SUMMARY: Most cancer patients do not die from primary tumors but from the metastatic disease. Before formation of lethal metastases, disseminated cancer cells (DCCs) may stay dormant for up to decades, shown in clinics as the minimal residual disease (MRD), for which effective therapies are unavailable. Immunotherapies have made significant progress. However, most of the research focuses on primary tumors. Much less is known for the metastatic disease, in particular for MRD. The overarching goal of this proposal is to uncover mechanisms involved in the interaction between dormant DCCs and the immune system and therefore reveal therapeutic opportunities for MRD before the development into a full-blown lethal disease. We will determine the specific phenotypes of dormant DCCs and the associated immune cells relative to outgrowing metastases using single-cell RNA-sequencing, mass cytometry (or CyTOF), and Nanostring technologies. An important preclinical model that will be used throughout the study is the MMTV-ErbB2 mice, which develop spontaneous dormant DCCs in secondary organs. Compared to widely used models involving transplantation of established tumors or vaccination, this model will better mimic real-life situations in patients, as the immune system co- evolve with the cancer cells since their inception. In Aim 1, we will determine if T cells inhibit the expansion of DCCs via induction of dormancy and the mechanisms involved. We will test if T cell depletion allows the reactivation of dormant DCCs in the MMTV-ErbB2 model but also an orthotopic model as a complement. We will also test if immune cytokines induce DCC dormancy in 3D organoid cultures and in vivo and determine the cellular source of these cytokines. In Aim 2, we will determine if DCCs use the dormancy program to induce immune-suppression/-evasion via regulation of immune checkpoints. Specifically, we will test if dormancy- inducing cytokines and transcription factors regulate expression of immunoregulatory factors. immune-evasion genes to determine if the dormancy program in general is used by DCCs to avoid immune response and therefore persist. We will also test if immune checkpoint inhibitors enable immune reactions against dormant DCCs. Together, these approaches will allow us to understand if the immune system induces DCC dormancy, if the dormancy program enables evasion of suppression of the immune response, and if intervention of these processes will eliminate dormant DCCs. Thus, this study should reveal therapeutic opportunities to treat MRD and thereby minimize the disease recurrence that kills the majority of cancer patients.