P4-07-03: Identification of Triple-Negative Primary Breast Cancer Xenograft Models with High Numbers of Circulating and Disseminated Tumor Cells.
Authors: Giuliano M , Christiny PI , Zhang X , Mao S , Contreras A , Lewis MT , Rimawi MF , Osborne CK , Schiff R , Trivedi MV 
Published: June 2012

Background: Primary breast cancer xenografts, in which tumors are grown directly from patients and which maintain their original genotype and phenotype, have the potential to facilitate the study of tumor biology and progression. These models can also be instrumental in the discovery of novel therapeutic targets especially for the triple-negative (ER-, PR- and HER2−negative, TN) breast cancer. TN breast cancer is associated with high numbers of circulating and disseminated tumor cells (CTCs and DTCs), which predict poor outcome in patients and may play a role in tumor progression. However, isolation and detection of human CTCs and DTCs in these xenograft models have been challenging even with EpCAM-based enrichment methods. The goal of this study was to determine if CTCs and DTCs could be identified using human pan-CK staining in a panel of triple-negative primary breast cancer xenograft lines, which could then be employed to study the biology of these cells and to test novel therapies.

Methods: We screened 13 stable primary transplantable xenograft lines (1-6 mice per line), established by directly transplanting ethnically diverse triple-negative tumor samples into the epithelium-free mammary fat pads of SCID/Beige mice, for the presence of CTCs and DTCs. The triple-negative status was maintained in these xenograft lines over serial passages. To detect CTCs, peripheral blood mononuclear cells (PBMCs) were isolated from the blood collected from the inferior vena cava either by Ficoll gradient or RBC lysis, with a typical yield of 500,000 PBMCs in 500 μl of blood. Subsequently, PBMCs were immunostained for the presence of CTCs, which were defined as the cells positive for cytoplasmic human pan-cytokeratin staining and nuclear (DAPI/hematoxylin) counter stain. We also flushed the femurs and tibias of 7 xenograft lines to harvest bone marrow cells (BMCs) for the detection of DTCs using the same staining procedure. A xenograft line was considered positive for CTCs or DTCs if they were detected in at least 25% of mice. The presence of lung metastases was assessed in all the xenograft lines by histological examination.

Results: We detected CTCs (range: 1–128/20,000 PBMCs) in 6 out of 13 xenograft lines (46%) and DTCs (range: 1–21/20,000 BMCs) in 5 out of 7 (71%) lines. Interestingly, 4 of the 5 DTC-positive lines also had detectable CTCs. High numbers of CTCs (&gt;20/20,000 PBMCs) were found in 3 xenograft lines, one of which also had high numbers of DTCs (&gt;20/20,000 BMCs). No human pan-CK+ cells were detected in PBMCs and/or BMCs from 5 control mice without tumors. Among 13 xenograft lines, lung metastases were found in 5 lines (38%), of which 3 had detectable CTCs or DTCs. Of the 3 xenograft lines containing high CTCs and/or DTCs, 2 had lung metastases.

Conclusion: In summary, human pan-CK staining can effectively detect CTCs and DTCs in isolated PBMCs and BMCs of mice bearing triple-negative primary breast cancer xenografts. These xenograft lines with detectable CTCs and DTCs may represent a valuable preclinical model for detailed characterization of human CTCs and DTCs and for the discovery of new therapeutic targets for the triple-negative breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-03.