Spatial profiling of benign and malignant melanocytic tumors via RNA-SMI (CosMx)

Melanoma clinical outcomes emerge from incompletely understood genetic mechanisms operating within the tumor and its microenvironment. Here, we utilized single-cell RNA-based spatial molecular imaging (RNA-SMI) in patient-derived archival tumors to reveal clinically relevant markers of malignancy progression and prognosis. We examined spatial gene expression of 203,472 cells inside benign and malignant melanocytic neoplasms, including melanocytic nevi, primary invasive and metastatic melanomas. Algorithmic cell clustering paired with intratumoral comparative 2D-analyses visualized synergistic, spatial gene signatures linking cellular proliferation, metabolism, and malignancy, validated by protein expression. Metastatic niches included upregulation of CDK2 and FABP5, which independently predicted poor clinical outcome in 473 melanoma patients via Cox regression analysis. More generally, our work demonstrates a framework for applying single-cell RNA-SMI technology toward identifying gene ..., NanoString® CosMx™ RNA Spatial Molecular Imaging (SMI)
The protocol used for NanoString® CosMx™ RNA Spatial Molecular Imaging (SMI) was based on the method previously described by He et al. (High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging. Nat Biotechnol 40, 1794-1806. 2022). 5-μm formalin-fixed, paraffin-embedded (FFPE) tissue sections were mounted on VWR Superfrost Plus Micro slides (cat# 48311-703) and baked at 60°C overnight to improve tissue-slide adherence. The slides were prepared for in-situ hybridization (ISH) by heat-induced epitope retrieval (HIER) at 100°C for 15 min using ER1 epitope retrieval buffer (Leica Biosystems product, citrate-based, pH 6.0). Following HIER, the tissues were digested with 3 µg/ml Proteinase K diluted in ACD Protease Plus (Advanced Cell Diagnostics, Inc.) at 40°C for 30 minutes. Slides were washed twice with diethyl pyrocarbonate (DEPC)-treated water (DEPC H2O) and incubated in 0.0005% dilu..., , # Spatial Profiling of Benign and Malignant Melanocytic Tumors via RNA-SMI (CosMx)

[https://doi.org/10.5061/dryad.ksn02v7b1](https://doi.org/10.5061/dryad.ksn02v7b1)

## Description of the data and file structure

\"Slide_1.zip\", \"Slide_2.zip\", \"Slide_3.zip\", \"Slide_4.zip\": 

Each .zip folder contains the RNA-SMI data pertaining to Slides 1-3, which examined 203,472 cells amongst ten melanocytic tumors (exact tumor identity per slide is outlined in fig. S1, with further slide information regarding microscopic field of view distribution outlined in fig. S2). Slide 4 contains the RNA-SMI data examining 84,312 cells including the nevus-melanoma mixed tumor featured in fig. 4 as well as four other melanocytic tumors (one melanoma, one cutaneous metastasis, and two nevi).

Specifically, each .zip folder contains the following files and folders:

**Files:**

Transcript file (tx_file.csv), which contains columns for:

* —fov (Field Of View (FOV) where transcript is located)

* —cell_ID (...