A Method for Quantifying Immune Infiltrates and Tertiary Lymphoid Structures in Murine Melanoma Tumors by Multiplex...

The presence of tertiary lymphoid structures (TLS) and elevated levels of tumor-infiltrating lymphocytes have been linked to improved survival outcomes in several solid cancers, including melanoma. Despite this association, the precise function and immunological impact of TLS remain incompletely understood, as do factors that drive their formation and which might potentially be leveraged to enhance patient outcomes. Functional studies using murine models may help clarify the biological roles of TLS; however, relatively few methodologies currently exist that allow for detailed assessment of the structural organization and functional characteristics of TLS within solid tumors. To advance understanding of the biology underlying TLS, this chapter describes multiplex immunohistochemistry panels designed for the identification, characterization, and quantification of TLS and intratumoral immune infiltrates in melanoma. Although these methods are presented in the context of melanoma, they are broadly applicable and can be used to investigate TLS across a wide range of human cancers.