Simple SummaryDifferent immunotherapies have been approved for the treatment of a multiplicity of cancers. However, a large proportion of patients do not respond or develop resistance, meaning that specified treatment combinations are required to enhance individual therapy efficiencies. A combined anti-PD-1/anti-LAG-3 therapy has already been approved for the treatment of melanoma patients. Here, we describe the checkpoint expression patterns and secretion of, e.g., TIM-3, LAG-3, galectin-9 and PD-(L)1/2 in breast cancer-specific humanized tumor mouse models. We quantitatively determine the breast cancer subtype-specific checkpoint co-expression and release. These data profoundly demonstrate the potential of humanized tumor mice as a significant mainstay for preclinical immunotherapeutic trials.AbstractCheckpoint blockade is particularly based on PD-1/PD-L1-inhibiting antibodies. However, an efficient immunological tumor defense can be blocked not only by PD-(L)1 but also by the presence of additional immune checkpoint molecules. Here, we investigated the co-expression of several immune checkpoint proteins and the soluble forms thereof (e.g., PD-1, TIM-3, LAG-3, PD-L1, PD-L2 and others) in humanized tumor mice (HTM) simultaneously harboring cell line-derived (JIMT-1, MDA-MB-231, MCF-7) or patient-derived breast cancer and a functional human immune system. We identified tumor-infiltrating T cells with a triple-positive PD-1, LAG-3 and TIM-3 phenotype. While PD-1 expression was increased in both the CD4 and CD8 T cells, TIM-3 was found to be upregulated particularly in the cytotoxic T cells in the MDA-MB-231-based HTM model. High levels of soluble TIM-3 and galectin-9 (a TIM-3 ligand) were detected in the serum. Surprisingly, soluble PD-L2, but only low levels of sPD-L1, were found in mice harboring PD-L1-positive tumors. Analysis of a dataset containing 3039 primary breast cancer samples on the R2 Genomics Analysis Platform revealed increased TIM-3, galectin-9 and LAG-3 expression, not only in triple-negative breast cancer but also in the HER2+and hormone receptor-positive breast cancer subtypes. These data indicate that LAG-3 and TIM-3 represent additional key molecules within the breast cancer anti-immunity landscape.