Project Summary/Abstract Breast cancer (BC) remains the second leading cause of cancer-related deaths in women in the United States and late-stage metastatic BC remains incurable. The recent approval of immune checkpoint inhibitors targeting PD-1 or PD-L1 in metastatic triple-negative BC (TNBC) demonstrates that immunotherapies may be an effective approach to decrease BC mortalities. However, over half of metastatic BC patients develop metastasis to the liver, a site that responds poorly to immunotherapies. Despite high prevalence and mortality rates, few research programs focus on BC liver metastasis and little is known about the impact of metastatic BC cells on the liver microenvironment. My K99/R00 proposal will fill this gap in knowledge by equipping me with the training to become an independent tenure-track faculty at a research-intensive institution with a program on BC metastasis, especially liver metastasis, research. My postdoctoral work supported by an NCI-T32 and NCI-F32 explored factors secreted by TNBC to support lung metastasis via immune suppression. My first-author paper showed that aggressive TNBC secrete cytokines to enhance the number of pro-tumor macrophages. Currently, I am testing the impact of TNBC heme metabolism by heme oxygenase-1 (HO-1) on immune suppression via its metabolite bilirubin (BR). I demonstrated for the first time that TNBC cells secrete BR to alter macrophage polarization and function. However, HO-1 and BR have never been studied in BC liver metastasis, even though my preliminary data and work from others showed that HO-1 and BR were elevated in BC patients with liver metastasis compared to those with metastasis to other sites. The overall goals of this proposal are to: 1) test the impact of tumor cell-HO-1 on immune cells in the metastatic liver via its regulation of suppressive cytokines and BR; 2) assess the effects of combined HO-1 and PD-1 inhibition on liver metastatic outgrowth; and 3) test the impact of BC liver metastasis metabolic reprogramming on HO-1 expression. During the mentored K99 phase, I will work with experts in animal procedures, liver disease, tumor immunology and metabolomics at the University of Colorado Anschutz Medical Campus (CU AMC). Under their guidance, I will test the effects of BC liver metastasis-HO-1 on checkpoint inhibitor resistance via promotion of T cell-mediated immune suppression (Aim 1). Throughout the K99 and R00 phases, I will also assess the impact of tumor cell-BR on liver cells including resident macrophages known as Kupffer cells (Aim 2). In the R00 phase, I will test the impact of BC liver metastasis-specific metabolic reprogramming via HIF-1α on HO-1 and determine if this further supports local immune suppression (Aim 3). With this research plan and my support system at CU AMC, I will advance the field of BC metastasis research and lay the foundation for my independent research career that will continue to assess the effects of organ-specific metabolic rew...