The functional and molecular definition of progenitors giving rise to blood vessel endothelium in vivo remains disputed. Upon investigating the overlap of seemingly divergent reports currently defining putative endothelial progenitor cells (EPCs) using single-cell RNA-sequencing and flow cytometry, Protein C Receptor (PROCR) and Platelet-Derived Growth Factor Receptor Alpha (PDGFRA) largely overlapped with previously characterized murine aorta’s CD34+CD31low endovascular progenitors (EVPs). Functional assays and lineage tracing in homeostatic aorta and excisional wounds demonstrated increased clonogenic capacity, engraftment potential, and ability to form differentiated endothelial (D) cells of PROCR+ PDGFRA+ EPCs, termed as refined endothelial progenitor cell (rEPC), as compared to PROCRnegPDGFRAneg EVPs. Similar PROCR and PDGFRA expression in normal human aorta, and increased clonogenic capacity of CD34+CD31lowPROCR+ endothelial cells from freshly isolated human term placenta were observed as compared to controls. Functional validation of human rEPCs is supported by PROCR enrichment, while PDGFRA co‑expression in human endothelial progenitor–like cells is supported at the transcriptomic level only. Thus, overlapping PROCR and PDGFRA expression in EVPs narrows the population with true functional progenitor capacity.

Topical 5-fluorouracil (5-FU) is an effective field therapy for actinic keratoses (AKs) with proven chemopreventive benefit; however, treatment-limiting local skin reactions frequently impair tolerability and adherence in clinical practice. Our objective is to determine whether adjunctive use of a prebiotic and panthenol-containing repair balm (BB5+ repair balm) improves tolerability and treatment adherence to topical 5-FU field therapy for AKs compared with standard of care alone. We conducted a single-blind, randomised controlled trial involving adults prescribed topical 5-FU for AKs on the face/scalp or hands/forearms. Participants were randomised 1:1 to standard of care (5-FU twice daily) or intervention (5-FU twice daily plus BB5+ repair balm applied 30 min after 5-FU), stratified by treatment site and immunocompetence. Primary outcomes were treatment adherence, (≥ 42 applications over four weeks) and worst local skin reaction (LSR) score using a validated severity scale. Secondary outcomes included LSR scores at weeks 1–4 and Dermatology Life Quality Index (DLQI). In all, 59 participants consented, and 50 completed the study (27 intervention, 23 control). Completion of ≥ 42 applications occurred in 59

Importance Therapies for individual keratinocyte carcinomas (KCs) do not prevent the onset of new KCs in a field of sun damage, and therefore the KC burden remains unchanged. Objective To investigate the association of immune checkpoint inhibitors (ICIs) with changes in field cancerization evaluated by the number of actinic keratoses (AKs) and KCs at baseline compared with 12 months after starting ICI therapy. Design, Setting, and Participants This prospective cohort study was performed at the outpatient oncology clinic of a single tertiary public hospital in Brisbane, Australia, from April 1, 2022, to November 30, 2023. Consecutive immunocompetent adults starting therapy with an inhibitor for programmed cell death 1 (PD-1) or programmed cell death ligand 1 (PDL-1) for any active cancer, with a planned treatment duration of at least 6 months, and who exhibited clinical AKs on their forearms were eligible. Those with immunosuppression, concurrent chemotherapy or radiotherapy, or recent topical fluorouracil use were excluded. Exposures Intravenous ICI therapy, either PD-1 or PDL-1 inhibitors with or without a cytotoxic T-lymphocyte–associated protein 4 inhibitor, with therapy duration determined by the treating oncologist. Main Outcomes and Measures Clinical AKs were counted and photographed before and 3, 6, and 12 months after starting ICI therapy. KC numbers were evaluated based on histopathology reports of all skin lesions excised 12 months before and after starting ICI therapy. Participants’ medical history, primary cancer tumor response using Response Evaluation Criteria in Solid Tumors, and adverse events were recorded. Results A total of 23 participants were recruited, of whom 17 (73.9%) were male, with a mean (SD) age of 69.7 (9.6) years. No participants withdrew; however, 4 died during the study due to disease progression. The mean (SD) AK number significantly decreased from 47.2 (33.8) at baseline to 14.3 (12.0) at 12 months ( P < .001). Younger patients (8 of 12 [66.7%] vs 4 of 12 [33.3%]; P = .007) and those with a history of blistering sunburn (12 of 12 [100%] vs 0; P = .005) were more likely to reduce their AK numbers by 65% or greater. KC total numbers decreased from 42 in the 12 months before starting ICI therapy to 17 in the 12 months after. The number of cutaneous squamous cell carcinomas decreased from 16 to 5 in the same period. Conclusions and Relevance This pilot cohort study found that ICIs used for any cancer were associated with a significant reduction of AKs, suggesting potential as an immunopreventive strategy for high-risk individuals. Given the known effects of other chemopreventive agents on KCs, further investigation into ICIs managing field cancerization is required, especially considering toxicity and cost.

Keratinocyte carcinomas such as basal cell carcinomas and squamous cell carcinomas are a major burden affecting morbidity and mortality in solid organ transplant recipients (SOTRs). Best treatment includes frequent skin checks for early detection and surgery for high incidence of skin cancers. Sirolimus is an immunosuppressive drug which may reduce the burden of skin cancer but may be poorly tolerated when given orally. Topical sirolimus has been proven effective at reducing the burden of skin cancers in animal models, and its safety has long been established in children with tuberous sclerosis. A recent 12-week phase II trial of topical sirolimus suggested it was safe and effective at reducing the early signs of skin cancer in the absence of major side effects. The aim of the SiroSkin trial is to determine whether topical sirolimus can fill a major gap in current therapies by reducing the onset and number of new skin cancers thus reducing burden of disease and cost-effectiveness. Protocol for a multi-centred phase III, participant- and clinician assessor-blinded, placebo-controlled randomised trial in SOTRs. A minimum 146 participants randomised 1:1 will be treated with 1

The development of new vascular structures is a pre-requisite for tumor growth and spread. This process is often disorganised and produces immature and leaky vessels and relies at least in part on the activity of endovascular progenitor cells (EVPs), residing in vessel walls and giving rise to mature endothelial cells in de novo blood vessel networks in the tumor. Sox9 is a transcription factor that is playing an important role in stem cell self-renewal and fate choice and is highly upregulated in EVPs. In this study, we aimed to explore how Sox9 activity in the endothelium affects tumor vascularisation, microenvironment, and metastasis. Indeed, Sox9 expression was upregulated in tumor endothelial cells of mice harbouring melanomas. Similarly, we observed the up regulation of SOX9 in human endothelial cells exposed to melanoma cell co-culture or conditioned medium resulting in increased colony formation and reduced maturity as revealed in tube formation assays. Endothelial-specific conditional knockout of Sox9 (Sox9fl/fl/Cdh5CreERt2/Rosa-YFP) resulted in a significant reduction in total endothelial cells in B16-F0 or HcMel12 melanoma tumors inoculated intradermally in both flow-cytometry, lineage tracing and immunostaining of tumor sections. Functionally, there was a significant reduction in tumour size and lung metastases after Sox9 deletion in the endothelium. Importantly, despite a major reduction in the number and area of CD31+ vessels there was a significant increase in pericyte coverage suggesting increased maturity of the remaining vessels upon Sox9 deletion in the endothelium. These changes in the endothelium translated into a reduction in hypoxia as demonstrated by decreased GLUT1 expression and reduced nuclear localisation of HIF1α. RNA sequencing of sorted tumor cells as well as spatial transcriptomics of tumor sections with endothelial-specific deletion of Sox9 versus controls confirmed the reduction in hypoxia and showed dramatic increases in CD4 and CD8 immune T cell infiltration in the centre of tumors as confirmed by immunostaining. In summary, endothelial-specific Sox9 deletion resulted in fewer and more mature de novo vessels in the centre of the tumor and reduced metastatic dissemination, suggesting strategies that target this pathway may restore the normal function of blood vessels in tumors and prevent disease progression.

Regenerative capabilities of the endothelium rely on vessel-resident progenitors termed endothelial colony forming cells (ECFCs). This study aimed to investigate if these progenitors are impacted by conditions (i.e., obesity or atherosclerosis) characterized by increased serum levels of oxidized low-density lipoprotein (oxLDL), a known inducer of Endothelial-to-Mesenchymal Transition (EndMT). Our investigation focused on understanding the effects of EndMT on the self-renewal capabilities of progenitors and the associated molecular alterations. In the presence of oxLDL, ECFCs displayed classical features of EndMT, through reduced endothelial gene and protein expression, function as well as increased mesenchymal genes, contractility, and motility. Additionally, ECFCs displayed a dramatic loss in self-renewal capacity in the presence of oxLDL. RNA-sequencing analysis of ECFCs exposed to oxLDL validated gene expression changes suggesting EndMT and identified SOX9 as one of the highly differentially expressed genes. ATAC sequencing analysis identified SOX9 binding sites associated with regions of dynamic chromosome accessibility resulting from oxLDL exposure, further pointing to its importance. EndMT phenotype and gene expression changes induced by oxLDL in vitro or high fat diet (HFD) in vivo were reversed by the silencing of SOX9 in ECFCs or the endothelial-specific conditional knockout of Sox9 in murine models. Overall, our findings support that EndMT affects vessel-resident endothelial progenitor’s self-renewal. SOX9 activation is an early transcriptional event that drives the mesenchymal transition of endothelial progenitor cells. The identification of the molecular network driving EndMT in vessel-resident endothelial progenitors presents a new avenue in understanding and preventing a range of condition where this process is involved.

The formation of de novo vascular structures is vital for melanoma growth and metastasis. Sox9 is a transcription factor that is highly upregulated in situations of EndMT such as wound fibrosis or atherosclerosis as well as in progenitors of the endothelium (EVPs), playing a crucial role in stem cell self-renewal and quiescence. In this study, we aimed to explore the role of Sox9 deletion in the endothelium on tumor vascularisation, metastasis, and melanoma gene expression. Firstly, we demonstrated thatSox9 expression was upregulated in tumor endothelial cells of mice harboring melanomas. Similarly, melanoma cells induced overexpression of SOX9 in human endothelial cells whether in co-culture or conditioned medium. Endothelial-specific conditional knockout of Sox9 (Sox9fl/fl/Cdh5CreERt2/Rosa-YFP) resulted in a significant reduction in total endothelial cells in B16-F0 or HcMel12 melanoma tumors inoculated subcutaneously. Immunofluorescence staining of tumor sections confirmed a significant reduction in the number and area of CD31+ vessels but revealed a significant increase in pericyte coverage suggesting increased maturity of the remaining vessels upon Sox9 deletion from the endothelium. Similarly, shRNA knockdown of SOX9 in human endothelial cells inhibited tube formation and cell migration in vitro. These changes in the endothelium translated into a reduction in hypoxia as demonstrated by GLUT1 expression and a reduction of glycolysis in melanoma cells as shown by RNAseq. Spatial transcriptomics of tumors with endothelial-specific deletion of Sox9 versus controls showed changes in the cell-cell interactions. Moreover, there was a significant reduction in lung metastases after Sox9 deletion in the endothelium. In summary, endothelial-specific Sox9 deletion resulted in fewer and more mature de novo vessels in the centre of the tumor and reduced metastatic dissemination, suggesting strategies that target this pathway may restore the normal function of blood vessels in tumors and prevent disease progression.

The main carcinogen for keratinocyte skin cancers (KCs) such as basal and squamous cell carcinomas is ultraviolet (UV) radiation. There is growing evidence that accumulation of mutations and clonal expansion play a key role in KC development. The relationship between UV exposure, epidermal mutation load, and KCs remains unclear. Here, we examined the mutation load in both murine (n = 23) and human (n = 37) epidermal samples. Epidermal mutations accumulated in a UV dose-dependent manner, and this mutation load correlated with the KC burden. Epidermal ablation (either mechanical or laser induced), followed by spontaneous healing from underlying epithelial adnexae reduced the mutation load markedly in both mouse (n = 8) and human (n = 6) clinical trials. In a model of UV-induced basal cell carcinoma, epidermal ablation reduced incident lesions by >80% (n = 5). Overall, our findings suggest that mutation burden is strongly associated with KC burden and represents a target to prevent subsequent KCs.

To the Editor: Immunosuppression in solid organ transplant recipients (SOTR) greatly increases the risk of keratinocyte cancer (KC), indisputably a significant burden in terms of morbidity, mortality, and cost. Switching calcineurin inhibitors to sirolimus is a major strategy that has resulted in a two-fold reduction in the risk of squamous cell carcinomas (SCC) but is poorly tolerated with many serious adverse events.1,2

Cutaneous field cancerization resulting from chronic ultraviolet radiation has a high burden of clinical and subclinical actinic damage. Field treatment with topical 5-fluorouracil (5-FU) is effective in treating field cancerization. Based on the concept of field cancerization, resulting from the accumulation of genetic alterations, we hypothesised that field therapies are effective at reducing future keratinocyte cancer formation through decreasing the epidermal mutation load. In our study, adult patients aged 45 years or older with evidence of photodamaged forearms were recruited. A 2mm (diameter) punch biopsy was collected from an area devoid of skin cancers and hyperkeratinisation on the dorsal forearm, to calculate the epidermal mutation burden before and after field treatment with topical 5-FU. A saliva sample was collected for reference genomic DNA. The number of keratotic lesions on the target forearm was assessed clinically before and after field treatment. A patient-reported symptom score was used to assess pain, erythema, itching, crusting, burning, scaling and swelling. Of the 12 patients recruited, the average percentage decrease in the number of keratotic lesions following 5-FU field treatment was 68% (-5 – 100%). Patients completed an average of 40 (21 – 56) applications of 5-FU. The average symptom score was 7.9 out of 21 (4 – 18). The change in epidermal mutation burden following field treatment was variable (P = 0.81). It was reduced in half of the patients, but this did not correlate with the clinical response nor symptoms. In conclusion, topical 5-FU as a field treatment was not associated with a decrease in epidermal mutation burden despite a clinical reduction in keratotic lesions on the photodamaged forearm and symptom severity. Instead of reducing field cancerization through decreasing the mutation load, an alternative mechanism of action of topical 5-FU is possible, such as through stimulation of local immune response.

Sun-exposure leads to field cancerization where all keratinocytes harbor mutations and can potentially develop into a carcinoma. Here, we investigated the relationship of skin mutation load with keratinocyte cancer risk in a case-control series. Finally, epidermal ablation was tested as an intervention to reduce mutation load in preventing skin cancers. Fifteen pairs of patients with high versus low cancer burden were recruited and matched for age, sex and phototype. Comparing the mutation load in the normal-looking forearm skin of these individuals, those with a high cancer burden had an increased mutation load (96.4 vs 76.4 mutations/Mb, p=.017) and a higher number of CC>TT UV specific mutation (15 vs 11, p=0.011). These findings suggest an association between sun exposure, skin mutation load and keratinocyte cancer burden. We therefore examined if reducing the mutation load would reduce the keratinocyte cancers burden. C57BL/6 wild type mice were exposed to a total of 20 weeks of chronic suberythermal UVB irradiation. The lower half of the dorsal epidermis was dermabrased after 10 weeks of irradiation whereas the upper half of the dorsal epidermis was left intact. Dermabrasion removed the entire interfollicular epidermis, leaving the hair follicles intact. Strikingly, the mutation load reduced from 19.5 in the non-dermabrased area to 2.25 mutations/Mb in the dermabrased area (p=0.014). To evaluate the efficacy of epidermal ablation on cancer formation, we utilised a UVB inducible Basal Cell Carcinoma (BCC) murine model (K14Cre/ER::Ptch1lox/+) and repeated the procedures above. Wholemount staining of a BCC biomarker, Keratin-17 (K17), revealed a significant reduction in the number of K17 patches in the dermabrased area (0.35 vs 0.07 patches/mm2). Overall, our results suggest that epidermal ablation can reduce cancer occurrence by lowering mutation load.

Ultra-Violet (UV) radiation from sunlight is the main carcinogen driving epidermal cancers of the skin. The accumulation of UV-induced mutations on sun-exposed areas leads to field cancerisation. Knowing the regenerative property of the skin, we investigated if epidermal ablation removes mutations on the epidermis and allows less mutated cells from the deeper part of the skin to repopulate the ablated surface. Patients with history of multiple epidermal cancers were subjected to epidermal ablation on their forearms with three different depths of laser (600nm, 400nm and fractional laser). Deep targeted DNA sequencing of 151 cancer related genes had revealed the mutation burdens on ablated and non-ablated epidermis. There were 12.9 mutations per Megabase (Muts/Mb) found in non-ablated control epidermis compared to 2.55 Muts/Mb in 600nm, 4.45 Muts/Mb in 400nm and 3.58 Muts/Mb in fractional laser ablated and regenerated epidermis (n=7). To evaluate the efficacy of epidermal ablation on epidermal cancers formation, we utilised a UVB inducible Basal Cell Carcinomas (BCC) murine model (K14Cre/ER::Ptch1lox/+ ). These mice were subjected to a total of 20 weeks of UVB radiation. Half of the dorsal epidermis was dermabrased after the first 10 weeks of radiation. Wholemount staining of a BCC biomarker, Keratin-17 (K17), revealed a significant reduction in the number of K17 patches in dermabrased area (0.35 patches/mm2 ± 0.04 compared to 0.07 patches/mm2 ± 0.03) suggesting dermabrsasion can be used to reduce BCC occurrence. Overall, our findings propose a potential technique to reverse the photo-damage process by removing the mutations accumulated on the epidermis. This study may also pave the way to a larger clinical trial of epidermal ablation as an adjuvant therapy for high-risk epidermal cancer patients.

Animal microbiota have complex interactions with hosts and environment that determines its composition. Yet the ability of hosts to determine their microbiota composition is less well studied. In this study, to investigate the role host genetics in determining skin microbiota, we used 30 different mouse strains from the recombinant inbred panel, the Collaborative Cross. Murine skin microbiota composition was strongly dependent on murine strain with > 50% of the variation explained by murine strain. In particular, a quantitative trait locus on chromosome 4 associates both with Staphylococcus abundance and principal-component multi-trait analyses. Additionally, excisional wound associated changes in microbiota composition were not uniform across mouse strains and were host-specific, the genetic background accounting for about 40% of the variation in microbiota. Genetic background also had the highest effect on the healing speed of wounds accounting for over 50% of the variation while mouse age and microbiota composition change accounted only for 20% and 5% of the healing speed despite reaching statistical significance. In conclusion, host genetics has a significant impact on the skin microbiota composition during both homeostasis and wound healing. These findings have long reaching implications in our understanding of associations between microbiota dysbiosis and disease.### Competing Interest StatementThe authors have declared no competing interest.

The endothelium possesses a profound regenerative capacity to adapt and reorganise in homeostasis and disease. The capacity to regenerate is increasingly attributed to a population of vessel-resident endovascular progenitor (EVP) cells that governs an endothelial hierarchy and have the ability to form vascular networks de novo. Using fate map analysis, we show that two transcription factors Sox9 and Rbpj specifically demarcate the EVP population and regulates progenitor fate choice differentiation. Conditional knock-out of Sox9 from the endothelium drove the depletion of EVP to a mature differentiated endothelial phenotype and enhanced Rbpj expression and Notch signalling. Additionally, skin wound analysis from Sox9 knock-out mice demonstrated a significant reduction in endothelial to mesenchymal transition (EndMT), reducing scar area. The converse was observed with Rbpj conditionally knocked-out from the vasculature, with enhanced Sox9 and key EndMT gene (Snail, Slug, Twist1, Twist 2, TGF-β) expression. Concurrently, vascular sonic hedgehog activation upregulates the expression Sox9 and is key in driving pathological EndMT and vascular fibrosis, resulting in over 3-fold increase in scar area in skin wound healing. In this scenario, we see EVP transitioning towards a mesenchymal fate; with increased Sox9, reduced Rbpj and enhanced EndMT gene expression. Importantly, using topical administration of siRNA against Sox9 on skin wounds significantly reduced scar area by blocking pathological EndMT. The understanding of how vascular resident EVP function opens exciting new avenues for more effective therapies in blocking fibrotic disease.