Spatial Correlation of the Extracellular Matrix to Immune Cell Phenotypes in the Tumor Boundary of Clear Cell Renal Cell Carcinoma Revealed by Cyclic Immunohistochemistry

The significance of the tumor microenvironment (TME) in predicting immunotherapy efficacy is increasingly acknowledged. However, the complexity of the TME necessitates novel technological approaches for the precise characterization of individual cell types, functional phenotypes, and heterocellular spatial interactions. This study utilizes a streamlined multiplex cyclic immunohistochemistry (cycIHC) protocol for detailed TME annotation. Unlike proprietary methods, cycIHC relies on iterative cycles of conventional immunohistochemistry, using off-the-shelf antibodies and reagents, followed by digitalization, chromogen removal, and antibody stripping. The method was combined with open-source tools for the coregistration of individual staining cycles. Using clear cell renal cell carcinoma (ccRCC) as a model, the protocol was applied for granular annotation of cellular and acellular structures in the tumor boundary zone. Our results demonstrate that the tumor periphery, particularly the pseudocapsule of ccRCC, is homogeneously organized across the 3D scale, yet exhibits distinct cellular distribution gradients of T and B cells. These patterns correspond to deposited extracellular matrix proteins, especially collagen types I and VI. Our findings indicate an instructive impact of extracellular matrix proteins on defining the spatial organization of immune cells in the TME of ccRCC. The developed cycIHC method facilitates detailed characterization of the TME and may enhance the understanding of tumor-immune cell interactions.