Development of a Multiplex Immunofluorescence Assay for Tumor Microenvironment Studies of Human and Murine Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine carcinoma. Checkpoint inhibitor immunotherapy plays an essential role in management of advanced MCC; however, predictors of immunotherapy response remain poorly defined. Syngeneic mouse models suitable for testing novel immunotherapy and combination therapy approaches are likely to soon become available and will require assays for evaluating the tumor microenvironment (TME). Multiplex immunofluorescence (mIF) is a powerful approach to characterize the TME for understanding immunotherapy responses and immune surveillance. In this method article, we provide detailed instructions on assay development for mIF, using as examples 2 new mIF panels for TME investigations of human and murine MCC tumors. Specifically, we demonstrate panels that allow simultaneous visualization of the Merkel cell master transcription factor SOX2 for tumor cell identification, alongside T-cell markers (CD3, CD8, and FOXP3), macrophage markers (F4/80 for mouse and CD163 for human tumors), together with the checkpoint marker PD-L1 for human tumors, and the myeloid-derived suppressor cell marker Arg1 for mouse tumors. We provide detailed protocols for investigators to incorporate these mIF panels into their investigations of human and murine MCC. We also provide fundamental guidance for mIF assay development that will be broadly useful for investigators who consider modifying the panels presented in this study or developing their own mIF panels.