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High-plex spatial RNA profiling reveals cell type-specific biomarker expression during melanoma development

ABSTRACT: Early diagnosis of melanoma is critical for improved survival. However, biomarkers of early melanoma evolution and their origin within the tumor and its microenvironment, including the keratinocytes, are poorly defined. To address this, we used spatial transcript profiling that maintains the morphological tumor context to measure expression of >1,000 RNAs in situ in patient-derived formalin-fixed, paraffin-embedded tissue sections in primary melanoma and melanocytic nevi. We profiled 134 200µm-diameter regions of interest enriched in melanocytes, neighboring keratinocytes or immune cells. This approach captured distinct expression patterns across cell types and tumor types during melanoma development. Unexpectedly, we discovered that S100A8 is expressed by keratinocytes within the tumor microenvironment during melanoma growth. Immunohistochemistry of 252 tumors showed prominent keratinocyte-derived S100A8 expression in melanoma but not in benign tumors and confirmed the same pattern for S100A8’s binding partner S100A9, suggesting that injury to the epidermis may be an early and easily detectable indicator of melanoma development Together, our results establish a framework for high-plex, spatial and cell type-specific resolution of gene expression in archival tissue applicable to the development of biomarkers and characterization of tumor-microenvironment interactions in tumor evolution. In this dataset, we capture the GeoMx DSP RNA profiling data associated with this study. The files here represent patients with melanocytic tumors (n = 16) which were profiled with the NanoString GeoMx pilot panel (n=4 patients, n=108 gene targets) or CTA developmental panel v1.0 (n=12 patients, n=1,412 gene targets). These panels were read out using Illumina sequencing according to manufacturer protocols. For access to high resolution (single channel, composite, or ROI) images please contact mkriner@nanostring.com.

ORGANISM(S): Homo sapiens

PROVIDER: GSE168013 | GEO | 2021/03/03

REPOSITORIES: GEO

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Project description:Tumor suppressor genes (TSGs) are sometimes inactivated by transcriptional silencing through promoter hypermethylation. To identify novel methylated TSGs in melanoma, we carried out global mRNA expression proﬁling on a panel of 12 melanoma cell lines treated with a combination of 5-Aza-2-deoxycytidine (5AzadC) and an inhibitor of histone deacetylase, Trichostatin A. Reactivation of gene expression after drug treatment was assessed using Illumina whole-genome microarrays. After qRT-PCR conﬁrmation, we followed up 8 genes (AKAP12, ARHGEF16, ARHGAP27, ENC1, PPP1R3C, PPP1R14C, RARRES1, and TP53INP1) by quantitative DNA methylation analysis using mass spectrometry of base-speciﬁc cleaved ampliﬁcation products in panels of melanoma cell lines and fresh tumors. PPP1R3C, ENC1, RARRES1, and TP53INP1, showed reduced mRNA expression in 35–59% of the melanoma cell lines compared to melanocytes and which was correlated with a high proportion of promoter methylation (>40–60%). The same genes also showed extensive promoter methylation in 6–25% of the tumor samples, thus conﬁrming them as novel candidate TSGs in melanoma. We sought to identify melanoma TSGs silenced by promoter methylation by carrying out an array-based analysis in a well-annotated panel of 12 cell lines after combined treatment with 5AzadC and an inhibitor of histone deacetylase, Trichostatin A (TSA). Expression profiles were generated for each cell line before and after drug treatment using Illumina Sentrix Human-6 Expression version 2 BeadChips. Genes reactivated in all 12 cell lines were removed from further analysis since they are likely responding to drug treatment as part of the ‘‘cellular stress response,’’ or due to promoter demethylation of genes normally silenced in the melanocytic lineage. Genes were further ﬁltered to identify those with an average of >4-fold increased expression in at least four samples in the panel of 12 lines and >10-fold increase in at least one of the cell lines.

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High-plex spatial RNA profiling reveals cell type-specific biomarker expression during melanoma development

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