Abstract 1447: Capture, culture and characterization of viable circulating tumor cells in a syngeneic breast cancer mouse model system.
Authors: Williams Anthony , Schrand Brett , Rawal Siddarth , Ao Zheng , Brenneman Randall , Gilboa Eli , Zheng Siyang , Datar Ram , Cote Richard 
Published: August 2014

Abstract
Circulating tumor cells (CTCs) play a critical role in metastasis. Functional characterization of CTCs is limited by a lack of available methods to enrich viable CTCs. We have developed a next-generation (next-gen) platform, modifying our current size-based CTC enrichment technology, for viable CTC capture. Here, we employed our next-gen microfilter device for viable CTC capture in a syngeneic breast cancer mouse model system to show that such cells can be cultured with high efficiency for further characterization.
Balb/c mice received subcutaneous injection of 4T1 (highly metastatic) or 4T07 (poorly metastatic) mouse mammary tumor cell lines originally derived from the same spontaneously arising tumor. Primary tumors were excised on day 21, digested and placed in culture, and viable CTCs were isolated using the next-gen microfilter device from 0.5ml whole blood draws for culture directly on-chip. Successfully established cell cultures were characterized by morphology, IHC for markers associated with epithelial-mesenchymal transition (EMT), and anchorage-independent growth to compare abilities to form tumorispheres.
Tumors developed in 100% (6/6) of mice injected; cultures were obtained from 100% dissociated primary tumors (n=3 4T1, n=3 4T07), and isolated CTCs yielded cultures from 33% of mice injected with both cell lines (1/3 each). While 4T07-CTC cultures demonstrate homogenous morphology similar to the corresponding primary tumor, 4T1-CTC cultures demonstrate two morphologically distinct subclones: “A," spheroid-like, different from the primary tumor, and “B," spindle-like, similar to the primary tumor. “A” cells demonstrate the ability to produce progeny with spherical as well as spindle-like morphology in vitro. EMT profiling revealed that both 4T1-CTC subclones demonstrate epithelial and mesenchymal expression characteristics, while 4T07-CTCs predominately show mesenchymal character. CTCs had a greater ability to form tumorispheres in anchorage-independent growth conditions than corresponding primary tumors, with 4T1 “A” having greater tumorisphere-forming ability than 4T1 “B”.
These studies demonstrate our ability to capture and culture viable CTCs from mice injected with tumor cells of varying metastatic capability. The characterization of CTCs may help identify functionally important subsets of CTCs. Molecular and functional analyses of CTCs are ongoing, and include epigenomic, transcriptomic and proteomic expression profiling, assessment of stem cell-like properties, and their mechanistic role in metastatic disease. Our work could be the basis to establish CTC cultures in human patients, and provide a transformative approach to the functional characterization of CTCs, leading to an exciting new technology for improved cancer research and individualized patient management.
Citation Format: Anthony Williams, Brett Schrand, Siddarth Rawal, Zheng Ao, Randall Brenneman, Eli Gilboa, Siyang Zheng, Ram Datar, Richard Cote. Capture, culture and characterization of viable circulating tumor cells in a syngeneic breast cancer mouse model system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1447. doi:10.1158/1538-7445.AM2013-1447