Analysing the inflammatory response to different skin lesions in zebrafish larvae. (A) Schematic to illustrate our cancer-wound comparison studies showing the region (black box) to be imaged in (B–E), for neutrophils (magenta) and macrophages (orange) as they respond to the different skin lesions: local wound (LW), healthy RFP-expressing goblet cells (HCs) or pre-neoplastic GFP-expressing goblet cells (PNCs); dotted lines indicate the “close proximity” zone from margins of each lesion type; 1 and 2 indicate immune cells in contact with the lesion or within “close proximity”, respectively, and 3 indicates immune cells outside this zone. (B–E) Multi-channel confocal movie frames of 6 dpf flank wounded Tg(lyz:DsRed;mpeg1:nls-Clover) (B,C), unwounded control Tg(kita:mCherry; lyz:DsRed;mpeg1:nls-Clover) (D) or cancerous Tg(kita:HRASG12V-GFP;lyz:DsRed;mpeg1:nls-Clover) (E) larvae prior to analysis of neutrophil (magenta) or macrophage (green nuclei) behaviour and their interactions with the respective skin lesions (white arrowheads). (B’–E’,B”–E”) Post-software images of the same larvae showing neutrophils (magenta) (B’–E’) or macrophage nuclei (green) (B”–E”) and their tracks in the vicinity of the lesion (white lines indicate lesion margins). (i–iii) Higher magnification views from (B,B’,B”) showing the “cell-to-cell” distance between neutrophil cytoplasmic (ii) or macrophage nuclear (iii) margins to lesion margins. (F,G,F’,G’) Graphs showing number and duration of neutrophil-lesion (F,F’) and macrophage-lesion (G,G’) interactions. (H,I,H’,I’) Graphs showing velocity and directionality ratio of neutrophils (H,H’) and macrophages (I,I’) quantified at the lesion site. Scale bars = 100 μm in (B–E), 50 μm in (Bi).

Alterations to the wound inflammatory response upon E. coli infection and consequences for tissue repair. (A) Schematic of the experimental timeline for locally infected flank wound studies showing the region (black box) to be imaged in (B,C,F–I). (B,C) Multi-channel confocal movie frames of flank wounded Tg(lyz:DsRed;mpeg1:nls-Clover) larvae at 0.5 hpw after local injection of control media (B) or E. coli(C), and prior to analysis of neutrophil (magenta) or macrophage (green nuclei) behaviour and their interactions with the wound. (B’,C’,B”,C”) Post-software images of the same larvae showing neutrophils (magenta) (B’,C’) or macrophage nuclei (green) (B”,C”) and their tracks towards the wound (white lines indicate wound margins). (D,E,D’,E’,D”,E”) Graphs showing number and individual/overall duration of neutrophil-wound (D,D’,D”) and macrophage-wound (E,E’,E”) interactions. (F,G) Multi-channel confocal images of flank wounded Tg(krt4:GFP;krt19:tdTomato-CAAX) larvae showing the disposition of the larval zebrafish skin superficial (green) and basal (magenta) cell layers at 0.5 hpw in control (F) or E. coli-injected fish (G). (H,I) Scanning electron micrographs of flank wounded larvae at 0.5 hpw after local injection of control media (H) or E. coli(I). (F’–I’,F”–I”,F”’–I”’) Higher magnification views of wound regions from (F–I) at 0.5 hpw (F’–I’), 8 hpw (F”–I”), and 24 hpw (F”’–I”’). (J) Graph showing rate of healing in infected vs. uninfected wounds. Scale bars = 100 μm in (B,C), 75 μm in (F,G), 50 μm in (H,I), 20 μm in (F’,G’,F”,G”,F”’,G”’), 10 μm in (H’,I’,H”,I”,H”’,I”’).

Altered cancer inflammatory response upon E. coli or Coley’s toxins infection. (A) Schematic of the experimental timeline for systemically infected cancer studies showing the region (black box) to be imaged in (B,C). (B,C) Multi-channel confocal movie frames of the flank of cancerous Tg(kita:HRASG12V-GFP;lyz:DsRed;mpeg1:nls-Clover) larvae at 2 dpi after systemic injection of control media (B) or E. coli(C), and prior to analysis of neutrophil (magenta) or macrophage (small green nuclei) behaviour and their interactions with pre-neoplastic cells (large green cells). (B’,C’,B”,C”) Post-software images of the same larvae showing neutrophils (magenta) (B’,C’) or macrophage nuclei (green) (B”,C”) and their tracks in the vicinity of pre-neoplastic clones (white lines indicate clonal margins). (D,E,D’,E’,D”,E”). Graphs showing number and individual/overall duration of neutrophil-cancer (D,D’,D”) and macrophage-cancer (E,E’,E”) interactions. (F) Schematic of the experimental timeline for locally infected cancer studies showing the region (black box) to be imaged in (G,H). (G–J,G’–J’,G”–J”) The same analysis was carried out for local infection as previously described for the systemic infection experiments. Scale bars = 100 μm.

Prolonged pro-inflammatory response and reduction in cancer cell numbers upon consecutive local injections of E. coli or Coley’s toxins. (A) Schematic of the experimental timeline for single/multiple locally infected cancer studies showing the region (black box) to be imaged in (B–E,G–J). (B–E) Multi-channel confocal images of the flank of cancerous Tg(kita:HRASG12V-GFP) larvae showing pre-neoplastic cells (green) at 3 dpfi after single vs. multiple local injections of control media (B,D) or E. coli(C,E). (F) Graph showing number of pre-neoplastic cells following each treatment. (G–J) Multi-channel confocal images of the flank of cancerous Tg(kita:HRASG12V-GFP;mpeg1:mCherry;tnfα:GFP) showing pre-neoplastic cells (green) and tnfα-positive macrophages (yellow) (tnfα-negative macrophages are red) at 1 dpfi (G,H) or 3 dpfi (I,J) after single vs. multiple local injections of E. coli. (G’–J’) Single-channel confocal images of the same larvae showing GFP-expressing cells (pre-neoplastic cells and tnfα-positive cells). (K) Graph showing the percentage of tnfα-positive macrophages at two timepoints for all of the injection regimes. Scale bars = 100 μm.