This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA-24-029 PROJECT SUMMARY Despite the critical influence of both aging and the immune system on carcinogenesis there is little understanding of the interplay between aging and anti-cancer immune responses. We hypothesize that immune responses to lung tumor decreases with age such that the immune system has less impact on tumor initiation and growth within the aged environment. To enable rapid analysis of gene function in cancer models in vivo across different environment including aging, we have integrated genetically engineered mouse models, multiplexed CRISPR-based somatic genome engineering, and tumor barcoding (including an optimized platform called Tuba-seqUltra). These approaches enable the quantification of the impact of many genes of interest on lung tumorigenesis in parallel with unprecedented resolution and ease. To quantify the impact of aging on the importance of diverse immune pathways on lung carcinogenesis, we will generate a focused Tuba-seqUltra lentiviral pool targeting many regulators of cancer immunity and generate autochthonous lung tumors of each genotype in parallel. These quantitative in vivo genetic approaches will uncover the interactions between age and anti-cancer adaptive and innate immune responses that control lung tumor initiation and growth. The quantitative data from Tuba-seqUltra requires equally robust analytical and statistical methods to maximize our ability to uncover the impact of aging on immune related pathways in lung cancer cells. We will refine our existing pipelines to quantify the impact of each immune-related perturbation in lung carcinogenesis and comparing their effects with age. This proposal will leverage the complementary strengths of our laboratories to assess the impact of immune responses in cancer across age. Our expertise in high-throughput in vivo cancer modeling (Winslow laboratory) and cancer immunology (Reticker-Flynn laboratory) will uniquely allow us to explore the interface between aging and immune responses to lung cancer. These highly quantitative in vivo experiments will transform our understanding of how aging changes the impact of adaptive and innate immune responses on lung tumor initiation and growth. These functional studies will provide key insights into the cancer-immune interactions that change with age and could have important implication for lung cancer prevention and treatment