Multiomic Spatial Phenotyping of the Tumor Immune Microenvironment at Single Cell Resolution

Abstract

Abstract The Tumor microenvironment (TME) is critical in cancer development, progression, and control. Immunological components within tumors, known as the tumor immune microenvironment (TIME), have been implicated in cancer progression. Effective strategies for cancer immunotherapies will require a deep understanding of factors that shape the TME and TIME. Here, we describe a spatial multiomics assay utilizing RNAscope™ ISH technology paired with high-plex whole-slide spatial phenotyping with the PhenoCycler ®-Fusion platform. This two-step approach is compatible with human FFPE tissues and enables characterization of the TIME more accurately by detecting RNA and protein markers on serial sections. We performed ultrahigh-plex spatial phenotyping on FFPE tumor tissue sections, using an antibody panel designed for immune cell phenotyping, evaluation of immune contexture and proliferation across the TME. On a serial section, we ran the RNAscope HiPlex v2 assay using the PhenoCycler-Fusion system. This assay consisted of a 12-plex immuno-oncology panel of RNA target probes, which detect macrophages, chemokines, and cytokines. In this proof-of-concept study, we demonstrate multiomic spatial phenotyping on the PhenoCycler-Fusion platform. Analysis of the resulting multiplex imaging data revealed structural organization of cells within the TME and activation states of immune cells. Together, this information provides a more complete functional map of immune cells within the TME and TIME and thereby enriches our understanding of tumor biology that may be deterministic of immunotherapy responsiveness.