MERFISH 2.0 enables high-sensitivity, customizable spatial transcriptomics for resolving immune and tumor programs in human cancers

High-plex, imaging-based spatial transcriptomics with single-cell resolution has substantially advanced our understanding of how heterogeneous tumor ecosystems shape immune architecture and function, target expression, and cell-cell interface dynamics within the human tumor microenvironment (TME). By measuring gene expression directly in intact human tissues, spatial transcriptomics provides detailed insights into immune-cell identity, spatial organization, and molecular programs that govern antitumor activity or immune suppression. Resolving transcripts in situ enables precise delineation of immune-cell states, spatial interactions, and regulatory circuits underlying antitumor responses and context-dependent cellular phenotypes. A key limitation of current spatial transcriptomics approaches is the ability to sensitively detect transcripts across complex and heterogeneous human tumors, particularly when studies require focused interrogation of specific pathways or cellular programs. MERFISH 2.0 overcomes these constraints with an enhanced chemistry and streamlined sample-processing workflow that delivers substantially higher transcript detection efficiency in both frozen and FFPE tissue, enabling robust profiling of panels up to 1,000 genes. Building on these improvements in sensitivity and workflow performance, MERFISH 2.0 also provides the flexibility to add up to 100 custom genes to Pre-designed Panels (PdP), enabling researchers to tailor experiments to specific hypotheses, tumor-intrinsic biology, or emerging clinical signatures. In this study, MERFISH 2.0 was applied to human tumor specimens using an 815-gene PdP with an immune-oncology focus, supplemented with custom gene content selected to capture key biological processes across diverse tumor types and disease stages. Implementation on MERSCOPE® Ultra™ slides generated high-resolution spatial maps that resolved the distribution of specific cell populations, the localization of surface and intracellular markers of interest, and the complex organization of immune infiltrates, stromal architecture, and tumor-intrinsic transcriptional programs within intact tissue environments. The ability to augment MERFISH 2.0 panels with tailored gene sets further expands its utility for dissecting molecular features and spatial patterns of the TME that influence pathway engagement and functional activity. By integrating enhanced sensitivity with customizable panel design, MERFISH 2.0 provides a powerful platform for resolving spatial determinants of immune function and tumor behavior, supporting mechanistic discovery and informing the development of next-generation precision medicine strategies.