Immune Checkpoint Profiling in Humanized Breast Cancer Mice Revealed Cell-Specific LAG-3/PD-1/TIM-3 Co-Expression and Elevated PD-1/TIM-3 Secretion

Different 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. Checkpoint 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.

Automated summary

Immunotherapies can be used to treat various cancers, but many patients do not respond well and develop resistance, so specific treatment combinations are needed to improve efficacy. Combined anti-PD-1/anti-LAG-3 therapy has been approved for melanoma patients. Humanized tumor mouse models can be used as an important tool for preclinical immunotherapeutic trials.