The wound inflammatory response exacerbates growth of pre-neoplastic cells and progression to cancer.
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.Our zebrafish studies led us to investigate the innate immune cell associations in ulcerated melanomas in human patients.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.
Affiliation: School of Biochemistry, University of Bristol, Bristol, UK.
- Neoplasms, Experimental/genetics/immunology*/pathology
- Precancerous Conditions/genetics/immunology*/pathology
- Wounds and Injuries/genetics/immunology*/pathology
- Cell Proliferation
- Mutation, Missense
- Zebrafish Proteins/genetics/immunology
- ras Proteins/genetics/immunology
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fig01: Tumours arise at sites of injuryA Unwounded 4-week-old kita:RasG12VeGFP juvenile fish followed over time.B Juvenile kita:RasG12VeGFP fish wounded by tail-fin resection (red line) every 2 weeks for 12 weeks.C Pigmentation of the tail fin as quantified by threshold analysis of the tail area (indicated by red dotted line) at each time point.D Graph illustrating the percent pigmentation over 12 weeks (mean ± SEM).E Number of tail fin tumours developed in wounded versus unwounded fish (n = 7 fish in unwounded and wounded group, respectively).F–F’’ An adult kita:RasG12VeGFP fish with a large tail melanoma prior to surgery (F), immediately after (F’) and 3 days post-surgery (F’’); the surgery left a small section of melanoma remaining on the ventral tail fin (highlighted by the red box).G Whole-mount immunohistochemistry of the excised unwounded tumour stained for LysC (red) to reveal neutrophils.H Immunohistochemistry of a section of the same excised tumour stained for phospho-histone H3 (pH3, white) to reveal the extent of proliferation.I Whole-mount immunohistochemistry of the tail with wounded tumour remnant 3 days post-surgery (green) illustrating accumulation of neutrophils (red) within the wounded tumour tissue (arrowhead).J, J’ A frozen section through the tail with wounded tumour remnant 3 days post-surgery (plane indicated by red dotted line in F’’) stained for phospho-H3 revealing how proliferating cells (white) appear to have increased in number in the remaining tumour. Equivalent bright-field section shown in (J’).K, K’ An adult kita:RasG12VeGFP; p53+/−; LysC:dsRed fish with an early-stage, flat, tail melanoma just post-punch biopsy at the tumour margin; punch biopsy tissue with green RasG12VeGFP tissue adjacent to healthy tissue is shown in (K’).L–L’’ Multichannel view of the repairing wound illustrating how, 24 h post-wounding, neutrophils (red) have been drawn to both tumour and healthy tissue wound edge. (L) and (L’’) are magnifications of the wound edges of non-tumour and tumour tissue, respectively.M–M’’ Multichannel view of the repairing wound at 3 days post-wounding, demonstrating that neutrophils have largely resolved away from the wound site in healthy tissue (M’) but remain highly concentrated in wounded tumour tissue (M’’).Data information: Scale bars represent 2 mm (A, B, F-F’’ and K), 50 μm (H, J and J’) and 200 μm (G, I, K’, L-L’’ and M-M’’).
In our previous studies, we observed that zebrafish expressing a mutant RasG12V oncogene in their skin tend to develop invasive melanomas at sites that are prone to friction and other damage, for example on the ventral fin and tail fin (Feng et al, 2010) (Supplementary Fig S1D and E). We wanted to test whether this correlation reflected a true causal association, and so we repeatedly wounded the tail fin of one group of juvenile RasG12V fish fortnightly for 3 months and compared tumour outcome with equivalent siblings that had not been wounded. By the second round of fortnightly wounds, we saw a clear increase in pigment intensity in the wounded tail fins (Fig1A and B), reflecting recruitment and/or local proliferation of pre-neoplastic melanocytes. This increase in pigmentation, as measured by threshold analysis (Fig1C and D), appeared to reach a plateau after five rounds of wounding, and we left the fish unwounded after six wounds. By 18 months after the initial wounding, we observed that 43% of all wounded fish had developed melanoma at the site of wounding while none of the unwounded fish exhibited fin tip melanomas (Fig1A, B and E), suggesting that chronic wounding can indeed increase the likelihood of cancer growth in tissues that already have a predisposition to developing melanoma. This chronic wounding study was repeated in adult 3- to 6-month-old fish and showed the same result (Supplementary Fig S2A–D).