PROJECT SUMMARY Metastasis is responsible for the majority of cancer-associated deaths. Dissemination to and colonization of lymph nodes (LNs) precedes metastasis to distant sites in most solid tumors. Our Center in Cancer Systems Biology (CCSB) addresses the central hypothesis that metastasis to distant sites requires systemic immune tolerance induced by early interactions between immune and malignant cells in the metastatic lymph nodes (LN). In Project 1, we have successfully uncovered mechanisms by which metastasis to LNs occurs and the effects of these metastases on the formation of distant metastases, through a murine model of melanoma and have been demonstrating similar phenomena in a murine model of HNSCC. For this cost extension, we will continue our work to evaluate the roles of additional immune subsets in the tolerance process in melanoma and HNSCC, and determine the roles of tumor clonal heterogeneity between primary tumors and LN metastases. In Project 2, we have successfully developed a novel multiplexed immunofluorescence in situ imaging platform, CODEX, to characterize tissue architecture and demonstrated the application of CODEX to fresh frozen and FFPE tissue samples of primary and murine samples of HNSCC and melanoma. For the cost extension, Project 2 aims to continue its work to achieve co-detection of RNA with protein antibodies in CODEX to study the chemokine and cytokine candidates identified in Projects 1 and 3. In Project 3, we developed an integrative single-cell analysis pipelines to study imaging and sequencing data generated in our center. Through this analysis we have identified potentially druggable chemokine ligand-receptor pairs related to lymph node metastasis and validated their functional significance in collaboration with the Project 1 team. Our completed work includes a novel machine learning cell type identification method “CELESTA” to facilitate new insights on spatial biology. For the cost extension, Project 3 aims to continue to apply our computational tools and analysis pipelines to investigate additional immune cell types and stroma cells to identify mediators of tissue architectures facilitating lymph node metastasis on HNSCC tissue samples. We will validate the consistency of the finding on an independent cohort of human HNSCC TMA (Core 3), which is annotated with survival outcomes and collaborate with Project 1 for functional studies. In addition, our Center proposes to continue our efforts in outreach and dissemination through virtual monthly seminars.