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Subcellular Raman Microspectroscopy Imaging of Nucleic Acids and Tryptophan for Distinction of Normal Human Skin Cells and Tumorigenic Keratinocytes

At present, tumor diagnostic imaging is commonly based on hematoxylin and eosin or immunohistochemical staining of biopsies, which requires tissue excision, fixation, and staining with exogenous marker molecules. Here, we report on label-free tumor imaging using confocal spontaneous Raman scattering microspectroscopy, which exploits the intrinsic vibrational contrast of endogenous biomolecular species. We present a chemically specific and quantitative approach to monitoring normal human skin cells (keratinocytes and fibroblasts) as well as the human HaCaT in vitro skin carcinogenesis model and the tumor-derived MET in vivo skin cancer progression model. Mapping the amplitudes of two spectrally well isolated Raman bands at 752 and 785 cm^–1 allowed for direct visualization of the distributions representative of tryptophan-rich proteins and nucleic acids, respectively, with subcellular spatial resolution. Using these Raman markers, it was feasible to discriminate between normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (NHDF) and to confine all tumorigenic cells from both the NHEK and NHDF. First evidence for the successful application of the proposed intracellular nucleic acid and tryptophan Raman signatures for skin cancer diagnosis was further demonstrated in an organotypic cutaneous squamous cell carcinomas model, allowing for the identification of tumor cells and their surrounding stroma in the tissue context

CPM values for gene COL1A1.

<p>Gene-wise counts per million (CPM) values directly derived from <i>summarizeOverlaps</i>.</p

Alignment depth for gene COL1A1.

<p>Align depth in genetic region COL1A1 after cutting out intronic regions and regions with low alignment depth. Group-wise mean alignment depth values have been smoothed using loess regression.</p

Gene-wise CPM values for ID1.

<p>Gene-wise <i>counts per million</i> (CPM) values derived from <i>summarizeOverlaps</i>.</p

Additional file 1: of Wild carrot pentane-based fractions suppress proliferation of human HaCaT keratinocytes and protect against chemically-induced skin cancer

Shebaby et al. Raw Data R4. (XLSX 56 kb

Enriched KEGG pathways in age MAR genes.

<p>Enriched KEGG pathways in age MAR genes.</p

Align depth estimates for gene ID1.

<p>The figure displays alignment depth in absolute numbers. Three lines estimate mean alignment depth for each age group (y = Young, m = Middle, o = Old).</p

CPM values for five genes.

<p>CPM (counts per million) values derived from <i>summarizeOverlaps</i> for Genes ATOH8, ID3, ID1, SMAD7 and FAM83G for all 54 samples.</p

Age, gender and UV-exposition related effects on gene expression in <i>in vivo</i> aged short term cultivated human dermal fibroblasts

<div><p>Ageing, the progressive functional decline of virtually all tissues, affects numerous living organisms. Main phenotypic alterations of human skin during the ageing process include reduced skin thickness and elasticity which are related to extracellular matrix proteins. Dermal fibroblasts, the main source of extracellular fibrillar proteins, exhibit complex alterations during <i>in vivo</i> ageing and any of these are likely to be accompanied or caused by changes in gene expression. We investigated gene expression of short term cultivated <i>in vivo</i> aged human dermal fibroblasts using RNA-seq. Therefore, fibroblast samples derived from unaffected skin were obtained from 30 human donors. The donors were grouped by gender and age (Young: 19 to 25 years, Middle: 36 to 45 years, Old: 60 to 66 years). Two samples were taken from each donor, one from a sun-exposed and one from a sun-unexposed site. In our data, no consistently changed gene expression associated with donor age can be asserted. Instead, highly correlated expression of a small number of genes associated with transforming growth factor beta signalling was observed. Also, known gene expression alterations of <i>in vivo</i> aged dermal fibroblasts seem to be non-detectable in cultured fibroblasts.</p></div