Fluorescent multiplexing of 3D spheroid FFPE with Roche automated stainer and reagents: assay optimization, imaging and future implications

Three-dimensional (3D) cell culture spheroids and aggregates are preferred over monolayer cell culture due to their architectural and functional similarity to solid tumors. To study expression profiles of key tumor biomarkers, physiologically relevant preclinical models of non-small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line‐derived 3D spheroids and aggregates were utilized. We performed multiplex immunofluorescence (mIF) staining, whole slide scanning and image analysis to enable simultaneous detection and quantification of multiple biomarkers on a single formalin-fixed paraffin embedded section.
We describe the workflow optimized in our lab at Roche Tissue Diagnostics to establish mIF assay for 3D spheroids and aggregates sections. Multiplex staining of 3D sections were performed on Roche DISCOVERY Ultra automated staining instruments with Roche reagent and fluorescent dyes. Biomarker panels were designed to examine the expression of frequently studies key markers of two cancer subtypes. The slides were scanned and multispectral images captured in Vectra 3 (Akoya Biosciences) fitted with filters compatible to the dyes. Quantitative assessment of the expressed biomarkers was performed through image analysis.