Knockdown of Simulated-Solar-Radiation-Sensitive miR-205-5p Does Not Induce Progression of Cutaneous Squamous Cell Carcinoma In Vitro.

Solar radiation is the main risk factor for cSCC development, yet it is unclear whether the progression of cSCC is promoted by solar radiation in the same way as initial tumorigenesis. Additionally, the role of miRNAs, which exert crucial functions in various tumors, needs to be further elucidated in the context of cSCC progression and connection to solar radiation. Thus, we chronically irradiated five cSCC cell lines (Met-1, Met-4, SCC-12, SCC-13, SCL-II) with a custom-built irradiation device mimicking the solar spectrum (UVB, UVA, visible light (VIS), and near-infrared (IRA)). Subsequently, miRNA expression of 51 cancer-associated miRNAs was scrutinized using a flow cytometric multiplex quantification assay (FirePlex®, Abcam). In total, nine miRNAs were differentially expressed in cell-type-specific as well as universal manners. miR-205-5p was the only miRNA downregulated after SSR-irradiation in agreement with previously gathered data in tissue samples. However, inhibition of miR-205-5p with an antagomir did not affect cell cycle, cell growth, apoptosis, or migration in vitro despite transient upregulation of oncogenic target genes after miR-205-5p knockdown. These results render miR-205-5p an unlikely intracellular effector in cSCC progression. Thus, effects on intercellular communication in cSCC or the simultaneous examination of complementary miRNA sets should be investigated.

Antimicrobial activity of NK cells to Trypanosoma cruzi infected human primary Keratinocytes.

Infection with the protozoan parasite Trypanosoma cruzi is causative for Chagas disease, which is a highly neglected tropical disease prevalent in Latin America. Humans are primary infected through vectorial transmission by blood-sucking triatomine bugs. The parasite enters the human host through mucous membranes or small skin lesions. Since keratinocytes are the predominant cell type in the epidermis, they play a critical role in detecting disruptions in homeostasis and aiding in pathogen elimination by the immune system in the human skin as alternative antigen-presenting cells. Interestingly, keratinocytes also act as a reservoir for T. cruzi, as the skin has been identified as a major site of persistent infection in mice with chronic Chagas disease. Moreover, there are reports of the emergence of T. cruzi amastigote nests in the skin of immunocompromised individuals who are experiencing reactivation of Chagas disease. This observation implies that the skin may serve as a site for persistent parasite presence during chronic human infection too and underscores the significance of investigating the interactions between T. cruzi and skin cells. Consequently, the primary objective of this study was to establish and characterize the infection kinetics in human primary epidermal keratinocytes (hPEK). Our investigation focused on surface molecules that either facilitated or hindered the activation of natural killer (NK) cells, which play a crucial role in controlling the infection. To simulate the in vivo situation in humans, an autologous co-culture model was developed to examine the interactions between T. cruzi infected keratinocytes and NK cells. We evaluated the degranulation, cytokine production, and cytotoxicity of NK cells in response to the infected keratinocytes. We observed a strong activation of NK cells by infected keratinocytes, despite minimal alterations in the expression of activating or inhibitory ligands on NK cell receptors. However, stimulation with recombinant interferon-gamma (IFN-γ), a cytokine known to be present in significant quantities during chronic T. cruzi infections in the host, resulted in a substantial upregulation of these ligands on primary keratinocytes. Overall, our findings suggest the crucial role of NK cells in controlling acute T. cruzi infection in the upper layer of the skin and shed light on keratinocytes as potential initial targets of infection.

Enrichment of Human Dermal Stem Cells from Primary Cell Cultures through the Elimination of Fibroblasts.

Dermal stem cells (DSCs), which are progenitor cells of melanocytes, are isolated from human foreskin and cultivated as mixed cultures containing both DSCs and fibroblasts in varying proportions. These contaminating fibroblasts may have an impact on the results of experimental studies and are a serious limitation for certain applications. The aim of the present study was to purify or enrich DSCs-an indispensable step towards future investigations. Applying different methods, we demonstrated that highly enriched DSCs with a good recovery rate can be obtained through positive selection with MACS® immunomagnetic cell sorting. These DSCs remain vital and proliferate constantly in culture, maintaining a high level of purity after enrichment. Other approaches such as treatment with Geneticin or selective detachment were not suitable to purify DSC-fibroblast co-cultures. Overall, enriched DSCs represent a novel and unique model to study the effects of UV radiation on the differentiation of DSCs into melanocytes and their potential relevance in the genesis of malignant melanoma.

UVB damage response of dermal stem cells as melanocyte precursors compared to keratinocytes, melanocytes, and fibroblasts from human foreskin.

Ultraviolet B (UVB) radiation induces mutagenic DNA photolesions in skin cells especially in form of cyclobutane pyrimidine dimers (CPDs). Protection mechanisms as DNA repair and apoptosis are of great importance in order to prevent skin carcinogenesis. In human skin, neural crest-derived precursors of melanocytes, the dermal stem cells (DSCs), are discussed to be at the origin of melanoma. Although they are constantly exposed to solar UV radiation, it is still not investigated how DSCs cope with UV-induced DNA damage. Here, we report a comparative study of the DNA damage response after irradiation with a physiological relevant UVB dose in DSCs in comparison to fibroblasts, melanocytes and keratinocytes isolated from human foreskin. Within our experimental settings, DSCs were able to repair DNA photolesions as efficient as the other skin cell types with solely keratinocytes repairing significantly faster. Interestingly, only fibroblasts showed significant alterations in cell cycle distribution in terms of a transient S phase arrest following irradiation. Moreover, with the applied UVB dose none of the examined cell types was prone to UVB-induced apoptosis. This may cause persistent genomic alterations and in case of DSCs it may have severe consequences for their daughter cells, the differentiated melanocytes. Altogether, this is the first study demonstrating a similar UV response in dermal stem cells compared to differentiated skin cells.

UV-type specific alteration of miRNA expression and its association with tumor progression and metastasis in SCC cell lines.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.