Investigating evolutionary antagonism between tumors and lymphocytes

PROJECT SUMMARY Metastasis accounts for the majority of cancer patient deaths and even though immune checkpoint blockade has proven effective in many malignancies, metastatic disease remains resistant to therapy. Cancer metastasis is an inefficient process, requiring cancer cells to disseminate from primary tumors to distant sites, surviving the stress of migration and evading clearance by circulating immune cells. As a result, metastasis is a selective event, leading to the establishment of metastatic tumors that often consist of just a few clones. Unlike the diverse primary tumor from which they derived, these clones must be capable of adapting to new tissue environments and evading continuous immune surveillance. We hypothesize that even amidst clonal selection during cancer metastasis, cells acquire diverse phenotypes upon subsequent outgrowth that allows them to adapt to their environment and evade innate and adaptive immune responses. Utilizing our family of lymph node metastatic cell lines, we will perform analyses of clonal diversity by viral integration site mapping, profiling diversity across nine rounds of in vivo selection. We will further perform single cell transcriptomics, allowing the diversity of phenotypes to be uncovered in relation to the clonal composition of each cell line. Expanding these analyses, we will perform analogous experiments using human head and neck squamous cell carcinoma specimens, validating whether amidst low genomic clonality tumors can support diverse phenotypic fates. As we have described previously, metastasis to lymph nodes can induce immune tolerance that permits further dissemination of cancer cells. We further hypothesize that these diverse phenotypes of metastatic cells drive unique lymphocyte phenotypes and clonal responses, such that the two antagonize one another. To address this hypothesis, we will perform phenotypic and immune repertoire diversity profiling by spectral flow cytometry and single cell genomics of murine tumors of varying clonality and the patient tumors already assessed for clonality and phenotype. The resulting relationship between tumor clonality, T cell phenotype, and T cell clonal diversity will address whether tumors and lymphocytes evolve antagonistically, adopting varied fates and clonal architectures in an attempt to outcompete the other side. We will finally perform similar immune analyses of a metastatic cell line evolved in the absence of lymphocyte pressure, validating whether ongoing coevolution of tumors and lymphocytes is occurring during cancer metastasis. These experiments will address the mechanisms of antagonism and adaption at the tumor-immune interface during cancer metastasis and identify potential avenues of therapeutic intervention to enhance immune responses against metastasis, such as delivery of vaccines containing diverse antigens to increase lymphocyte diversity.