Fig. 1. Single-cell RNA-based SMI generates maps of gene expression within benign, malignant, and metastatic melanocytic neoplasms.

(A) FFPE samples are sectioned and undergo SMI (18). Native RNA is hybridized in situ to gene-specific antisense oligonucleotides fused to multiplexed readout domains, which undergo cyclical reactions with four different fluorophores (Alexa Fluor 488, ATTO 532, Dyomics-605, or Alexa Fluor 647) or null, nonfluorescent domain (represented in white). The readout domain length and number of iterative cycles are plexed to allow detection of ~1000 unique genes. Slides are then stained with DAPI to detect nuclei and other IHC markers, which facilitate automated cell segmentation in silico. Segmented cells and nuclei generate a “map” onto which RNA transcripts are assigned. Bottom left panels show epidermis above melanocyte-containing tissue with 11 differentially colored gene transcripts, with DAPI shown in white. The solid white arrows show a detected RNA transcript outside a cell border, which may represent a z-axis out-of-plane RNA or extracellular RNA. (B) H&E and RNA imaging of an intradermal nevus and cutaneous melanoma metastasis. The solid outlined, rectangular boxes in panels show highlighted insets below. The panels show the expected increased detection of melanoma marker PRAME amongst the S100B melanocytes of metastatic melanoma as well as the expected increase in S100A8 (black open arrow) in epidermal keratinocytes (marked by KRT14) overlying the malignant proliferation. Closed arrows show mitotic cells. Open arrow shows malignant melanocytes juxtaposed to the epidermis. (C) UMAP projections of gene expression amongst 203,472 cells analyzed in 10 patient-derived tumors. S100B expression includes melanocytes, a subset of which express melanoma marker PRAME (black versus green dashed boxes); the red dashed box shows epidermal keratinocytes that express KRT14, a subset of which express S100A8 (blue dashed ellipse).