Effect of wound size and immune infiltrate on tumorigenesis

To test whether wound size, wound closure rate or inflammatory response to wounding influenced tumour incidence, full-thickness skin wounds of different sizes (2, 4, 5, 6 and 8 mm2) were made on back skin of WT and InvEE (Inv) mice, and papilloma formation at the wound site was monitored. Wounds in InvEE and WT littermates healed at the same rate but only InvEE mice developed tumours (Supplementary Fig. 1a,b). Although onset of tumour formation was independent of wound size, there was a linear correlation between wound size and tumour incidence (R2=0.91381; Fig. 1a,b). Wound size and total immune cell infiltrate (CD45+ cells) were correlated in both WT and InvEE skin, but CD45+ cells were significantly more abundant in InvEE skin both before wounding9 and at the time of wound closure (Fig. 1c and Supplementary Fig. 1c,d). There were even more CD45+ cells in the tumour stroma than in newly closed 8 mm2 InvEE wounds (Fig. 1c). These results indicate that the degree of inflammation remaining once the acute response to injury has resolved correlates with the extent of the primary insult and subsequent tumour incidence.

Figure 1: Pro-inflammatory signalling and wound-induced tumour formation in InvEE mice. (a) Correlation between wound diameter and papilloma incidence at wound site in InvEE mice 30 days post wounding (n>20 mice per condition). (b) Incidence of tumours in InvEE mice in 2 mm2 (n=28 mice), 5 mm2 (n=27 mice) or 8 mm2 (n=33 mice) wounds (*0.01<P<0.05; ****P<0.0001; one-way analysis of variance (ANOVA)). (c) Quantification of CD45+ leukocytes in different sized wounds at time of wound closure (n≥4 mice per condition, at least three microscopic fields were quantified per mouse; *0.01<P<0.05, **0.01<P<0.001, ***0.0001<P<0.001, ****P<0.0001; two-way ANOVA). (d) Quantitative PCR normalized to glyceraldehyde 3-phosphate dehydrogenase of NF-κB target gene mRNAs in 8-week-old InvEE epidermis relative to transgene negative littermates (log 2-fold upregulation relative to WT; n=3 mice per condition). Data are means±s.d. of biological and technical triplicates (P<0.0001 for each individual gene product; unpaired t-test). (e–g) Serum levels of TSLP (e), TNF-α (f) and IL-6 (g) in control, tumour-free and tumour-bearing InvEE mice, assessed by ELISA (n=4 mice per condition; *0.01<P<0.05, **0.01<P<0.001, ***0.0001<P<0.001; unpaired t-test). Data are means±s.e.m.; pap: papilloma. Full size image

As nuclear factor-κB (NF-κB) is an important mediator of inflammation-associated cancer10, we analysed expression of NF-κB target genes in InvEE and control epidermis. All 16 of the genes examined were significantly upregulated in InvEE epidermis relative to WT epidermis (P<0.0001 for each individual gene product; Fig. 1d). The effects were systemic, as levels of thymic stromal lymphopoietin (TSLP), tumour-necrosis factor (TNF)-α and interleukin (IL)-6 were elevated in serum of tumour-free InvEE mice and increased further in tumour-bearing animals (Fig. 1e–g).

Tumour formation requires haematopoietic TNFR signalling

TNF-α is well known for its context-dependent pro- and anti-tumorigenic roles11 and downstream TNF-α signalling is mediated by TNFR-1 and TNFR-2. Mice deficient in both receptors are resistant to skin cancer induced by chemical carcinogens12. To examine whether wound-induced tumorigenesis was dependent on TNFR signalling specifically in leukocytes, sub-lethally irradiated InvEE mice were reconstituted with TNFR-1/-2−/− (TNFR−/−) bone marrow (BM) and subsequently wounded. Successful engraftment was verified by Y chromosome-fluorescence in situ hybridization (Y-FISH) in spleens of reconstituted mice as previously described8 (Supplementary Fig. 2a).

TNFR−/− chimeric mice were highly resistant to wound-induced tumour formation (Fig. 2a). Only 8.3% of TNFR−/− chimeric mice developed papillomas, compared with 50% of control chimeras, and time of tumour onset was delayed in TNFR−/− chimeric mice (Fig. 2a). Although wounds closed more rapidly in TNFR−/− chimeras (Supplementary Fig. 2b), in agreement with observations on TNFR-1−/− mice13, the epidermis remained thickened and hyperproliferative, consistent with the ability of MEK1 to stimulate keratinocyte proliferation in the absence of other cell types7 (Fig. 2b). Serum levels of TNF-α were markedly reduced in tumour-free but not tumour-bearing TNFR−/− chimeras (Supplementary Fig. 2c), consistent with MEK1 activation in epidermal tumour cells driving NF-κB activation14.

Figure 2: Role of TNFR signalling in wound-induced tumours. (a) Incidence of papillomas in TNFR-1/-2−/− (TNFR−/−/Inv; n=12) and control (Inv/Inv; n=12) BM chimeras (****P<0.0001; Fisher’s exact test). (b) Epidermal thickness in healed and unwounded skin, defined as the distance from basal to upper granular layer and measured at ten sites per mouse (wound Inv/Inv: n=4; wound TNFR−/−/Inv: n=8; skin Inv/Inv: n=8; skin TNFR−/−/Inv: n=10). (c–g) Infiltration of different immune cell populations in wound beds and tumour stroma of post-wounded InvEE and TNFR−/− chimeric skin (*0.01<P<0.05; unpaired t-test). (d,f) Histological sections were stained with antibodies to CD3 (red in d) and CD4 (green in d) or CD11b (red in f) and F4/80 (green in f) and double positive cells were quantified (c,e). Magnified views are represented on the right. (g) Mast cells were quantified following toluidine blue staining. (c,e,g) n≥3 mice per condition and ≥3 fields were quantified per section. Means±s.e.m. are shown. (d,f) Nuclei were stained with 4,6-diamidino-2-phenylindole (blue); dotted lines indicate basement membrane. Scale bars, 300 μm. Full size image

The tumour-protective effect of TNFR ablation in radiosensitive leukocytes correlated with changes in the skin immune cell infiltrate. CD4+ T cells were markedly reduced in wounds and papillomas of TNFR−/− BM chimeras (Fig. 2c,d). When irradiated InvEE mice were reconstituted with BM from mice expressing enhanced green fluorescent protein under the control of the β-actin cytomegalovirus (CMV) promoter and subsequently wounded, both the wounds and wound-induced tumours were heavily infiltrated with F4/80+ macrophages (Supplementary Fig. 2d). Macrophage (F4/80+ CD11b+) and mast cell numbers were similar in healed wounds of TNFR−/− and control chimeras but significantly reduced in tumour stroma of TNFR−/− chimeras (Fig. 2e–g).

Epidermal γδ T cells infiltrated wounds of both TNFR−/− and control chimeras to the same extent. They were never present within the tumour epithelium, but did accumulate in adjacent epidermis (Supplementary Fig. 2e), suggesting that the previously observed reduction in tumours on γδ T-cell ablation is an indirect effect of reduced macrophage recruitment8. TNFR ablation in the BM did not affect numbers of dendritic cells (CD207+ CD11c+), NK or NKT cells infiltrating wounds or tumours (Supplementary Fig. 2f–h). B cells (CD19+) were not detectable in unwounded skin or healed wound beds15 and there was no difference in the stromal B-cell content of TNFR−/− and control chimeric tumours (Supplementary Fig. 2i).

We conclude that TNFR ablation in leukocytes protected mice from developing tumours. It also led to a selective reduction in CD4+ T cells in wounded skin and a reduction in several immune cell subsets in tumour stroma.

MyD88 and TLR-5 signalling mediate tumour formation

MyD88 (Myeloid Differentiation primary response gene 88) is a master regulator of innate signalling events as it is the key adaptor for most TLRs, IL-1R1 and IL-18R16,17,18. Loss of MyD88 prevents tumour formation in various tissues19,20,21,22. Given that MyD88 controls TNF-α production, we analysed the effect of reconstituting InvEE mice with MyD88−/− radiosensitive leukocytes. BM chimeras lacking MyD88 in the haematopoietic compartment exhibited a striking protection against wound-induced tumour formation (Fig. 3a).

Figure 3: MyD88 and TLR-5 signalling on radiosensitive leukocytes is required for tumour formation. (a–e) Incidence of papillomas at wound site. (a) MyD88−/− (MyD88−/−/Inv; n=20) and control BM chimeras (Inv/Inv; n=20; ****P<0.0001; Fisher’s exact test). (b) IL-1R1−/− (IL-1R1−/−/Inv; n=28) and control BM chimeras (Inv/Inv; n=16; P=0.4312; Fisher’s exact test). (c) TLR-2/−4−/− (TLR-2/-4−/−/Inv; n=29), TLR-9−/− (TLR-9−/−/Inv; n=10) and control BM chimeras (Inv/Inv; n=30; P>0.05; one-way analysis of variance (ANOVA)). (d) TLR-7/-8−/− (TLR-7/−8−/−/Inv; n=9), TLR-5−/− (TLR5−/−/Inv; n=13; ***0.0001<P<0.001; one-way ANOVA) and control BM chimeras (Inv/Inv; n=20). (e) TRIF−/− (TRIF−/−/Inv; n=12) and control BM chimeras (Inv/Inv; n=14; P>0.05; Fisher’s exact test). (f) Rate of wound healing in TLR5−/− and control BM chimeras (n=12 per condition; P>0.05; unpaired t-test). Full size image

Although InvEE keratinocytes express elevated levels of IL-1α and administration of the IL-1 receptor antagonist Kineret decreases tumour formation8,9, no differences in wound-induced tumour formation were observed between IL-1R1−/− BM and control chimeras (Fig. 3b). We therefore examined the effects of deleting TLRs. Replacement of the radiosensitive haematopoietic compartment with TLR-2/-4−/− or TLR-9−/− cells did not affect tumour formation (Fig. 3c), in contrast to the role of TLR-4 on haematopoietic and non-haematopoietic cells in chemically induced skin carcinogenesis23. InvEE mice reconstituted with TLR-7/-8−/− BM exhibited accelerated wound closure (Supplementary Fig. 3a) but no difference in tumour incidence was observed (Fig. 3d). TLR-3 and TLR-4 can signal via TIR-domain-containing adapter-inducing interferon-β (TRIF), instead of MyD88. However, reconstitution with TRIF−/− BM cells had no effect on papilloma formation (Fig. 3e).

Ablation of TLR-5 in radiosensitive leukocytes markedly reduced the number of tumours that developed on wounding (Fig. 3d). Wound closure rates were similar in TLR-5−/− and control BM chimeras, suggesting that the dynamics of wound closure does not affect wound-induced tumour formation (Fig. 3f).

These findings reveal the significance of an innate MyD88-TLR-5-sensing axis specifically in BM-derived leukocytes that drives wound-induced tumour initiation.

Bacterial products mediate tumour initiation in InvEE mice

As flagellin (Fla), the sole known TLR-5 ligand24, is the main protein constituent of bacterial flagella, we analysed whether lowering the microbial content of the skin would affect wound-induced tumour incidence. When mice were treated with the broad-spectrum antibiotic enrofloxacin (enr), either by administration in drinking water or topical application, the skin bacterial load was decreased (Supplementary Fig. 3c, d) and wound-induced tumour formation was greatly reduced (Fig. 4a,c). Tumour size in antibiotic-treated mice was greatly reduced (Fig. 4b), an effect that was previously observed in intestinal tumours25. No reduction in tumour initiation was observed when mice were topically treated with methicillin (met; Fig. 4c), a narrow-spectrum antibiotic that targets Gram-positive bacteria that are highly abundant on the skin, including the non-flagellated species Staphylococcus aureus and Staphylococcus epidermidis.

Figure 4: Microbial products promote tumour formation in InvEE mice. (a,c,d,f,g) Incidence of papilloma formation following wounding (a,c,d,g) or intradermal injection (f,g). (a) Mice were untreated (n=13) or treated with orally administered enrofloxacin (Enr) antibiotic (n=15) starting 8 days before wounding (P<0.0001; Fisher’s exact test). (b) Tumour growth in untreated (n=8) and oral antibiotic-treated (n=4) mice (*0.01<P<0.05; unpaired t-test). Data are means±s.e.m. (c) Mice were treated topically with vehicle (Veh; acetone; n=12), Enr (n=21) or methicillin (Met; n=12; ***0.0001<P<0.001; one-way analysis of variance (ANOVA)). (d) PBS (n=10), 1 μg flagellin (Fla; n=8) or 4 μg Fla (n=10) was applied in wound at time of wounding (****P<0.0001; one-way ANOVA). (e) Wounds treated with flagellin or PBS were photographed 7 days after wounding. (f) Papilloma incidence in mice that received intradermal injections of PBS (n=19), 1 μg Fla (n=8) or 4 μg Fla (n=10; ***0.0001<P<0.001; one-way ANOVA). (g) Papilloma incidence in TLR-5−/− BM chimeras (n=8) and control (n=10) chimeras that were wounded or injected (n=10) with 4 μg Fla (***0.0001<P<0.001; one-way ANOVA). (h) Immunofluorescence labelling of InvEE skin with antibody to flagellated E. coli K12 strain. Wounded skin was collected 17 days after wounding. Unwounded skin was either untreated or treated (+Abx) for 8 days with oral Enr. Dotted line represents epidermal–dermal boundary. Scale bars, 300 μm. Full size image

In mice topically treated with antibiotics, there was no significant reduction in faecal bacterial load, excluding involvement of the gut microbiome in wound-induced skin carcinogenesis (Supplementary Fig. 3e). Broad-spectrum antibiotic treatment resulted in a transient increase in wound closure in WT but not InvEE mice (Supplementary Fig. 3f).

In contrast to the tumour-suppressive effects of TLR-5 ablation and antibiotic treatment, topical application of flagellin to InvEE wounds increased tumour incidence in a dose-dependent manner (Fig. 4d) and delayed wound closure (Fig. 4e and Supplementary Fig. 3b). Strikingly, intradermal injection of flagellin was sufficient to induce small tumours in InvEE mice in the absence of wounding (Fig. 4f). WT mice never developed tumours after administration of flagellin to wounds or intradermal injection (data not shown). Flagellin did not induce tumours when injected into TLR-5−/− BM chimeras. When flagellin was administered to wounds of TLR-5−/− BM chimeras, tumour formation was greatly diminished compared with control chimeras treated with flagellin (Fig. 4g).

Flagellated bacterial strains are commensals on murine skin26. When we labelled unwounded InvEE skin with an antibody to the flagellated Escherichia coli (E. coli) strain K12, we observed strong immunoreactivity in hair follicles, sebaceous glands and cornified skin layers (Fig. 4h), in agreement with a previous report26. As expected, E. coli labelling was markedly reduced in antibiotic-treated skin. All epidermal layers stained positive for K12 E. coli in InvEE healed wound beds and papillomas (Fig. 4h).

Taken together, these data demonstrate that exposure to bacterial flagellin sensed by TLR-5 on radiosensitive leukocytes promotes tumour formation in InvEE mice.

Role of TLR-5 signalling in carcinogen-induced tumours

To validate our observations in a second experimental setting, we induced tumours in WT mice via the classic two-stage DMBA/TPA (7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate) chemical carcinogenesis protocol in which DMBA induces H-Ras mutations and TPA causes chronic inflammation, promoting tumour development27. Irradiated WT mice were reconstituted with WT (control) or TLR-5−/− BM and topically treated with DMBA. Mice subsequently received repeated applications of TPA with or without prior wounding (Fig. 5a). Wound closure was accelerated in TLR-5−/− BM chimeras treated with TPA (Supplementary Fig. 4a).

Figure 5: TLR-5 signalling in leukocytes promotes DMBA/TPA wound-induced tumorigenesis. (a) Schematic of DMBA/TPA wound-induced tumorigenesis protocol. (b,c) Incidence of papilloma formation in WT mice reconstituted with TLR-5−/− (WT/TLR5−/−) or control (WT/WT) BM and treated with DMBA and TPA with (b) or without (c) wounding. (b) n=10 WT/WT mice; n=9 TLR5−/−/WT mice; ****P<0.0001; unpaired t-test. (c) n=10 WT/WT mice; n=9 TLR5−/−/WT chimeras; **0.01<P<0.001; unpaired t-test. (d) Back skin of representative wounded WT/WT and WT/TLR5−/− mice, 4, 14 and 18 weeks after start of TPA treatment. (e) Average total number of tumours and tumour size measured weekly after first TPA treatment. n>8 mice per condition. Full size image

Mice treated with DMBA, but not TPA, and subsequently wounded, did not develop tumours (data not shown). Control chimeras that were wounded before TPA treatment developed tumours after 2 weeks of promotion, which is significantly faster than mice treated with TPA only (Fig. 5). There was a substantial delay in the development of DMBA/TPA-induced tumours in wounded TLR-5−/− BM chimeras relative to wounded WT chimeras (Fig. 5b). The tumour-protective effect of TLR-5−/− BM was also apparent in non-wounded DMBA/TPA-treated mice, albeit less marked (Fig. 5c). TLR-5−/− BM reconstitution did not reduce the final number of papillomas that formed (Supplementary Fig. 4b,c) but decreased tumour size considerably (Fig. 5d,e).

We conclude that TLR-5-mediated signalling is involved in tumour initiation in two different skin cancer models.

Upregulation of HMGB1 in wound-induced tumours

To investigate the relevance of mouse wound-induced tumour formation to human skin cancer, we analysed SCCs from RDEB patients. RDEB is a rare skin blistering condition in which repetitive cycles of wounding and repair predispose the skin to the development of SCCs6.

HMGB1 is a nuclear danger-associated molecular pattern that is passively released from necrotic cells and actively secreted by inflammatory cells28,29. HMGB1 is upregulated in RDEB patients30,31 and HMGB1 serum levels correlate with RDEB disease severity30. HMGB1 is also upregulated in a mouse RDEB model and mediates recruitment of BM-derived cells in injured tissue31. Furthermore, HMGB1 is induced in epithelial cells upon exposure to flagellin32. We therefore investigated HMGB1 as a candidate biomarker linking human and mouse wound-associated skin cancer.

In lesional skin from RDEB patients, HMGB1 was highly upregulated compared with normal human skin and there was strong immunoreactivity for HMGB1 in epidermis and dermis (Fig. 6a; n=6 patients per group). There was an even greater increase in HMGB1 immunoreactivity in RDEB SCCs (Fig. 6a; n=6 patients). Although HMGB1 was mainly nuclear in normal human skin, we observed cytoplasmic HMGB1 in lesional skin and SCCs from RDEB patients (Fig. 6a), which is indicative of HMGB1 secretion in these inflammatory conditions29. Consistent with these findings, HMGB1 was elevated in unwounded InvEE skin relative to WT (Fig. 6b,c) and further increased on wounding and in wound-induced papillomas (n=8; Fig. 6b,c). HMGB1 expression was significantly downregulated in skin of unwounded TLR-5−/−/Inv relative to Inv/Inv BM chimeras and the absence of TLR-5 prevented HMGB1 upregulation on wounding (Fig. 6c). The reduced immunolabelling in skin correlated with a reduction in serum HMGB1 levels in wounded mice (Fig. 6d).

Figure 6: HMGB1 expression in RDEB and InvEE tumours (a–c) Paraffin sections were labelled with anti-HMGB1 (red) and counterstained with 4,6-diamidino-2-phenylindole (blue). Dotted line denotes basement membrane. Arrows denote wound edge. Scale bars, 200 μm. (a) Normal human skin, lesional RDEB skin and RDEB SCCs. (b) WT skin, InvEE healed wound bed (26 days post wounding) and wound-induced papilloma. (c) Unwounded and wounded InvEE/InvEE and InvEE/TLR5−/− BM chimeras. (d) Serum levels of HMGB1 in non-tumour-bearing InvEE/InvEE and InvEE/TLR5−/− BM chimeras at day 26 post wounding, assessed by ELISA (n=9 mice per condition; *0.01<P<0.05; Mann–Whitney U-test). Data are means±s.e.m. Full size image

We conclude that HMGB1 is upregulated in wound-associated mouse and human skin tumours and that HMGB1 levels are regulated by leukocytic TLR-5 signalling.