Abstract LB-305: Circulating tumor cells as a novel model to test efficacy of individualized therapy in breast cancer

Abstract

Abstract Background: Recent reports suggest that circulating tumor cells (CTCs) mimic cancer progenitor cells, which are commonly resistant to various treatment modalities and are capable of invasion and tumor initiation at a single cell level. We hypothesized that CTCs isolated from metastatic breast cancer (MBC) patients can be propagated in 3-D in vitro systems, representing a clinically relevant model to study MBC biology and to test efficacy of multiple anti-cancer drugs. Methods: CTCs were isolated from MBC patient peripheral blood by depleting leucocytes and red blood cells. Enriched CTCs were immunostained for detection as CD45-negative (-) and nuclear counter stain-positive (+) cells, and for expression of estrogen receptor (ER) and HER-2. In order to obtain a large and continuous supply of CTCs for our culture studies, we screened 19 primary transplantable xenograft lines, established by directly transplanting human breast tumors into epithelium-free mammary fat pads of SCID/Beige mice, for their production of CTCs. The CTCs were isolated together with mouse mononuclear blood cells by either gradient centrifugation or red blood cell lysis, and identified by human pan-cytokeratin immunostaining. Viable CTCs isolated from both patients and xenograft mice were plated in mammosphere conditions either alone or in co-culture with mammary fibroblasts tagged with green fluorescent protein (GFP). the propagated CTCs were identified as GFP-, cytokeratin 19+ cells. Results: To date, we have collected and processed blood samples from 14 MBC patients. CTCs were detected in 5 out of the 8 samples (62%) already analyzed. Interestingly, in 2 out of 4 patients (50%) with positive CTC counts and ER+ primary tumor, all the isolated CTCs were ER-. Moreover, we found HER-2+ CTCs in 1 out of 4 patients (25%) with HER-2 negative disease. CTC count and characterization of the additional collected samples is ongoing and will be presented at the conference. In the mice bearing primary xenografts, we detected CTCs in 13 out of 19 lines (68%) and in 26 out of 54 mice (48%). When CTCs isolated from either human patients or primary xenografts were cultured alone, no mammospheres were observed. In contrast, when CTCs were cultured in direct contact with fibroblasts we observed the formation of mixed CTC/fibroblast spheres that may reproduce the tumor cell/stroma interaction observed in vivo. Moreover, in the presence of GFP+ fibroblasts, the numbers of GFP- cells and cell aggregates were clearly higher at all time points compared to the single culture. Conclusions: Our study suggests that viable CTCs isolated from human patients and primary xenograft bearing mice can be propagated in vitro in presence of mammary fibroblasts. The successful development of a CTC-based cell culture platform will provide a unique model to better understand the biological mechanisms of the metastatic process and to guide treatment decisions in breast cancer. 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 LB-305. doi:1538-7445.AM2012-LB-305