Abstract 6302: A CellProfiler pipeline for RNAscope analyzing multiple markers across adjacent tissue sections spatially

Understanding the spatial distribution of cancer cells and how they interact with other cell types in the tumor microenvironment (TME) is crucial to elucidate the mechanisms of cancer development and progression. The identification of cell types in the TME relies on the distinct expression profiles of different cell types. Compared to sequencing-based spatial transcriptomics analysis, multiplex nucleic acid in-situ hybridization (ISH) at a single-molecule level is more focused and cost-effective. Based on ISH technology, RNAscope developed novel approaches to detect multiple gene expression profiles on tissue sections at a single-molecule level. It is highly sensitive and specific and can simultaneously quantify gene expression at sub-cellular levels. Although the technology has been applied to study spatial gene expression in various cancer types, few have applied it to elucidate the association between different markers in different cell compartments within the TME.This protocol describes an image analysis pipeline for multiplex fluorescent signals detected by RNAscope technology. It was developed based on a previous CellProfiler pipeline, facilitating spatial expression analyses across different cell compartments through a flexible, user-friendly interface for non-computational biologists. We describe specific steps to analyze the expression and spatial arrangement of four endogenous housekeeping genes as quality controls (QCs): POLR2A, PPIB, HPRT1, and UBC. We further apply these pipelines to analyze target genes in breast cancer samples, comparing those with and without skin metastasis. The analyses were demonstrated in one sample each using formalin-fixed paraffin-embedded tissue (FFPET) and fresh-frozen tissue (FFT) sections with the ISH steps followed the manufacturer’s protocol. Citation Format: Meng-Shin Shiao, Natini Jinawath, Katherine Copeland, Ariestya Indah Permata Sari. A CellProfiler pipeline for RNAscope analyzing multiple markers across adjacent tissue sections spatially [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6302.