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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

Live imaging reveals neutrophils distracted away from wounds due to competing signals from adjacent pre-neoplastic cellsA, A’ Five-days post-fertilisation larva illustrating the region of flank where we image and wound (A). Wounds (yellow circle) are made in the centre of the flank just above the cloaca (arrow) in all larval experiments (A’).B–B’’ Stills from a time-lapse movie of a larva with RasG12VeGFP pre-neoplastic cell clones but no wound.C–C’’’ Equivalent time-lapse stills of a control, laser-wounded larva with no pre-neoplastic cell clones at 90 min post-wounding (wound indicated with yellow dotted line).D–D’’’ Stills from a time-lapse movie of a larva with RasG12VeGFP pre-neoplastic cell clones (green), again wounded 90 min before.E Graph comparing the number of LysC:dsRed+ neutrophils recruited over a 2-h period to the equivalent flank region of unwounded RasG12VeGFP larvae (n = 11), versus wounded WT larvae (n = 3), wounded RasG12VeGFP larvae (n = 5) and wounded RasG12VeGFP larvae treated with DPI inhibitor (n = 7). ***P ≤ 0.001.F Graph comparing the percentage of Ras+ pre-neoplastic cells that receive contacts with neutrophils during the 2-h period of the movie in unwounded (n = 11) versus wounded (n = 5) larvae. ***P ≤ 0.001.G–J Unwounded WT sibling; LysC:dsRed+ larva (n = 10 per time point) for comparison of clone growth with (H) laser-wounded WT sibling: LysC:dsRed+ larva (n = 15 per time point), (I) unwounded Ras+; LysC:dsRed+ larvae (n = 15 per time point) and (J) laser-wounded Ras+; LysC:dsRed+ (n = 20 per time point). Larvae were harvested and fixed between 3 dpf and 7 dpf (i.e. between 3 and 96 h post-wounding), and stained with anti-L-plastin and anti-RFP antibodies to distinguish neutrophils (yellow) and macrophages (red).K–N Graphs showing the numbers of neutrophils and macrophages in the flanks of unwounded WT siblings (K), unwounded Ras+ larvae (L), wounded WT siblings (M) and wounded Ras+ larvae (N).O–Q Graphs indicating the total number of innate immune cells (O), macrophages (P) and neutrophils (Q) recruited over time in wounded and unwounded WT and unwounded and wounded Ras+ larvae.R Graph showing the number of pre-neoplastic cells receiving contact by immune cells in unwounded/wounded larvae over time (P = 0.0052 for 5 days post-wound).Data information: In (K–R) x-axes denote time post-wound (h). All scale bars represent 50 μm. (B, C and D) show stills of the time-lapse movie; (C’ and D’) show tracks of five immune cells as they migrate after entering the wound; (B’’, C’’ and D’’) show the footprints of all LysC:dsRed immune cells throughout the movie. (C’’’ and D’’’) illustrate the migration of neutrophils from when they enter the centre of the wound. All graphs display mean ± SEM.
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fig02: Live imaging reveals neutrophils distracted away from wounds due to competing signals from adjacent pre-neoplastic cellsA, A’ Five-days post-fertilisation larva illustrating the region of flank where we image and wound (A). Wounds (yellow circle) are made in the centre of the flank just above the cloaca (arrow) in all larval experiments (A’).B–B’’ Stills from a time-lapse movie of a larva with RasG12VeGFP pre-neoplastic cell clones but no wound.C–C’’’ Equivalent time-lapse stills of a control, laser-wounded larva with no pre-neoplastic cell clones at 90 min post-wounding (wound indicated with yellow dotted line).D–D’’’ Stills from a time-lapse movie of a larva with RasG12VeGFP pre-neoplastic cell clones (green), again wounded 90 min before.E Graph comparing the number of LysC:dsRed+ neutrophils recruited over a 2-h period to the equivalent flank region of unwounded RasG12VeGFP larvae (n = 11), versus wounded WT larvae (n = 3), wounded RasG12VeGFP larvae (n = 5) and wounded RasG12VeGFP larvae treated with DPI inhibitor (n = 7). ***P ≤ 0.001.F Graph comparing the percentage of Ras+ pre-neoplastic cells that receive contacts with neutrophils during the 2-h period of the movie in unwounded (n = 11) versus wounded (n = 5) larvae. ***P ≤ 0.001.G–J Unwounded WT sibling; LysC:dsRed+ larva (n = 10 per time point) for comparison of clone growth with (H) laser-wounded WT sibling: LysC:dsRed+ larva (n = 15 per time point), (I) unwounded Ras+; LysC:dsRed+ larvae (n = 15 per time point) and (J) laser-wounded Ras+; LysC:dsRed+ (n = 20 per time point). Larvae were harvested and fixed between 3 dpf and 7 dpf (i.e. between 3 and 96 h post-wounding), and stained with anti-L-plastin and anti-RFP antibodies to distinguish neutrophils (yellow) and macrophages (red).K–N Graphs showing the numbers of neutrophils and macrophages in the flanks of unwounded WT siblings (K), unwounded Ras+ larvae (L), wounded WT siblings (M) and wounded Ras+ larvae (N).O–Q Graphs indicating the total number of innate immune cells (O), macrophages (P) and neutrophils (Q) recruited over time in wounded and unwounded WT and unwounded and wounded Ras+ larvae.R Graph showing the number of pre-neoplastic cells receiving contact by immune cells in unwounded/wounded larvae over time (P = 0.0052 for 5 days post-wound).Data information: In (K–R) x-axes denote time post-wound (h). All scale bars represent 50 μm. (B, C and D) show stills of the time-lapse movie; (C’ and D’) show tracks of five immune cells as they migrate after entering the wound; (B’’, C’’ and D’’) show the footprints of all LysC:dsRed immune cells throughout the movie. (C’’’ and D’’’) illustrate the migration of neutrophils from when they enter the centre of the wound. All graphs display mean ± SEM.

Mentions: To gain a more dynamic impression of how wounding may influence the behaviour of innate immune cells in the vicinity of cancer cells, we made a series of laser wounds adjacent to clones of pre-neoplastic cells on the flanks of zebrafish larvae (Fig2A and A’) which are amenable to live imaging because of their translucency. Our previous studies have shown how fluorescently labelled neutrophils and macrophages are recruited to pre-neoplastic goblet cells expressing mutant RasG12V and GFP (Feng et al, 2010). These innate immune cells are recruited by stochastic pulses of hydrogen peroxide (Feng et al, 2010), the same signal that has been shown to draw neutrophils to wounds (Niethammer et al, 2009), and they remain at one pre-neoplastic clone for brief periods before moving on to visit adjacent clones (Fig2B, Supplementary Movie S1). Tissue wounding triggers an acute, rapid recruitment of large numbers of neutrophils to the wound, but rather than remaining predominately within the wound site for up to 3 or 4 h, as in control wounded fish with no burden of pre-neoplastic cells (Fig2C, Supplementary Movie S2), many of these immune cells are distracted from the wound and “visit” the nearby pre-neoplastic cells (Fig2D, Supplementary Movie S3); this is most clearly visualised by “footprints” of neutrophil tracks from 90 min to three hours post-wounding which extend well beyond the wound site in fish carrying a pre-neoplastic cell burden, whereas they remain in the vicinity of the wound in fish without pre-neoplastic cells (Fig2C’’ and D’’). If wound-triggered hydrogen peroxide release is blocked by treatment with DPI, then few, if any, neutrophils are drawn to the wound (Fig2E), and consequently, many fewer visits to nearby pre-neoplastic cells are seen (Supplementary Movie S4). Because wounding standardly draws many more neutrophils to the flank than are normally present (Fig2E), this leads to considerably more opportunity for contacts with pre-neoplastic cells. Indeed, in this period following wounding, we observe 64.2% of Ras+ cells adjacent to a wound receive neutrophil contacts compared to only 26% of pre-neoplastic cells in a comparable region of an unwounded larvae (Fig2F). Contacts ranged from less than one minute to more than 90 min, and we have used them as a proxy for neutrophil/pre-neoplastic cell interactions although we have no evidence that physical contacts between these two lineages are necessary for one cell to influence the other. To follow neutrophil and macrophage recruitment over a substantially longer period, we fixed Ras+ larvae and their WT siblings at various time points up to 5 days post-wounding (Fig2G–J). To distinguish macrophages, we immunostained larvae with L-plastin, which is known to be a pan-leucocyte marker in zebrafish larvae, and considered cells which were L-plastin+ but LysC− to be macrophages (Feng et al, 2010; Jones et al, 2013). Over the 5 days post-wounding, the number of neutrophils recruited to flank pre-neoplastic cells in wounded Ras+ larvae was significantly higher (P = 0.007) than in unwounded Ras+ larvae (Fig2P). In contrast, despite the large numbers of macrophages recruited to the wound by 24 h post-wounding, there appears to be no significant increase in macrophage recruitment to nearby pre-neoplastic cells when comparing Ras+ unwounded and Ras+ wounded larvae over the total time course (Fig2Q; P = 0.1019). However, we do observe increased recruitment of macrophages to wounds in Ras+ larvae compared to their WT siblings (Fig2Q; P = 0.0493), and once recruited, they appear to persist in the area around the wound for longer than in WT siblings due to the presence of the pre-neoplastic cells (Fig2M and N). From the earliest time points examined in fixed larvae—3 h after wounding—we observe a significant increase in the number of pre-neoplastic cells receiving contacts from immune cells (P = 0.0052 for 5 days post-wound); the number of these contacts peaks by 24 h post-injury and is maintained for at least 3 days post-wounding (Fig2R).


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)

Live imaging reveals neutrophils distracted away from wounds due to competing signals from adjacent pre-neoplastic cellsA, A’ Five-days post-fertilisation larva illustrating the region of flank where we image and wound (A). Wounds (yellow circle) are made in the centre of the flank just above the cloaca (arrow) in all larval experiments (A’).B–B’’ Stills from a time-lapse movie of a larva with RasG12VeGFP pre-neoplastic cell clones but no wound.C–C’’’ Equivalent time-lapse stills of a control, laser-wounded larva with no pre-neoplastic cell clones at 90 min post-wounding (wound indicated with yellow dotted line).D–D’’’ Stills from a time-lapse movie of a larva with RasG12VeGFP pre-neoplastic cell clones (green), again wounded 90 min before.E Graph comparing the number of LysC:dsRed+ neutrophils recruited over a 2-h period to the equivalent flank region of unwounded RasG12VeGFP larvae (n = 11), versus wounded WT larvae (n = 3), wounded RasG12VeGFP larvae (n = 5) and wounded RasG12VeGFP larvae treated with DPI inhibitor (n = 7). ***P ≤ 0.001.F Graph comparing the percentage of Ras+ pre-neoplastic cells that receive contacts with neutrophils during the 2-h period of the movie in unwounded (n = 11) versus wounded (n = 5) larvae. ***P ≤ 0.001.G–J Unwounded WT sibling; LysC:dsRed+ larva (n = 10 per time point) for comparison of clone growth with (H) laser-wounded WT sibling: LysC:dsRed+ larva (n = 15 per time point), (I) unwounded Ras+; LysC:dsRed+ larvae (n = 15 per time point) and (J) laser-wounded Ras+; LysC:dsRed+ (n = 20 per time point). Larvae were harvested and fixed between 3 dpf and 7 dpf (i.e. between 3 and 96 h post-wounding), and stained with anti-L-plastin and anti-RFP antibodies to distinguish neutrophils (yellow) and macrophages (red).K–N Graphs showing the numbers of neutrophils and macrophages in the flanks of unwounded WT siblings (K), unwounded Ras+ larvae (L), wounded WT siblings (M) and wounded Ras+ larvae (N).O–Q Graphs indicating the total number of innate immune cells (O), macrophages (P) and neutrophils (Q) recruited over time in wounded and unwounded WT and unwounded and wounded Ras+ larvae.R Graph showing the number of pre-neoplastic cells receiving contact by immune cells in unwounded/wounded larvae over time (P = 0.0052 for 5 days post-wound).Data information: In (K–R) x-axes denote time post-wound (h). All scale bars represent 50 μm. (B, C and D) show stills of the time-lapse movie; (C’ and D’) show tracks of five immune cells as they migrate after entering the wound; (B’’, C’’ and D’’) show the footprints of all LysC:dsRed immune cells throughout the movie. (C’’’ and D’’’) illustrate the migration of neutrophils from when they enter the centre of the wound. All graphs display mean ± SEM.
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fig02: Live imaging reveals neutrophils distracted away from wounds due to competing signals from adjacent pre-neoplastic cellsA, A’ Five-days post-fertilisation larva illustrating the region of flank where we image and wound (A). Wounds (yellow circle) are made in the centre of the flank just above the cloaca (arrow) in all larval experiments (A’).B–B’’ Stills from a time-lapse movie of a larva with RasG12VeGFP pre-neoplastic cell clones but no wound.C–C’’’ Equivalent time-lapse stills of a control, laser-wounded larva with no pre-neoplastic cell clones at 90 min post-wounding (wound indicated with yellow dotted line).D–D’’’ Stills from a time-lapse movie of a larva with RasG12VeGFP pre-neoplastic cell clones (green), again wounded 90 min before.E Graph comparing the number of LysC:dsRed+ neutrophils recruited over a 2-h period to the equivalent flank region of unwounded RasG12VeGFP larvae (n = 11), versus wounded WT larvae (n = 3), wounded RasG12VeGFP larvae (n = 5) and wounded RasG12VeGFP larvae treated with DPI inhibitor (n = 7). ***P ≤ 0.001.F Graph comparing the percentage of Ras+ pre-neoplastic cells that receive contacts with neutrophils during the 2-h period of the movie in unwounded (n = 11) versus wounded (n = 5) larvae. ***P ≤ 0.001.G–J Unwounded WT sibling; LysC:dsRed+ larva (n = 10 per time point) for comparison of clone growth with (H) laser-wounded WT sibling: LysC:dsRed+ larva (n = 15 per time point), (I) unwounded Ras+; LysC:dsRed+ larvae (n = 15 per time point) and (J) laser-wounded Ras+; LysC:dsRed+ (n = 20 per time point). Larvae were harvested and fixed between 3 dpf and 7 dpf (i.e. between 3 and 96 h post-wounding), and stained with anti-L-plastin and anti-RFP antibodies to distinguish neutrophils (yellow) and macrophages (red).K–N Graphs showing the numbers of neutrophils and macrophages in the flanks of unwounded WT siblings (K), unwounded Ras+ larvae (L), wounded WT siblings (M) and wounded Ras+ larvae (N).O–Q Graphs indicating the total number of innate immune cells (O), macrophages (P) and neutrophils (Q) recruited over time in wounded and unwounded WT and unwounded and wounded Ras+ larvae.R Graph showing the number of pre-neoplastic cells receiving contact by immune cells in unwounded/wounded larvae over time (P = 0.0052 for 5 days post-wound).Data information: In (K–R) x-axes denote time post-wound (h). All scale bars represent 50 μm. (B, C and D) show stills of the time-lapse movie; (C’ and D’) show tracks of five immune cells as they migrate after entering the wound; (B’’, C’’ and D’’) show the footprints of all LysC:dsRed immune cells throughout the movie. (C’’’ and D’’’) illustrate the migration of neutrophils from when they enter the centre of the wound. All graphs display mean ± SEM.
Mentions: To gain a more dynamic impression of how wounding may influence the behaviour of innate immune cells in the vicinity of cancer cells, we made a series of laser wounds adjacent to clones of pre-neoplastic cells on the flanks of zebrafish larvae (Fig2A and A’) which are amenable to live imaging because of their translucency. Our previous studies have shown how fluorescently labelled neutrophils and macrophages are recruited to pre-neoplastic goblet cells expressing mutant RasG12V and GFP (Feng et al, 2010). These innate immune cells are recruited by stochastic pulses of hydrogen peroxide (Feng et al, 2010), the same signal that has been shown to draw neutrophils to wounds (Niethammer et al, 2009), and they remain at one pre-neoplastic clone for brief periods before moving on to visit adjacent clones (Fig2B, Supplementary Movie S1). Tissue wounding triggers an acute, rapid recruitment of large numbers of neutrophils to the wound, but rather than remaining predominately within the wound site for up to 3 or 4 h, as in control wounded fish with no burden of pre-neoplastic cells (Fig2C, Supplementary Movie S2), many of these immune cells are distracted from the wound and “visit” the nearby pre-neoplastic cells (Fig2D, Supplementary Movie S3); this is most clearly visualised by “footprints” of neutrophil tracks from 90 min to three hours post-wounding which extend well beyond the wound site in fish carrying a pre-neoplastic cell burden, whereas they remain in the vicinity of the wound in fish without pre-neoplastic cells (Fig2C’’ and D’’). If wound-triggered hydrogen peroxide release is blocked by treatment with DPI, then few, if any, neutrophils are drawn to the wound (Fig2E), and consequently, many fewer visits to nearby pre-neoplastic cells are seen (Supplementary Movie S4). Because wounding standardly draws many more neutrophils to the flank than are normally present (Fig2E), this leads to considerably more opportunity for contacts with pre-neoplastic cells. Indeed, in this period following wounding, we observe 64.2% of Ras+ cells adjacent to a wound receive neutrophil contacts compared to only 26% of pre-neoplastic cells in a comparable region of an unwounded larvae (Fig2F). Contacts ranged from less than one minute to more than 90 min, and we have used them as a proxy for neutrophil/pre-neoplastic cell interactions although we have no evidence that physical contacts between these two lineages are necessary for one cell to influence the other. To follow neutrophil and macrophage recruitment over a substantially longer period, we fixed Ras+ larvae and their WT siblings at various time points up to 5 days post-wounding (Fig2G–J). To distinguish macrophages, we immunostained larvae with L-plastin, which is known to be a pan-leucocyte marker in zebrafish larvae, and considered cells which were L-plastin+ but LysC− to be macrophages (Feng et al, 2010; Jones et al, 2013). Over the 5 days post-wounding, the number of neutrophils recruited to flank pre-neoplastic cells in wounded Ras+ larvae was significantly higher (P = 0.007) than in unwounded Ras+ larvae (Fig2P). In contrast, despite the large numbers of macrophages recruited to the wound by 24 h post-wounding, there appears to be no significant increase in macrophage recruitment to nearby pre-neoplastic cells when comparing Ras+ unwounded and Ras+ wounded larvae over the total time course (Fig2Q; P = 0.1019). However, we do observe increased recruitment of macrophages to wounds in Ras+ larvae compared to their WT siblings (Fig2Q; P = 0.0493), and once recruited, they appear to persist in the area around the wound for longer than in WT siblings due to the presence of the pre-neoplastic cells (Fig2M and N). From the earliest time points examined in fixed larvae—3 h after wounding—we observe a significant increase in the number of pre-neoplastic cells receiving contacts from immune cells (P = 0.0052 for 5 days post-wound); the number of these contacts peaks by 24 h post-injury and is maintained for at least 3 days post-wounding (Fig2R).

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