorsal to the cloaca (A’–D’). Larvae were left to grow for 3 days before being fixed and analysed for pre-neoplastic cell number (n = 30 larvae in each group). EdU accumulation (purple) is shown at low magnification (C and C’) and a representative pre-neoplastic cell clone is shown (D and D’).E Graph illustrating pre-neoplastic cell numbers in unwounded versus wounded larvae (n = 27 of each group). Data from three independent experiments showed the same level of significance. ***P ≤ 0.001.F–G’ Images of larvae injected with control morpholino at the one-cell stage, and either left unwounded (F, high magnification: G) or laser-wounded at 3 dpf (F’, high magnification: G’) and fixed at 5 dpf.H–J’ Larvae injected with a combination of PU-1 and GCSF morpholinos at the one-cell stage, left unwounded (H, high magnification: I) or laser-wounded at 3 dpf (H’, high magnification: I’) and fixed at 5 dpf. Images of larvae injected with irf8 morpholino, and either unwounded (J) or laser-wounded at 3 dpf (J’) before subsequent fixation at 5 dpf.K, K’ Larvae injected with GCSF morpholino, and either left unwounded (K) or laser-wounded at 3 dpf (K’) and fixed at 5 dpf.L, M Graphs to show the total number of Ras+ cells and clones (respectively) at 5 dpf after PU-1 and GCSF morpholinos.N, O Graphs showing the number of Ras+ cells at 5 dpf after irf8 morpholino (N) or after GCSF morpholino injection (O).Data information: All scale bars represent 50 μm except (D) and (D’) which represent 15 μm. n = 15–20 larvae in each group (L–O). All graphs display mean ± SEM.

Mentions: To determine what effect this increased inflammation might have on pre-neoplastic cell proliferation, we counted the number of pre-neoplastic cells 3 days post-wounding in a series of domains extending up to 250 μm away from the wound centre and corresponding approximately to the region of increased inflammation. We see a significant increase in pre-neoplastic cell number in comparison with equivalent flank regions of unwounded larvae (Fig3A, B and E) in bands extending 50–150 μm and 150–250 μm from the wound centre (Supplementary Fig S4), and this is corroborated by a significant increase in EdU-positive pre-neoplastic cells in 2-days post-wounding larvae (Fig3C and D, Supplementary Fig S5A), where the thymidine analogue EdU has been incorporated into the DNA of proliferating cells. This effect is local, rather than systemic, because we see no significant increase in numbers of Ras+ cells in the epithelium beyond 250 μm from the wound centre (Supplementary Fig S4), or overlying the yolk or in the head (Supplementary Fig S5). Moreover, only wounds that are sufficiently large to trigger a wound inflammatory response over 48 h appear to trigger a pre-neoplastic proliferative response; small wounds show no increase above background, unwounded, levels (Supplementary Fig S5C). To further test whether this increase is a consequence of the wound inflammatory response, we transiently delayed innate immune cell development by injection of PU.1 and GCSF morpholinos at the one-cell-stage embryo. This led to an almost complete depletion of neutrophils and macrophages until 4 days post-fertilisation (dpf) (Feng et al, 2012) and, as a consequence, we observed a significant decrease in pre-neoplastic cell numbers, suggesting reduced proliferation both in unwounded control fish, as previously reported (Feng et al, 2010), and in 2-days post-wounded fish, by comparison with fish with a normal wound inflammatory response (Fig3L).


The wound inflammatory response exacerbates growth of pre-neoplastic cells and progression to cancer.

Antonio N, Bønnelykke-Behrndtz ML, Ward LC, Collin J, Christensen IJ, Steiniche T, Schmidt H, Feng Y, Martin P - EMBO J. (2015)

Wounding leads to increased proliferation of pre-neoplastic cellsA–D’ RasG12VeGFP larvae were left unwounded (A–D) or laser-wounded (yellow dotted line) at 2 dpf just dorsal to the cloaca (A’–D’). Larvae were left to grow for 3 days before being fixed and analysed for pre-neoplastic cell number (n = 30 larvae in each group). EdU accumulation (purple) is shown at low magnification (C and C’) and a representative pre-neoplastic cell clone is shown (D and D’).E Graph illustrating pre-neoplastic cell numbers in unwounded versus wounded larvae (n = 27 of each group). Data from three independent experiments showed the same level of significance. ***P ≤ 0.001.F–G’ Images of larvae injected with control morpholino at the one-cell stage, and either left unwounded (F, high magnification: G) or laser-wounded at 3 dpf (F’, high magnification: G’) and fixed at 5 dpf.H–J’ Larvae injected with a combination of PU-1 and GCSF morpholinos at the one-cell stage, left unwounded (H, high magnification: I) or laser-wounded at 3 dpf (H’, high magnification: I’) and fixed at 5 dpf. Images of larvae injected with irf8 morpholino, and either unwounded (J) or laser-wounded at 3 dpf (J’) before subsequent fixation at 5 dpf.K, K’ Larvae injected with GCSF morpholino, and either left unwounded (K) or laser-wounded at 3 dpf (K’) and fixed at 5 dpf.L, M Graphs to show the total number of Ras+ cells and clones (respectively) at 5 dpf after PU-1 and GCSF morpholinos.N, O Graphs showing the number of Ras+ cells at 5 dpf after irf8 morpholino (N) or after GCSF morpholino injection (O).Data information: All scale bars represent 50 μm except (D) and (D’) which represent 15 μm. n = 15–20 larvae in each group (L–O). All graphs display mean ± SEM.
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fig03: Wounding leads to increased proliferation of pre-neoplastic cellsA–D’ RasG12VeGFP larvae were left unwounded (A–D) or laser-wounded (yellow dotted line) at 2 dpf just dorsal to the cloaca (A’–D’). Larvae were left to grow for 3 days before being fixed and analysed for pre-neoplastic cell number (n = 30 larvae in each group). EdU accumulation (purple) is shown at low magnification (C and C’) and a representative pre-neoplastic cell clone is shown (D and D’).E Graph illustrating pre-neoplastic cell numbers in unwounded versus wounded larvae (n = 27 of each group). Data from three independent experiments showed the same level of significance. ***P ≤ 0.001.F–G’ Images of larvae injected with control morpholino at the one-cell stage, and either left unwounded (F, high magnification: G) or laser-wounded at 3 dpf (F’, high magnification: G’) and fixed at 5 dpf.H–J’ Larvae injected with a combination of PU-1 and GCSF morpholinos at the one-cell stage, left unwounded (H, high magnification: I) or laser-wounded at 3 dpf (H’, high magnification: I’) and fixed at 5 dpf. Images of larvae injected with irf8 morpholino, and either unwounded (J) or laser-wounded at 3 dpf (J’) before subsequent fixation at 5 dpf.K, K’ Larvae injected with GCSF morpholino, and either left unwounded (K) or laser-wounded at 3 dpf (K’) and fixed at 5 dpf.L, M Graphs to show the total number of Ras+ cells and clones (respectively) at 5 dpf after PU-1 and GCSF morpholinos.N, O Graphs showing the number of Ras+ cells at 5 dpf after irf8 morpholino (N) or after GCSF morpholino injection (O).Data information: All scale bars represent 50 μm except (D) and (D’) which represent 15 μm. n = 15–20 larvae in each group (L–O). All graphs display mean ± SEM.
Mentions: To determine what effect this increased inflammation might have on pre-neoplastic cell proliferation, we counted the number of pre-neoplastic cells 3 days post-wounding in a series of domains extending up to 250 μm away from the wound centre and corresponding approximately to the region of increased inflammation. We see a significant increase in pre-neoplastic cell number in comparison with equivalent flank regions of unwounded larvae (Fig3A, B and E) in bands extending 50–150 μm and 150–250 μm from the wound centre (Supplementary Fig S4), and this is corroborated by a significant increase in EdU-positive pre-neoplastic cells in 2-days post-wounding larvae (Fig3C and D, Supplementary Fig S5A), where the thymidine analogue EdU has been incorporated into the DNA of proliferating cells. This effect is local, rather than systemic, because we see no significant increase in numbers of Ras+ cells in the epithelium beyond 250 μm from the wound centre (Supplementary Fig S4), or overlying the yolk or in the head (Supplementary Fig S5). Moreover, only wounds that are sufficiently large to trigger a wound inflammatory response over 48 h appear to trigger a pre-neoplastic proliferative response; small wounds show no increase above background, unwounded, levels (Supplementary Fig S5C). To further test whether this increase is a consequence of the wound inflammatory response, we transiently delayed innate immune cell development by injection of PU.1 and GCSF morpholinos at the one-cell-stage embryo. This led to an almost complete depletion of neutrophils and macrophages until 4 days post-fertilisation (dpf) (Feng et al, 2012) and, as a consequence, we observed a significant decrease in pre-neoplastic cell numbers, suggesting reduced proliferation both in unwounded control fish, as previously reported (Feng et al, 2010), and in 2-days post-wounded fish, by comparison with fish with a normal wound inflammatory response (Fig3L).

Bottom Line: There is a long-standing association between wound healing and cancer, with cancer often described as a "wound that does not heal".In an adult model of chronic wounding in zebrafish, we show that repeated wounding with subsequent inflammation leads to a greater incidence of local melanoma formation.We find a strong correlation between neutrophil presence at sites of melanoma ulceration and cell proliferation at these sites, which is associated with poor prognostic outcome.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, University of Bristol, Bristol, UK.

Show MeSH
Related in: MedlinePlus