Innate sensing of microbial products promotes wound-induced skin cancer

ABSTRACT The association between tissue damage, chronic inflammation and cancer is well known. However, the underlying mechanisms are unclear. Here we characterize a mouse model in which

constitutive epidermal extracellular-signal-regulated kinase-MAP-kinase signalling results in epidermal inflammation, and skin wounding induces tumours. We show that tumour incidence

correlates with wound size and inflammatory infiltrate. Ablation of tumour necrosis factor receptor (TNFR)-1/-2, Myeloid Differentiation primary response gene 88 or Toll-like receptor

(TLR)-5, the bacterial flagellin receptor, but not other innate immune sensors, in radiosensitive leukocytes protects against tumour formation. Antibiotic treatment inhibits, whereas

injection of flagellin induces, tumours in a TLR-5-dependent manner. TLR-5 is also involved in chemical-induced skin carcinogenesis in wild-type mice. Leukocytic TLR-5 signalling mediates

upregulation of the alarmin HMGB1 (High Mobility Group Box 1) in wound-induced papillomas. HMGB1 is elevated in tumours of patients with Recessive Dystrophic Epidermolysis Bullosa, a disease

characterized by chronic skin damage. We conclude that in our experimental model the combination of bacteria, chronic inflammation and wounding cooperate to trigger skin cancer. SIMILAR

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ANTIBACTERIAL DEFENCE Article Open access 04 August 2021 INTRODUCTION The association between skin wounding, inflammation and cancer is well established1,2. For example, Marjolin’s ulcers

are aggressive squamous cell carcinomas (SCCs) that specifically develop on areas of previous skin trauma3. Keloid scarring is a consequence of aberrant wound healing and is also described

as benign fibrotic tumour formation4. In addition, malignancies at wound sites are often overlooked in chronic ulcers in diabetic and elderly patients5. Another context in which the

association between skin wounding and cancer is well established is Recessive Dystrophic Epidermolysis Bullosa (RDEB). This inherited skin blistering disease is characterized by repetitive

cycles of wounding and repair and is linked with a high incidence of SCC formation. Almost 100% of RDEB patients will develop at least one SCC6. However, despite the clear link between skin

damage and cancer, little is known about the underlying mechanisms. We previously described a mouse model of wound-induced skin cancer that mimics key features of human hyperproliferative

skin conditions. Wounded human skin and psoriatic lesions are characterized by misexpression of β1 integrin extracellular matrix receptors in the differentiating epidermal cell layers, and

consequent upregulation of extracellular signal-regulated kinase-MAP-kinase signalling7. When this is modelled in transgenic mice by expression of constitutively active MAP-kinase kinase 1

under the control of the involucrin promoter (InvEE mice), there is chronic skin inflammation and epidermal hyperproliferation, and mice develop benign tumours (papillomas) on wounding8,9.

We previously identified a pro-tumorigenic role for macrophages and peripheral γδT cells in this model8. Here we set out to identify the molecular signalling events that underlie the link

between chronic inflammation, tissue damage and skin cancer. We have found a previously unknown role for Toll-like receptor (TLR)-5, the receptor for bacterial flagellin, in skin tumour

formation, both in the InvEE mouse model and in wild-type (WT) mice treated with chemical carcinogens. We further show that TLR-5 plays a role in upregulation of the alarmin HMGB1 (High

Mobility Group Box 1) in wound-induced mouse tumours and demonstrate that HMGB1 is also elevated in tumours of RDEB patients. RESULTS 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

(_R_2=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. 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. 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). 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_. 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). 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). 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. DISCUSSION Host–microbe interactions are crucial for tissue homeostasis and evidence showing

cancer-promoting effects of the commensal microbiome in various organs is accumulating33,34,35. Our studies establish, for the first time, a role for innate sensing of flagellated bacteria

in wound-induced skin cancer. We demonstrate that TLR-5 ablation in BM-derived leukocytes reduced tumour incidence in mice and injection of the TLR-5 ligand flagellin induced papillomas in a

TLR-5-dependent manner. Our analysis of RDEB tumours points to the relevance of our findings to human skin cancer. HMGB1, which is induced in epithelial cells in response to flagellin32,

was strongly upregulated in wound-associated tumours in mice and RDEB patients. Ablation of TLR-5 in BM cells led to significantly reduced murine HMGB1 serum levels. The skin microbiome

composition is different across body surfaces36 and directs local immune responses26,37. The human microbiome project reference genome database reveals that intact skin contains flagellated

bacterial species (based on the presence of _FliC_ and _FliA_ genes; http://www.hmpdacc.org/catalog/38). Consistent with this, a flagellated _E. coli_ strain is present on InvEE skin26.

Although most bacteria are kept at bay by the skin barrier, in a wound situation the moist and metabolite-rich environment can promote growth of opportunistic bacteria, including

energetically expensive flagellated species39. Profiling the microbial configurations in chronic diabetic wounds and leg ulcers has indicated an increased representation of _E. coli_,

_Pseuodomonas aeruginosa_, _Shigella_ and other species comprising flagellated strains, whereas Staphylococci, a genus that does not comprise flagellates, are negatively correlated with

ulcer duration40,41. Consistent with the observations in human skin, topical application of a broad-spectrum antibiotic was tumour protective in InvEE mice, whereas an antibiotic that

targets Staphylocci was not. These observations lead us to propose that a key, and previously unrecognized, driver of wound-induced skin cancer is an increased exposure of leukocytes to

flagellated bacteria. Both of the mouse models in which we demonstrated a role for leukocytic TLR-5 sensing in wound-induced skin cancer are characterized by chronic inflammation. In InvEE

mice, the inflammation results from epidermal expression of constitutively active MEK1 and in WT mice inflammation is induced by TPA treatment. TLR-5 ablation in BM does not abolish the

inflammatory infiltrate in InvEE skin or TPA-treated WT skin. Our studies indicate that in the context of chronic inflammation, TLR-5 signalling in leukocytes can tip the balance between

normal wound repair and tumour formation. The tumour-inhibitory effect of ablating TNFR or MyD88 in radiosensitive leukocytes in InvEE mice was greater than that of TLR-5 ablation (Figs 2a

and 3a,d; 0.01<_P_<0.05; one-way analysis of variance), suggesting that additional components of the immune system that converge on NF-κB may contribute (Fig. 1d). Our findings raise

the possibility that the incidence of SCCs in non-healing ulcers and skin from RDEB patients could be reduced by systemic antibiotics treatment and the use of flagellin-specific targeting

strategies in wound-induced malignancies might present an interesting clinical avenue. METHODS MICE InvEE mice9 were maintained on an F1 genetic background (CBA × C57Bl/6) and kept

heterogeneous for the MEK1 transgene. Transgene negative littermates were used as WT controls and in DMBA/TPA experiments. All donor mice in the BM transplantation experiments were on a

C57/Bl6 genetic background. TLR-5−/− and TNFR-1/-2−/− mice were purchased from Jackson Laboratories. TLR-2/-4−/− mice were obtained from Simon Clare, IL-1R1−/− mice from Nancy Rothwell,

TRIF−/− mice from Frederic Geissmann, TLR-7/-8−/− mice from Lena Alexapoulou, MyD88−/− mice from Caetano Reis e Sousa and TLR-9−/− mice from Kinya Otsu. Animal procedures were subject to

local ethical approval and performed under a UK Government Home Office Licence. Sample sizes were determined on the basis of prior power calculations. No mice died as a direct result of

wounding or tumour formation. Mice that died as a result of myeloablation were excluded from analysis. WOUNDING AND BM RECONSTITUTION Full-thickness wounds were made on the back skin by

using 2, 4, 5, 6 or 8 mm2 punch biopsy needles (Stiefel Instruments) under analgesia and general anaesthesia in 8- to 16-week-old age- and sex-matched InvEE and control littermates.

Statistical power was calculated using the resource equation and animals were randomly assigned to treatment groups. No animals were excluded from any experiment. Tumour formation and size

were measured by two independent researchers, who were blinded to group allocations. In BM transplantation experiments8, 8- to 16-week-old female recipient mice were treated with acidified

water at least 10 days before irradiation. Allogenic BM transplants were performed 24 h after myeloablative total body irradiation42 (two times 5 Gy, separated by 3 h) of InvEE mice. Donor

BM was isolated from the tibia and femur of male mice. BM reconstitution was performed by intravenous injection of 5 × 106 BM cells in 200 μl of PBS 24 h after irradiation. Chimerism was

confirmed using Y-chromosome _in situ_ hybridization (Cytocell, probe AMPOYR) on spleens of reconstituted mice8. Mice were wounded 4 to 6 weeks post BM reconstitution and papilloma formation

was monitored for 50 to 60 days post wounding. Each InvEE BM reconstitution experiment was repeated two to three times. TREATMENT WITH ANTIBIOTICS AND FLAGELLIN Mice were treated with the

broad-spectrum antibiotic enrofloxacin by oral administration (5 ml Baytril per liter drinking water) starting 8 days before wounding and continuing until the end of the experiment. For

topical applications, shaved back skin of mice was treated daily with 200 μl of vehicle (acetone) or antibiotics in acetone starting 8 days before wounding. Antibiotics used were 0.25%

enrofloxacin or 0.2 mg ml−1 methicillin. Skin swabs were taken on day 10 post wounding and plated on lysogeny broth (LB) and blood agar plates to verify bacterial depletion. 16S rRNA

quantification was performed on skin biopsies taken at the time of wounding. 1 or 4 μg flagellin (high-purity flagellin, EnzoLifeSciences) was injected once intradermally in back skin in 100

 μl PBS; control mice were injected with 100 μl PBS. Flagellin was applied once topically to 8 mm2 full-thickness skin wounds at time of wounding. QUANTITATIVE PCR Epidermis was separated

from the underlying dermis by scraping skin after incubation at 60 °C for 7 s in RNase-free water. Isolated epidermis was homogenized in lysis buffer using ceramic beads. RNA extraction was

performed according to the manufacturer’s instructions using a QIAGEN RNeasy Kit (Qiagen). Complementary DNA was generated using reverse transcriptase III (Invitrogen). Quantitative

real-time PCR reactions were set up using gene-specific primer sets (see Supplementary Table 1) and reactions were performed on a 7900HT real-time PCR machine (Applied Biosystems) on

biological triplicates. MICROBIOTA QUANTIFICATION Bacterial load was quantified in skin biopsy samples (8 mm2) and in faeces collected from mice 8 days after antibiotic treatment. Samples

were digested with Ready-Lyse Lysozyme Solution (Epidentre Biotechnologies) in lysis buffer (20 mM Tris, pH 8.0, 2 mM EDTA, 1.2% Triton X-100, DNA-free water). Samples were homogenized using

5 mm Stainless Steel Beads (Qiagen) in a Precellys homogenizer twice at 6,000 r.p.m. for 50 s. DNA was extracted using a Purelink Genomic DNA kit (Invitrogen). For 16S rRNA quantification,

the following primers were used: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 5′-CTGCTGCCTCCCGTAGGAGT-3′. Data for skin bacterial load were normalized against the relative mouse genomic DNA amplicon

quantification. Primers used were 5′-TTAGCAGTTTGGCACAGCTAGG-3′ and 5′-CTAGGTTGGCAAGGAATTGTGG-3′. ELISA Blood was collected by cardiac puncture and left to clot for 2 h. Serum was collected

after centrifugation for 20 min at 4 °C. TNF-α, IL-6 and TSLP levels were quantified using Quantikine ELISA kits (R&D systems). HMGB1 serum levels were measured by ELISA (Shino-test

Corporation). DMBA/TPA TWO-STAGE CARCINOGENESIS Chemical carcinogenesis experiments were performed as previously described27. In brief, back skin of mice was shaved and treated once with 100

 nmol (25 μg) DMBA in 200 μl acetone 5 weeks after BM reconstitution. Seven days later, mice were either wounded with an 8 mm2 full-thickness skin biopsy punch or treated with 6 nmol (3.7 

μg) TPA. After 2 days, all mice were treated three times weekly with TPA for 15 weeks. Tumour incidence and burden were assessed once a week by two independent researchers who were blinded

to the group allocations. IMMUNOFLUORESCENCE STAINING Frozen sections were fixed in acetone/methanol (1:1) or 4% paraformaldehyde/PBS pH 7.4 and blocked with 2% BSA, 0.02% fish skin gelatin

and 10% goat serum for 1 h in PBS at room temperature. Paraffin sections were subjected to heat-mediated antigen retrieval (citrate buffer, pH6). When fluorochrome-conjugated primary

antibodies were used, sections were incubated overnight at 4 °C in antibody solution containing 4',6-diamidino-2-phenylindole, washed in PBS and then mounted using the DAKO mounting

reagent (DAKO). In the case of the unconjugated primary antibodies (HMGB1, Abcam, ab79823; anti-_Escherichia coli_ antibody, DAKO, B0357), incubation was overnight at 4 °C followed by 2 h

incubation at room temperature in secondary antibody (AlexaFluor 555 goat-anti-rabbit) containing 4,6-diamidino-2-phenylindole. Alexafluor 647-, 660- or 488-conjugated antibodies to CD3

(BioLegend, clone 17.A2), CD4 (eBioscience, clone RM4-5), F4/80 (eBioscience, clone BM8), CD19 (eBioscience, eFluor 660), CD11b (eBioscience, clone M1/70), CD11c (Cambridge Bioscience), γδ

TCR (BD Pharmingen, clone GL3), NK1.1 (BioLegend, clone PK136) and CD207 (eBioscience, eFluor 660) were used. IMMUNOHISTOCHEMISTRY Dewaxed paraffin sections were subjected to heat-mediated

antigen retrieval (citrate buffer, pH6). Mast cells were quantified by counting toluidine blue-stained cells on paraffin skin sections. CD45 (BD Pharmingen, clone 30-F11) antibody was used

on paraffin sections at 1:100 dilution. Quantification of immune cell infiltration was performed within the wound and adjacent to the wound (maximum distance from wound/pap: 4 mm) or in

tumour stroma. HUMAN SAMPLES All human samples were collected after informed, written consent and in accordance with Helsinki guidelines, in compliance with the UK Human Tissue Act. The

study protocol was approved by the Universities of Monterrey and New South Wales. STATISTICS Statistical analyses were performed using GraphPad Prism v6. Specific tests included Fisher’s

exact tests, one or two-way analysis of variance, unpaired _t_-tests and Mann–Whitney tests. *0.01<_P_<0.05, **0.01<_P_<0.001, ***0.0001<_P_<0.001, ****_P_<0.0001.

ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Hoste, E. _et al._ Innate sensing of microbial products promotes wound-induced skin cancer. _Nat. Commun._ 6:5932 doi: 10.1038/ncomms6932

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repopulating cells. _Blood_ 93, 1916–1921 (1999). CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Watt lab members for input. We thank Ludmila Sabolcikova, Matthew

Clayton, Jennifer Arthur, Claire Pearce, Ellie and Mark Pryor and the CRUK Cambridge Research Institute Histopathology Lab for technical assistance. We thank Lena Alexopoulou, Caetano Reis

e Sousa, Simon Clare, Frederic Geissmann, Kinya Otsu and Nancy Rothwell for supplying mutant mice or bone marrow. We acknowledge financial support from the MRC, Wellcome Trust, Cancer

Research UK and European Union (HEALING EU-FP7). The research was funded/supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy's and St

Thomas' NHS Foundation Trust and King's College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.

AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Cancer Research UK Cambridge Research institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK Esther Hoste, Esther N. Arwert & 

Giacomo Donati * Centre for Stem Cells and Regenerative Medicine, King’s College London, 28th Floor, Tower Wing, Guy’s Campus, London, SE1 9RT, UK Esther Hoste, Giacomo Donati & Fiona M.

Watt * Cancer Clinical Academic Group, Guy's and St Thomas' NHS Trust, Bermondsey Wing, Guy's Hospital, Great Maze Pond, London, SE1 9RT, UK Rohit Lal * Division of Cancer

Research, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK Andrew P. South * Department of Dermatology and Cutaneous Biology, Thomas Jefferson University,

Philadelphia, Pennsylvania 19107, USA Andrew P. South * Basic Science Department, Medicine School, University of Monterrey, Mexico, Nuevo Leon 64849 Julio C. Salas-Alanis * Department of

Dermatology, St George Hospital, University of New South Wales, Sydney, New South Wales 2217, Australia, Dedee F. Murrell Authors * Esther Hoste View author publications You can also search

for this author inPubMed Google Scholar * Esther N. Arwert View author publications You can also search for this author inPubMed Google Scholar * Rohit Lal View author publications You can

also search for this author inPubMed Google Scholar * Andrew P. South View author publications You can also search for this author inPubMed Google Scholar * Julio C. Salas-Alanis View author

publications You can also search for this author inPubMed Google Scholar * Dedee F. Murrell View author publications You can also search for this author inPubMed Google Scholar * Giacomo

Donati View author publications You can also search for this author inPubMed Google Scholar * Fiona M. Watt View author publications You can also search for this author inPubMed Google

Scholar CONTRIBUTIONS E.H. conception and design, acquisition of data, analysis and interpretation of data, drafting and revising the article; E.N.A., R.L., G.D. acquisition of data,

analysis of data; A.P.S., J.C.S.-A., D.F.M. acquisition of data; F.M.W. conception and design, analysis and interpretation of data, drafting and revising the article. CORRESPONDING AUTHOR

Correspondence to Fiona M. Watt. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION

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promotes wound-induced skin cancer. _Nat Commun_ 6, 5932 (2015). https://doi.org/10.1038/ncomms6932 Download citation * Received: 28 October 2014 * Accepted: 21 November 2014 * Published: 09

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