Abstract 2822: Establishing the use of Vortex technology for investigating circulating tumor cells in mouse models of breast cancer
Authors: Ramani Vishnu , Gupta Rakhi , Triboulet Melanie , Renier Corinne , Crouse Steve C. , Sollier-Christen Elodie , Jeffrey Stefanie S. 
Published: July 2017

Abstract
Background: Circulating tumor cells (CTCs) are precursors of metastatic disease and are an important indicator of the disease progression and outcome of many cancers. Though several recent studies have advanced our understanding of CTCs, many critical areas of CTC biology remain largely unexplored. Murine models of cancer offer a unique opportunity to address this issue. However, small sample volumes, low number of CTCs, difficulty to access ports for blood collection are challenges that diminish the utility of mouse models for studying CTCs. In our previous studies, we had validated the use of Vortex technology for the label-free capture of CTCs from blood samples of different cancers. In this current study, we now report our recent, unpublished data on the adaptation of Vortex platform for enriching and characterizing murine and human tumor cells from mouse blood.
Methods: By spiking tumor cells into small volumes of mouse blood (200-400 µl) from cardiac puncture, we tested the impact of i) different dilutions of mouse blood (10X, 20X, 40X); ii) varying numbers of tumor cells; iii) different cell types (MDA-MB-231, 4T1 and EMT6); and iv) sample recycling on capture efficiency and purity. Blood was processed through the Vortex plastic chip and, to accurately identify and enumerate the enriched tumor cells, we developed a staining protocol that labels human- or murine-specific cytokeratin, vimentin, and CD45.
Results: Our results reveal that over a range of dilutions tested, just after one cycle, 10X dilution of mouse blood was superior, yielding a capture efficiency of 38.83% and a purity of 33% for MDA-MB-231 cells, compared to 20X (34.16% efficiency, 31.1% purity), and 40X (31.83% efficiency, 31% purity) dilutions. We are currently testing whether recycling the blood flow-through would increase the recovery. Even when as many as ~7500 tumor cells were spiked in mouse blood, the Vortex platform was able to successfully enrich tumor cells with high consistency and purity. Importantly, we were now able to successfully isolate human and murine tumor cell lines with varying levels of epithelial cell adhesion molecule (EpCAM) expression (4T1 - high, MDA-MB-231 - moderate to low) spiked into mouse blood. Experiments confirming the viability and growth rate of cancer cells isolated from mouse blood using MTT assay will also be presented.
Conclusion: In summary, our results reveal the use of the Vortex technology for studying CTCs in murine models of cancer, with the ability to handle volumes of blood as low as 200uL while having a capacity to capture extremely large number of CTCs. While this study was focused on breast cancer, our workflow is easily adapted to other malignancies. Studies are underway to grow viable CTCs from tumor bearing animals and isolated by this platform for subsequent growth in 3D culture systems for future biologic and drug testing studies.
Citation Format: Vishnu Ramani, Rakhi Gupta, Melanie Triboulet, Corinne Renier, Steve C. Crouse, Elodie Sollier-Christen, Stefanie S. Jeffrey. Establishing the use of Vortex technology for investigating circulating tumor cells in mouse models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2822. doi:10.1158/1538-7445.AM2017-2822