Abstract 4279: Human HER2-overexpressing mouse breast cancer cell lines derived from MMTV.f.HuHER2 mice: characterization and use in a model of metastatic breast cancer
Authors: Park Sunju , Sgouros George 
Published: July 2016

Abstract
Preclinical evaluation of therapeutic agents against metastatic breast cancer require cell lines and animal models that recapitulate clinical metastatic breast cancer as much as possible. We have previously used cell lines derived from the neu-N transgenic model to investigate anti-neu targeting of metastatic breast cancer using an alpha-emitter labeled antibody reactive with the rat variant of HER2/neu expressed by the neu-N model. To investigate alpha-emitter targeting of metastatic breast cancer using clinically relevant, commercially available anti-HER2/neu antibodies, we have developed cell lines derived from HuHER2 mice (MMTV.f.HuHER2 obtained from Genetech). HuHER2 mice develop breast cancer spontaneously and with a higher frequency than normal mice.
We extracted primary mammary gland tumors, purified the epithelial breast cancer cells, and established 7 different HuHER2 cell lines. We also established 2 different cell lines from spontaneous lung metastases (HuHER2-L1 and -L2). We evaluated HuHER2 protein expression in the cell lines by western blot analysis. Cell surface receptor expression was evaluated by immunofluorescence (IF) staining. We performed qRT-PCR to assess phenotypic measures of aggressiveness and metastatic propensity. The following were evaluated: ER, PR, Twist1, Vimentin and E-Cadherin. Sensitivity to trastuzumab antibody, in vitro, was also assessed by evaluating changes in cellular metabolism via the MTT assay.
HuHER2 protein was overexpressed in all of the isolated cell lines. One of the cell lines (denoted HuHER2-6) had approximately 1.5-8 times more protein expression compared to the other 6 cell lines developed. The two cell lines derived from spontaneous lung metastases showed approximately 1.5-fold greater HER2 protein expression than HuHER2-6. HuHER2-6 cell surface HER2 receptor staining by IF was similar to that of BT-474, a high HuHER2 expressing cell line. The 2 lung cell lines also showed comparable cell-surface staining. ER mRNA levels assessed by qRT-PCR were 20% and 0.05% of the level measured in normal mammary gland for the HuHER2-6 and HuHer2-L2 lines, respectively. The PR levels were also substantially lower (&lt;0.01%) relative to normal mammary gland (MG) tissue. The HuHER2-6 line expressed 1.4 times more HuHER2/neu mRNA than BT-474. The mRNA for E-Cadherin in HuHER2-L1 and -L2 was 20% of MG tissue. The mRNA for TWIST1 and Vimentin were similarly elevated 3-fold and 1.5-fold, respectively, relative to MG. At trastuzumab concentrations ranging from 10 to 500 ug/ml, cell metabolism was decreased to 50%.
These lines show all the hallmarks of highly aggressive, metastatic breast cancer and are being used to establish a left cardiac ventricle injection model of widespread HER2/neu positive metastatic breast cancer to evaluate combination therapy with alpha-particle emitter labeled HER2/neu reactive antibodies.
Citation Format: Sunju Park, George Sgouros. Human HER2-overexpressing mouse breast cancer cell lines derived from MMTV.f.HuHER2 mice: characterization and use in a model of metastatic breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4279.