A Quantitative Pathology Approach to Analyze the Development of Human Cancer-Associated Tertiary Lymphoid Structures

: Tertiary lymphoid structures (TLS) develop in the human tumor microenvironment and correlate with prolonged survival in most cancer types. We recently demonstrated that TLS development follows sequential maturation stages and culminates in the generation of a germinal center (GC) reaction. This maturation process is crucial for the prognostic relevance of TLS in lung and colorectal cancer patients.The mechanisms underlying TLS development in various inflammatory conditions or their functional relevance in tumor immunity are not fully understood. Investigating which cell types and soluble mediators orchestrate lymphoid neogenesis in human tissues requires a method that allows simultaneous detection of multiple markers.Here, we describe a quantitative pathology approach to identify and quantify different TLS maturation stages in combination with other parameters. This approach consists of seven-color immunofluorescence protocol using tyramide signal amplification combined with multispectral microscopy and quantitative data acquisition from histological images.