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Conversely, our results show that Pfs contain cancer cells more within the epidermis. Rfs are clearly predisposed to differentiate into CAFs upon SCC signals, assisting invasion and EMT.

Human dermis can be morphologically divided into the upper papillary and lower reticular dermis. Previously, we demonstrated that papillary (PFs) and reticular (RFs) fibroblasts show distinct morphology and gene expression profiles. Moreover, they differently affect tumor invasion and epithelial-to-mesenchymal transition (EMT) in in vitro 3D-organotypic cultures of cutaneous squamous cell carcinoma (cSCC). In this study, we examined if these distinct effects of PFs and RFs can be extrapolated in other epithelial/non-epithelial tumors such as melanoma and head and neck squamous cell carcinoma (HNSCC). To this end, 3D-Full-Thickness Models (FTMs) were established from melanoma (AN and M14) or HNSCC cell lines (UM-SCC19 and UM-SCC47) together with either PFs or RFs in the dermis. The interplay between tumor cells and different fibroblasts was investigated. We observed that all the tested tumor cell lines showed significantly stronger invasion in RF-FTMs compared to PF-FTMs. In addition, RF-FTMs demonstrated more tumor cell proliferation, EMT induction and basement membrane disruption. Interestingly, RFs started to express the cancer-associated fibroblast (CAF) biomarker α-SMA, indicating reciprocal interactions eventuating in the transition of RFs to CAFs. Collectively, in the melanoma and HNSCC FTMs, interaction of RFs with tumor cells promoted EMT and invasion, which was accompanied by differentiation of RFs to CAFs.

Porokeratosis is characterized by the formation of cornoid lamella. Porokeratosis ptychotropica (PP) is a rare variant of porokeratosis, where the dyskeratotic skin changes are mainly located around the gluteal cleft or genital area. Several causal mutations have been reported in members of the mevalonate pathway. Among them, to our knowledge, all cases of PP were caused by mutations in the MVK gene, which encodes mevalonate kinase (MVK). A-52-yearold man presented with mildly pruritic, symmetrical, hyperkeratotic, brownish plaques with subtle fine scaling and smaller satellite plaques affecting buttocks and partially the intergluteal cleft. Histological examination revealed verrucous epidermal hyperplasia with hyperkeratosis alternating with columns of parakeratosis or cornoid lamella consistent with PP. Direct sequencing analysis of exons and intron-exon boundaries of MVK revealed the heterozygous for the frameshift mutation c.314_318delinsT (p.Glu105Valfs*26) in the exon 6 of MVK. Immunofluorescence analysis revealed that KRT6 expression is accelerated in affected lesion compared with unaffected lesion. MVK is located on chromosome 12q24 and encodes the peroxisomal enzyme mevalonate kinase, which plays an important role in the mevalonate pathway. The pathway is a vital for multiple cellular processes, providing cells with essential bioactive molecules. Although the genetic defects are known, the pathogenic mechanism remains to be elucidated, and treatment of the disease are mainly supportive, with poor efficacy. Our study showed that MVK and KRT6 might play a crucial role in the pathogenesis of PP.

Lesional skin in mycosis fungoides (MF) is populated primarily by benign and neoplastic T-cells. PUVA is an effective therapy for MF but its effects on these two populations are uncharacterized. We studied skin biopsies from 8 MF patients before and after 12-16 weeks of PUVA using high throughput TCR sequencing and nanostring-based gene expression profiling. Patients had an average mSWAT score of 19.7 before therapy and experienced an average 88% improvement in mSWAT scores after therapy. The total number of T-cells in skin decreased by 50% after therapy, from a mean 150,435 T-cells/100ng DNA to 76,200. However, the percentage of malignant clones was not significantly different despite evident clinical improvement. Clonal malignant T-cells made up 44% of the total T-cell population before therapy and 40% after. Effective PUVA therapy was associated with large shifts in the benign T-cell population. 49% of benign clones in skin present before therapy were eliminated by PUVA, and large numbers of new benign T-cells not previously present in skin were recruited into skin following therapy. After therapy, newly recruited benign T-cell clones made up 52% of the total T-cell population and clones present before therapy made up only 8%. A linear regression analysis demonstrated that clinical improvement correlated with turnover of benign T-cell clones (R¼0.8, p¼0.01) but not with reduction of malignant T-cells (R¼0.13, p¼0.4). Nanostring analyses demonstrated reduction in STAT6, suggesting reduced Th2 responses, and increases in genes associated with cytotoxicity, innate cell and NK cell activation. These studies suggest that PUVA may induce a shift in benign T-cell populations away from a phenotype that supports malignant T-cell activation and towards a phenotype that restrains it. In summary, PUVA exerts its effects by modulating benign T-cell populations, in a way that may deprive malignant T-cells of necessary activation signals.

Keloids are fibroproliferative scars that invade the surrounding tissue beyond the original wound site. The cellular and molecular mechanism for the pathogenesis of keloid scars are still unknown. We investigated the role of the transcription factor Twist, a key regulator in epithelial-to-mesenchymal transition, its upstream regulators, and downstream targets, which are associated with self-renewal, proliferation and invasion, in keloid tissue. Multicolour immunofluorescence microscopy was performed on frozen tissue sections of keloid scars, normal scars and normal skin staining for Twist, and related molecules such as Bmi1, active b-catenin and inhibitor of differentiation 1 (Id1). Staining of these markers was quantified using Metamorph software. Endothelial and mesenchymal cells were identified and inflammatory infiltrates were investigated using markers for T cells, B cells, and macrophages. Our results reveal remarkable upregulation of Twist expression in keloids compared to normal scar and normal skin. Furthermore, we show a significant increase in expression of Bmi1, active