Ultraviolet A and B (UVA 320-400 nm and UVB 280-320 nm) induced cyclobutane-pyrimidine dimers (CPDs) are the most critical lesions caused by environmental sun exposure. Here we show that CPD removal is accelerated when, in addition to UV, cells are simultaneously exposed to water-filtered near-infrared (nIR, 750-1600 nm). The described effect is dose-dependent on the nIR-dose and is found in skin keratinocytes and fibroblasts. Accelerated removal of CPDs, which coincides with chromatin relaxation and faster CPD recognition, occurs after nIR exposure. While nIR alone does not affect cellular survival, co-exposure to UVB leads to reduced cellular survival and an increased number of mutations. Increasing single strand break levels (SSB) occur transiently after nIR exposure and independent of reactive oxygen species (ROS) formation. These data suggest that the rate-limiting step in the NER repair process - damage recognition - is facilitated by nIR-induced chromatin relaxation, causing the accumulation of unnatural high levels of SSBs and single stranded DNA, unfavourable for the cell fate resulting in reduced survival and increased mutation rates. Since nIR modulates the UV-dependent damage response, risk estimation of solar radiation-induced DNA damage should not only consider the UV components but also include the nIR fraction of the solar spectrum.

MicroRNAs (miRNAs) control organogenesis in mammals by inhibiting translation of mRNA. Skin is an excellent model to study the role of miRNAs in epidermis and the mesenchyme. Previous research demonstrated miRNA-29 family functions in skin; however, the mRNA targets and the downstream mechanisms of miRNA-29-mediated regulation are missing. We used the miRNA crosslinking and immunoprecipitation method to find direct targets of miRNA-29 in keratinocytes and fibroblasts from human skin. miRNA-29 inhibition using modified antisense oligonucleotides in 2D and 3D cultures of keratinocytes and fibroblasts enhanced cell-to-matrix adhesion through autocrine and paracrine mechanisms of miRNA-29-dependent tissue growth. We reveal a full transcriptome of human keratinocytes with enhanced adhesion to the matrix, which supports regeneration of the epidermis and is regulated by miRNA-29. Impact statement The functions of small, therapeutically targetable microRNA molecules identified in our study can provide a new approach to improve wound healing by restoring and enhancing the inner molecular mechanisms of a cell and its surrounding matrix. We also provide a plethora of new mRNA targets for follow-up studies of cell adhesion and extracellular matrix formation in humans.

Psoriasis is a major inflammatory skin disease for which a causal therapy is still not available. The pro-inflammatory cytokines interleukin(IL)-1β and IL-36γ are key drivers of the disease phenotype, but the mechanisms underlying their regulation in psoriasis remain poorly understood. Generation of IL-1β activity is regulated by protein complexes, termed inflammasomes. We activated the NLRP1 inflammasome in human keratinocytes cultivated in three-dimensional skin equivalents. NLRP1 activation induced histological and molecular features that are highly reminiscent of psoriasis. Mechanistically, the phenotype was dependent on IL-1, which triggered a pro-inflammatory epidermal-dermal crosstalk. This included induction of expression of IL-36γ, which, together with IL-1β, was released from keratinocytes through NLRP1-induced gasdermin D pores. The in vivo relevance of these findings is reflected by the expression of the NLRP1 sensor and signs of inflammasome activation in lesional skin of psoriatic patients. Finally, we discovered endogenous cytoplasmic double stranded (ds) RNA, recently associated with cellular perturbations in psoriasis, as a novel activator of the NLRP1 inflammasome in human keratinocytes. Our results identify a novel endogenous double-stranded RNA-mediated NLRP1-IL-1-IL-36γ signaling axis relevant in psoriasis and suggest targeting of this pathway as a promising treatment strategy. ### Competing Interest Statement The authors have declared no competing interest.

MicroRNAs (miRNAs) control organogenesis in mammals but their role in specific cell types is not fully explored. miRNAs exert their function by binding mRNAs and inhibiting translation. Skin is an excellent model to study the role of miRNAs in primary cells of epidermal (keratinocytes) and mesodermal (fibroblasts) origin, because the growth of these cells is tightly controlled at translation. Previous research demonstrated that miRNA-29 family functions during skin repair, however, the exact mRNA targets and the downstream mechanisms of miRNA-29-mediated regulation of cell growth is missing. Here, we use miRNA crosslinking and immunoprecipitation (miRNA-CLIP) method to find the direct targets of miRNA-29 in keratinocytes and fibroblasts from human skin. We uncover previously unrecognized roles of miRNA-29 in protein folding and RNA processing, common to all cell types tested, and determine the cell-specific role of miRNA-29. Using modified anti-sense oligonucleotides (ASO) in 2D and 3D cultures of keratinocytes and fibroblasts, we enhanced cell-to-matrix adhesion and found an autocrine and paracrine mechanism of miRNA-29-dependent cell growth. Our results include a comprehensive list and functional analyses of mRNAs directly bound by miRNA-29 keratinocytes and fibroblasts, determined by miRNA-CLIP and ASO-mediated inhibition of miRNA-29 followed by RNA-seq. We reveal a full transcriptome of human keratinocytes with enhanced adhesion to the wound matrix, which supports regeneration of the epidermis and is regulated by miRNA-29. The functions of miRNA-29 identified in this study can provide a new approach to improve cutaneous repair by restoring and enhancing the endogenous mechanisms through the stage-specific delivery of miRNA-29 ASO. ### Competing Interest Statement The authors have declared no competing interest.

Vδ1 T cells are a subpopulation of γδT cells found in human dermis. Much less is known regarding their role and function in skin health and disease than regarding the roles of murine skin-resident γδT cells. In this study, we report the successful integration of Vδ1 T cells into long-term fibroblast-derived matrix skin equivalents. We isolated Vδ1 T cells from human blood, where they are rare, and established conditions for the integration and maintenance of the freshly isolated Vδ1 T cells in the skin equivalents. Plated on top of the dermal equivalents, almost all Vδ1 T cells migrated into the dermal matrix where they exerted their influence on both the dermal equivalents and the epithelium. Vδ1 T cells contributed to epidermal differentiation of HaCaT cells as indicated by histology, expression of epidermal differentiation markers, and RNA-sequencing expression profile. When complemented with the carcinoma-derived SCC13 cells instead of HaCaT, our data suggest a role for Vδ1 T cells in slowing growth of the tumor cells, as indicated by reduced stratification and changes in gene expression profiles. Together, we demonstrate the successful establishment of human Vδ1 T cell-competent skin equivalents and skin carcinoma equivalents and provide evidence for molecular and functional consequences of the Vδ1 T cells on their respective environment.

The BRAF inhibitor vemurafenib, approved for treating patients with BRAF V600E-mutant and unresectable or metastatic melanomas, rapidly induces cutaneous adverse events, including hyperkeratotic skin lesions and cutaneous squamous cell carcinomas (cSCC). To determine, how vemurafenib would provoke these adverse events, we utilized long-term in vitro skin equivalents (SEs) comprising epidermal keratinocytes and dermal fibroblasts in their physiological environment. We inserted keratinocytes with different genetic background [normal keratinocytes: NHEK, HaCaT (p53/mut), and HrasA5 (p53/mut+Hras/mut)] to analyze effects depending on the stage of carcinogenesis. We now show that vemurafenib activates MEK-ERK signaling in both, keratinocytes, and fibroblasts in vitro and in the in vivo-like SEs. As a consequence, vemurafenib does not provide a growth advantage but leads to a differentiation phenotype, causing accelerated differentiation and hyperkeratosis in the NHEK and normalized stratification and cornification in the transformed keratinocytes. Although all keratinocytes responded very similarly to vemurafenib in their expression profile, particularly with a significant induction of MMP1 and MMP3, only the HrasA5 cells revealed a vemurafenib-dependent pathophysiological shift to tumor progression, i.e., the initiation of invasive growth. This was shown by increased proteolytic activity allowing for penetration of the basement membrane and invasion into the disrupted underlying matrix. Blocking MMP activity, by the addition of ilomastat, prevented invasion with all corresponding degradative activities, thus substantiating that the RAS-RAF-MEK-ERK/MMP axis is the most important molecular basis for the rapid switch towards tumorigenic conversion of the HrasA5 keratinocytes upon vemurafenib treatment. Finally, cotreatment with vemurafenib and the MEK inhibitor cobimetinib prevented MEK-ERK hyperactivation and with that abolished both, the epidermal differentiation and the tumor invasion phenotype. This suggests that both cutaneous adverse events are under direct control of vemurafenib-dependent MEK-ERK hyperactivation and confirms the dependence on preexisting genetic alterations of the skin keratinocytes that determine the basis towards induction of tumorigenic progression.

To understand the importance of terrestrial solar exposure on human skin, not only individual spectral components need to be considered in biomedical studies, but also the relevance of the combined action profile of the complete solar spectrum (cSS) must be established. We therefore developed a novel irradiation device that combines the emission of four individual lamps (UVB, UVA, VIS and nIR) to achieve exposure from 280 to 1400 nm with individual controllable lamps. The integrated irradiance of each spectral band is similar to the solar spectrum. The lamps can be utilised individually or in any desired combination. Here we present the design, realisation, and validation of this irradiation device as well as biological results on cellular metabolism (MTT assay), cell cycle alterations, and clonogenic growth in HaCaT cells after exposures to the individual spectral bands as well as their simultaneous combinations. Thereby, we demonstrate that UVB combined with UVA is the main determinant for the metabolic activity within cSS. Also, UVB-dependent effects dominate cell cycle regulation in cSS, whilst UVA and nIR have little influence. Lastly, also clonogenic growth is dominated by the UVB action profile in cSS, despite nIR showing modulatory activity when applied in combination with UVB. Together, this highlights the regulatory influence of the different spectral bands on the three biological endpoints and demonstrates their modulation when being part of the complete solar spectrum.

Aging is associated with progressive skin fragility and a tendency to tear, which can lead to severe clinical complications. The transcription factor NRF2 is a key regulator of the cellular antioxidant response, and pharmacological NRF2 activation is a promising strategy for the prevention of age-related diseases. Using a combination of molecular and cellular biology, histology, imaging and biomechanical studies we show, however, that constitutive genetic activation of Nrf2 in fibroblasts of mice suppresses collagen and elastin expression, resulting in reduced skin strength as seen in aged mice. Mechanistically, the "aging matrisome" results in part from direct Nrf2-mediated overexpression of a network of microRNAs that target mRNAs of major skin collagens and other matrix components. Bioinformatics and functional studies revealed high NRF2 activity in aged human fibroblasts in 3D skin equivalents and human skin biopsies, highlighting the translational relevance of the functional mouse data. Together, these results identify activated NRF2 as a promoter of age-related molecular and biomechanical skin features.

The skin microbiota plays a prominent role in health and disease; however, its contribution to skin tumorigenesis is not well understood. We comparatively assessed the microbial community compositions from excision specimens of the main human non-melanoma skin cancers, actinic keratosis (AK), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). Keratinocyte skin tumors are characterized by significantly different microbial community compositions, wherein AK and SCC are more similar to each other than to BCC. Notably, in SCC, which represents the advanced tumor entity and frequently develops from AK, overabundance of Staphylococcus aureus, a known skin pathogen, was noted. Moreover, S. aureus overabundance was significantly associated with increased human β-defensin-2 (hBD-2) expression in SCC. By challenging human SCC cell lines with S. aureus, a specific induction of hBD-2 expression and increased tumor cell growth was seen. Increased proliferation was also induced by directly challenging SCC cells with hBD-2. Together, our data indicate that a changed microbial community composition in SCC, specified by S. aureus overabundance, might promote tumor cell growth via modulation of hBD-2 expression.

Three-dimensional (3D) in vitro skin and skin cancer models have become an invaluable tool in skin research. They go back to 1979, when Bell and colleagues reported on the establishment of a fibroblast-dependent collagen tissue (Bell, Ivarsson, & Merrill, 1979). On top of such tissue a stratified and differentiated epidermis could be established (Bell, Merrill, & Solomon, 1979). Hydrogel-based dermal equivalents have been generated ever since and upon co-culture with normal human skin keratinocytes, these constructs were then termed skin equivalents. Due to a number of deficiencies, the most important one being their restricted survival time, new developments helped to circumvent premature fibroblast activation and tissue destruction. By avoiding collagen for the dermal equivalent (DE), we proposed, a scaffold-based DE, allowing fibroblasts to reorganize the primary fibrin solution into an "authentic" dermal matrix (Boehnke et al., 2007; Stark et al., 2004, 2006). With this, our goal of a long-term skin equivalent-successful cultivation for several months-was achieved. Nevertheless, also this model presented limitations. One being its opaqueness made it difficult to image the intact tissue. Another draw-back was that tumor cells upon invasion used the scaffold as a guardrail leaving behind an unspecific invasion pattern. All this could be avoided by an approach, the fibroblast-derived matrix-based model, based on the work by Ahlfors and Billiar (2007) We here provide a protocol for this type of model, thereby providing the basis for future work in the field of skin research.

UV exposure is the main risk factor for development of cutaneous squamous cell carcinoma (cSCC). While early detection greatly improves cSCC prognosis, locally advanced or metastatic cSCC has a severely impaired prognosis. Notably, the mechanisms of progression to metastatic cSCC are not well understood. We hypothesized that UV exposure of already transformed epithelial cSCC cells further induces changes which might be involved in the progression to metastatic cSCCs and that UV-inducible microRNAs (miRNAs) might play an important role. Thus, we analyzed the impact of UV radiation of different quality (UVA, UVB, UVA + UVB) on the miRNA expression pattern in established cell lines generated from primary and metastatic cSCCs (Met-1, Met-4) using the NanoString nCounter platform. This analysis revealed that the expression pattern of miRNAs depends on both the cell line used per se and on the quality of UV radiation. Comparison of UV-induced miRNAs in cSCC cell lines established from a primary tumor (Met-1) and the respective (un-irradiated) metastasis (Met-4) suggest that miR-7-5p, miR-29a-3p and miR-183-5p are involved in a UV-driven pathway of progression to metastasis. This notion is supported by the fact that these three miRNAs build up a network of 81 potential target genes involved e.g. in UVA/UVB-induced MAPK signaling and regulation of the epithelial–mesenchymal transition. As an example, PTEN, a target of UV-upregulated miRNAs (miR-29a-3p, miR-183-5p), could be shown to be down-regulated in response to UV radiation. We further identified CNOT8, the transcription complex subunit 8 of the CCR4-NOT complex, a deadenylase removing the poly(A) tail from miRNA-destabilized mRNAs, in the center of this network, targeted by all three miRNAs. In summary, our results demonstrate that UV radiation induces an miRNA expression pattern in primary SCC cell line partly resembling those of metastatic cell line, thus suggesting that UV radiation impacts SCC progression beyond initiation.

We here present the spontaneously immortalised cell line, HaSKpw, as a novel model for the multistep process of skin carcinogenesis. HaSKpw cells were established from the epidermis of normal human adult skin that, without crisis, are now growing unrestricted and feeder-independent. At passage 22, clonal populations were established and clone7 (HaSKpwC7) was further compared to the also spontaneously immortalized HaCaT cells. As important differences, the HaSKpw cells express wild-type p53, remain pseudodiploid, and show a unique chromosomal profile with numerous complex aberrations involving chromosome 20. In addition, HaSKpw cells overexpress a pattern of genes and miRNAs such as KRT34, LOX, S100A9, miR21, and miR155; all pointing to a tumorigenic status. In concordance, HaSKpw cells exhibit reduced desmosomal contacts that provide them with increased motility and a highly migratory/invasive phenotype as demonstrated in scratch- and Boyden chamber assays. In 3D organotypic cultures, both HaCaT and HaSKpw cells form disorganized epithelia but only the HaSKpw cells show tumorcell-like invasive growth. Together, HaSKpwC7 and HaCaT cells represent two spontaneous (non-genetically engineered) "premalignant" keratinocyte lines from adult human skin that display different stages of the multistep process of skin carcinogenesis and thus represent unique models for analysing skin cancer development and progression.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin tumor in humans. Although current therapies are sufficient to clear the tumor in many cases, the overall risk of cSCC metastasis is still 5%. Alternative treatment options could help to overcome this situation. Here we focused on the role of the Hedgehog (HH) signaling pathway and its interplay with epidermal growth factor receptor (EGFR) signaling in cSCC. The analyses revealed that, despite lack of Sonic HH (SHH) expression, a subset of human cSCC can express GLI1, a marker for active HH signaling, within distinct tumor areas. In contrast, all tumors strongly express EGFR and the hair follicle stem cell marker SOX9 at the highly proliferative tumor-stroma interface, whereas central tumor regions with a more differentiated stratum spinosum cell type lack both EGFR and SOX9 expression. In vitro experiments indicate that activation of EGFR signaling in the human cSCC cell lines SCL-1, MET-1, and MET-4 leads to GLI1 inhibition via the MEK/ERK axis without affecting cellular proliferation. Of note, EGFR activation also inhibits cellular migration of SCL-1 and MET-4 cells. Because proliferation and migration of the cells is also not altered by a GLI1 knockdown, GLI1 is apparently not involved in processes of aggressiveness in established cSCC tumors. In contrast, our data rather suggest a negative correlation between Gli1 expression level and cSCC formation because skin of Ptch +/- mice with slightly elevated Gli1 expression levels is significantly less susceptible to chemically-induced cSCC formation compared to murine wildtype skin. Although not yet formally validated, these data open the possibility that GLI1 (and thus HH signaling) may antagonize cSCC initiation and is not involved in cSCC aggressiveness, at least in a subset of cSCC.

Background T-lymphocytes are involved in tumor progression and regression. Actinic keratoses (AK) are atypical proliferations of keratinocytes of the skin. Some AK progress into invasive cutaneous squamous cell carcinomas (cSCC). Keratoacanthomas (KA) are either classified as a cSCC subtype or a benign tumor with histologic resemblance to well-differentiated cSCC as it is supposed to regress spontaneously. In contrast, cSCC represent malignant tumors that may metastasize. Objectives To compare the T-lymphocyte profiles of AK, KA and cSCC in relation to PD-L1 expression. Methods Tissue micro-arrays of 103 cases of AK, 43 cases of KA and 106 cases of cSCC were stained by immunohistochemistry for E-cadherin, CD3, CD4, CD8, FOXp3, and the receptor-ligand pair PD-1/PD-L1. Immunohistological scores were computationally determined to assess PD-L1 expression as well as the expression profiles of T-lymphocytes. Results AK had lower numbers of CD3+ and PD-1+ cells compared to KA and lower numbers of CD3+, CD8+ and PD-1+ cells in comparison with cSCC. KA showed significantly higher numbers of CD4+ and FOXp3+ cells as well as lower numbers of CD8+ cells in comparison with invasive cSCC. cSCC expressed significantly more PD-L1 in comparison with AK and KA. Among cSCC PD-L1 expression was higher in moderately and poorly-differentiated cSCC than in well-differentiated cSCC. Increased PD-L1 expression also correlated with increased numbers of CD4+, CD8+ and FOXp3+ cells in cSCC. Conclusions Tumor-associated T-lymphocyte infiltrates showed significant differences between AK, KA and invasive cSCC. PD-L1 expression correlated with invasion of T-cell infiltrates in invasive cSCC.

Sunlight encompasses UVB, UVA, visible light (VIS) and infrared (IR) radiation. Historically, the impact of these wavelength regions on human skin cells was studied one by one and it was shown that each of them per se can affect human skin. However, human skin is exposed to a combination of all of them. We therefore hypothesize that the stress response of human skin to the complete sunlight spectrum has been evolutionary optimized and differs from that induced by single wavelength regions. We have developed an irradiation device which allows simultaneous exposure to UVB+UVA+VIS+IRA at doses relevant for sunlight, to selected combinations or single wavelength regions thereof, and which avoids cross-contaminations between them. Since UVB-induced sunburn cell formation in mice was reported to be decreased if the proportion of contaminating UVA radiation in the emitted spectrum was increased we thought to validate our new source by studying apoptosis in HaCaT keratinocytes. We found that UVB-induced apoptosis was significantly reduced if cells were exposed to a combination of UVB+UVA, UVB+UVA+IRA, or UVB+UVA+VIS+IRA. This effect was caused by UVA rays in the emission spectrum, UVA-dose dependent, and observed at physiologically relevant UVA:UVB ratios. In contrast, VIS and IRA did not modify UVA+UVB-induced apoptosis. UVA reduced UVB-induced apoptosis by affecting the extrinsic apoptosis pathway because activation of caspase (Cas)-8, BID and Cas-3 were reduced, whereas intrinsically induced cleavage of Cas-9 remained unchanged. UVA also reduced UVB-induced death-receptor trimerization indicating that it might interact with UVB signaling at the level of the cell membrane. Our results emphasize the importance of studying the impact of the complete sunlight spectrum rather than that of single wavelength regions on human skin and that the here reported irradiation source may be well suited to do so.

Nonmelanoma skin cancer is the most prevalent cancer worldwide, and a number of cases are steadily increasing. The incidence of skin squamous cell carcinoma, which is the second most frequent tumor in this group of tumors, is markedly increasing in immunosuppressed transplant recipients and under certain cancer treatment modalities. Unfortunately, the aggressiveness of these tumors is also increasing, and once metastasized, the therapies to successfully eradicate these tumors are lacking, thus making research advancements vital. We firstly discuss what is currently known about cutaneous squamous cell carcinomas (cSCCs), an important consideration when designing relevant model systems for further investigation. We continue by reviewing recent studies, which have utilized different models and highlight the importance/requirement for human 3D models to combat this devastating tumor type.