Abstract 1368: Investigating the tumorigenicity of disseminated tumor cells

Abstract

Abstract Background: Tumor cells that disseminate to the bone marrow (disseminated tumor cells, DTCs) have been identified in 30% of stage I - III breast cancer (BC) patients and independently predict outcome. Because DTCs are a rare, heterogeneous population of cells, little is known regarding their characteristics; such as their clinical significance with regard to disease progression, and targets that can be utilized for their eradication. DTC Identification is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM), and cytokeratin (CK). However, EpCAM and CK can be down-regulated during epithelial–mesenchymal transition (EMT). To date, no published report has assessed the tumorigenicity of DTCs isolated from patient samples using an in vivo animal model. The purpose of this study was to investigate the tumorigenicity of DTCs in vivo. Methods: Thirteen clinical stage I-III BC patients provided informed consent to participate in an IRB-approved study involving collection of bone marrow at the time of primary surgery. Following Ficoll centrifugation, bone marrow aspirates were cultured using breast cancer stem cell enriching conditions for 21 days. At 21 days, viability of floating cells with high nuclear: cytoplasmic ratios were confirmed with Tryphan blue staining and cytokeratin expression was identified using pan-cytokeratin immunostaining. 200 cells were co-injected with Matrigel into the mammary fat pads (3 mice/patient) of female nude mice. Mice were monitored daily for tumor formation. Results: Viable floating cells with large nuclear: cytoplasmic ratios were observed in 10/13 cases. Both CK+ and CK- DTCs were identified in 8 out of 10 cases, and CK- DTCs only were identified in 2 out of 10 cases. One of the 10 samples was tumorigenic in vivo; all 3 mice injected with this patient's cells developed breast tumors within ≤ 55 days. This sample was obtained from a 40-year old T2N2 patient with a grade 2 estrogen receptor positive, progesterone receptor negative, Her2/neu negative tumor. Interestingly, CK expression was not detected in this patient's cultured cells. Conclusions: DTCs can be isolated and propagated using breast cancer stem cell enriching conditions, enabling the characterization and assessment of DTCs that are in the process of (or have undergone) EMT. In addition, the tumorigenetic potential of DTCs can be assessed on a patient-by-patient basis by employing an in vivo mouse model. Further studies are needed to develop targeted therapies for the eradication of micrometastatic disease in stage I-III BC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1368. doi:1538-7445.AM2012-1368