Characterization of HER2-Positive Murine Breast Cancer Models for Investigating HER2-Targeted Therapy and Immunotherapy

Background/Objectives: Human epidermal growth factor receptor 2 (HER2) -positive breast cancer is linked to poorer overall survival and a higher risk of brain metastases compared to HER2-negative breast cancer. Current preclinical studies lack robust HER2+ metastatic syngeneic mouse models for investigating targeted and immunomodulatory therapies. This study aims to develop effective HER2+ mouse models to investigate response dynamics to HER2-targeted therapy and immunotherapy. Methods: The human HER2 gene (WT or mutant p. A775G776insYVMA, GFP-tagged at the C-terminus) was introduced into triple-negative breast cancer (TNBC) mouse mammary carcinoma cells with known metastatic potential (4T1 and EO771) via lentiviral transduction. HER2 expression and phosphorylation were analyzed using Western blotting and immunohistochemistry. Tumors were treated with HER2-targeted therapy (trastuzumab and tucatinib), immune checkpoint blockade (anti-PD-1 and anti-CTLA-4), and anti-HER2 antibody–drug conjugate (ADC) to evaluate treatment efficacy. Metastatic potential was assessed with brain fluorescence imaging. Statistical analysis included ANOVA and Kaplan–Meier tests. Results: Newly established lines demonstrated expression of HER2+, with HER2YVMA lines showing higher phosphorylation than HER2WT lines. Cells were tumorigenic, demonstrating in vivo tumor take rates at 100% for 4T1-HER2 and 15–30% for EO771-HER2. HER2 overexpression led to a 30% increase in spontaneous brain metastasis in the 4T1-HER2 models. Trastuzumab alone did not reduce primary tumor size but significantly reduced brain GFP signal by 17% ± 8% and 26% ± 7% in the 4T1-HER2WT and 4T1-HER2YVMA models, respectively. Combinational therapies with anti-HER2 therapy and immune checkpoint blockade effectively suppressed primary tumor growth and prolonged survival in EO771-HER2YVMA model. T-Dxd, but not T-DM1, demonstrated partial treatment response in the EO771-HER2WT model. Conclusions: HER2+ syngeneic tumor models were developed that spontaneously metastasize to the brain and demonstrate variable responses to immunotherapies and ADCs. These models are valuable for advancing molecular imaging modalities for HER2+ brain metastasis, studying blood–brain barrier penetration of HER2-targeted drugs, and exploring the combination of therapies, including immunotherapy.