Delahny Deivendran

Here, to understand the homeostatic mechanisms governing melanocytes, we interrogate the mutational landscapes, gene-expression profiles and morphological features of 297 clonal expansions of epidermal melanocytes from 31 donors. We show that a population of melanocytes with low mutation burden persists in sun-exposed epidermis. These cells are smaller, less dendritic, and exhibit stem-like expression profiles when compared to melanocytes carrying high mutation burdens. Using single-cell spatial transcriptomics, we show that melanocytes inferred to have low mutation burdens localize to both hair follicles and interfollicular epidermis, whereas melanocytes with high mutation burdens are largely restricted to epidermis. We propose that melanocytes in the hair follicle occupy a privileged niche, protected from ultraviolet radiation, but replenish the epidermis following photodamage. This study highlights the value of incorporating mutational information into cell atlases. Cells can change their positions over time, but mutations provide a historical record of processes that were operative on each cell.

Tanning bed users have a significantly increased risk of melanoma, but it remains unclear how indoor tanning drives melanomagenesis. To better understand the etiology of melanoma associated with tanning bed use, we described the patterns of melanoma in patients with quantifiable tanning bed usage and performed exome sequencing of melanocytes from normal skin of a subset of these patients. Tanning bed users were more likely to have melanoma on body sites with low cumulative levels of sun damage and to have multiple melanomas. The melanocytes in skin from tanning bed users had higher mutation burdens and higher proportions of cells with pathogenic mutations—these differences were most prominent over body sites that experience comparatively less exposure to natural sunlight. We conclude that tanning bed radiation induces melanoma by increasing the mutation burden of melanocytes and by mutagenizing a broader field of melanocytes than are typically exposed to natural sunlight.

Cutaneous squamous cell carcinomas (cSCCs) arise from keratinocytes in the skin, but the molecular changes driving this transformation remain unclear. To better understand this process, we perform multi-omic profiling of keratinocytes, actinic keratoses, and cSCCs. Single-cell mutational analyses reveal that most keratinocytes have remarkably low mutation burdens; however, keratinocytes with TP53 or NOTCH1 mutations exhibit substantially higher burdens. These findings suggest that keratinocytes can withstand high dosages of cumulative ultraviolet radiation, but certain pathogenic mutations break these adaptive mechanisms, inducing a mutator phenotype. Mutational profiling of cSCCs adjacent to actinic keratoses reveals TERT promoter and CDKN2A mutations emerge in actinic keratoses, whereas additional mutations that inactivate ARID2 and activate the mitogen-activated protein kinase pathway delineate the transition to cSCC. Surprisingly, actinic keratoses are often not related to their neighboring cSCC. Spatial analyses reveal gene expression heterogeneity, including checkpoint molecule enrichment at invasive fronts, highlighting tumor and immune cell interactions.

To better understand the homeostatic mechanisms governing melanocytes, we performed deep phenotyping of clonal expansions of single melanocytes from human skin. In total, we interrogated the mutational landscapes, gene expression profiles, and morphological features of 297 melanocytes from 31 donors. To our surprise, a population of melanocytes with low mutation burden was maintained in sun damaged skin. These melanocytes were more stem-like, smaller, less dendritic and displayed distinct gene expression profiles compared to their counterparts with high mutation burdens. We used single-cell spatial transcriptomics (10X Xenium) to reveal the spatial distribution of melanocytes inferred to have low and high mutation burdens (LowMut and HighMut cells), based on their gene expression profiles. LowMut melanocytes were found in hair follicles as well as in the interfollicular epidermis, whereas HighMut melanocytes resided almost exclusively in the interfollicular epidermis. We propose that melanocytes in the hair follicle occupy a privileged niche, protected from UV radiation, but periodically migrate out of the hair follicle to replenish the interfollicular epidermis after waves of photodamage. More broadly, our study illustrates the advantages of a cell atlas that includes mutational information, as cells can change their cellular states and positional coordinates over time, but mutations are like scars, providing a historical record of the homeostatic processes that were operative on each cell.

We performed multi-omic profiling of epidermal keratinocytes, precancerous actinic keratoses, and squamous cell carcinomas to understand the molecular transitions during skin carcinogenesis. Single-cell mutational analyses of normal skin cells showed that most keratinocytes have remarkably low mutation burdens, despite decades of sun exposure, however keratinocytes with TP53 or NOTCH1 mutations had substantially higher mutation burdens. These observations suggest that wild-type keratinocytes (i.e. without pathogenic mutations) are able to withstand high dosages of cumulative UV radiation, but certain pathogenic mutations break these adaptive mechanisms, priming keratinocytes for transformation by increasing their mutation rate. Mutational profiling of squamous cell carcinomas adjacent to actinic keratoses revealed TERT promoter and CDKN2A mutations emerging in actinic keratoses, whereas additional mutations inactivating ARID2 and activating the MAPK-pathway delineated the transition to squamous cell carcinomas. Surprisingly, actinic keratoses were often not related to their neighboring squamous cell carcinoma, indicating that collisions of unrelated neoplasms are common in the skin. Spatial variation in gene expression patterns was common in both tumor and immune cells, with high expression of checkpoint molecules at the invasive front of tumors. In conclusion, this study catalogues the key events during the evolution of cutaneous squamous cell carcinoma.

Background Tanning bed users have a significantly increased risk of melanoma, but it remains unclear how indoor tanning drives melanomagenesis. Tanning bed radiation is often thought of as a substitute for natural UV radiation despite differences in the maximum doses, UV content, body sites exposed, and patterns of melanoma that arise. Methods To better understand the epidemiologic trends and etiology of melanoma associated with tanning bed use, we described the patterns of melanoma in patients with quantifiable tanning bed usage and performed exome sequencing of 182 melanocytes from normal skin of a subset of these patients. Results Tanning bed users were more likely than non-users to have melanoma on body sites with low cumulative levels of sun damage and were more likely to have multiple melanomas. The melanocytes in normal appearing skin from tanning bed users had higher mutation burdens, a higher proportion of melanocytes with pathogenic mutations, and distinct mutational signatures. These differences were most prominent over body sites that experience comparatively less exposure to natural sunlight. Conclusions We conclude that tanning bed radiation induces melanoma by increasing the mutation burden of melanocytes and by mutagenizing a broader field of melanocytes than are typically exposed to natural sunlight. The unique signatures of mutations in skin cells of tanning users may be attributable to the distinct spectra of radiation emitted from solariums.

Spatial transcriptomic technologies, such as the Visium platform, measure gene expression in different regions of tissues. Here, we describe new software, STmut, to visualize somatic point mutations, allelic imbalance, and copy number alterations in Visium data. STmut is tested on fresh-frozen Visium data, formalin-fixed paraffin-embedded (FFPE) Visium data, and tumors with and without matching DNA sequencing data. Copy number is inferred on all conditions, but the chemistry of the FFPE platform does not permit analyses of single nucleotide variants. Taken together, we propose solutions to add the genetic dimension to spatial transcriptomic data and describe the limitations of different datatypes.