Fluorescent multiplexing of 3D spheroids: Analysis of biomarkers using automated immunohistochemistry staining platform and multispectral imaging

In preclinical cancer studies, three‐dimensional (3D) cell spheroids and aggregates are preferred over monolayer cell cultures due to their architectural and functional similarity to solid tumors. We performed a proof‐of‐concept study to generate physiologically relevant and predictive preclinical models using non–small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line‐derived 3D spheroids and aggregates. Distinct panels were designed to determine the expression profiles of frequently studied biomarkers of the two cancer subtypes. The lung adenocarcinoma panel included ALK, EGFR, TTF‐1, and CK7 biomarkers, and the colon and colorectal adenocarcinoma panel included BRAF V600E, MSH2, MSH6, and CK20. Recent advances in immunofluorescence (IF) multiplexing and imaging technology enable simultaneous detection and quantification of multiple biomarkers on a single slide. In this study, we performed IF staining of multiple biomarkers per section on formalin‐fixed paraffin‐embedded 3D spheroids and aggregates. We optimized protocol parameters for automated IF and demonstrated staining concordance with automated chromogenic immunohistochemistry performed with validated protocols. Next, post‐acquisition spectral unmixing of the captured fluorescent signals were utilized to delineate four differently stained biomarkers within a single multiplex IF image, followed by automated quantification of the expressed markers. This workflow has the potential to be adapted to preclinical high‐throughput screening and drug efficacy studies utilizing 3D spheroids from cancer cell lines and patient‐derived organoids. The process allows for cost, time, and resource savings through concurrent staining of several biomarkers on a single slide, the ability to study the interactions of multiple expressed proteins within a single region of interest, and enable quantitative assessment of biomarkers in cancer cells. This study describes a multiplexed immunofluorescence staining process aimed at exploring the expression of multiple cancer biomarkers in three‐dimensional (3D) spheroid‐derived formalin‐fixed paraffin‐embedded sections on an automated immunohistochemistry platform. We demonstrate the simultaneous detection of several key expressed biomarkers in non–small cell lung adenocarcinoma and colorectal adenocarcinoma cells on a single slide. In addition, multispectral imaging allows quantification of fluorescent signals and visualization of colocalized biomarker