Automated Quantification of Tertiary Lymphoid Structures in Human Tumor Samples Using Immunofluorescence and AI-Powered Analysis Pipeline

The tumor microenvironment (TME) is a complex entity comprising not only tumor cells but also immune, stromal, and endothelial cells. Preclinical and clinical studies indicate that the density, localization, function, and organization of immune infiltration can influence survival probability and treatment response in many cancers. Among these cell organizations, the clustering of T and B cells into tertiary lymphoid structures (TLS) has been associated with favorable clinical outcomes. In this protocol, we propose a protocol for 7-plex immunofluorescence staining for identifying six cell types enriched in TLS in formalin-fixed paraffin-embedded (FFPE) human tumor sections. Additionally, we provide a detailed methodology for quantifying these cell subtypes using the Halo-AI analysis software. This approach will enable a more precise and detailed characterization of TLS in the TME, opening new avenues for understanding their role in anticancer treatment response.