Abstract LB-34: Microfilter capture of live circulating tumor cells from the blood of mice bearing tumor xenografts: A new model for the study of cancer dissemination

Abstract

Abstract Introduction: Cancer dissemination traditionally has been studied by characterizing cancer cells from metastatic sites relative to cancer cells from primary tumors, because technical limitations precluded direct analysis of the actual disseminating cells. We recently reported a novel slot microfilter platform capable of capturing and analyzing live circulating tumor cells (CTC) from peripheral blood. Microfilter capture is uniquely suited for the study of cancer dissemination, because it does not rely on the binding of epithelial cell adhesion molecules which may be downregulated in key populations of disseminating cells. In the current study, we used the slot microfilter platform to capture CTC from the blood of mice inoculated with tumor xenografts and to characterize the captured CTC relative to the parental cancer cells. Methods: GFP-labeled SCC351 human squamous cell carcinoma cells were inoculated subcutaneously into NOD-SCID/IL2Rγ K/O mice. After formation of subcutaneous xenografts, 500 μl of blood was collected by intracardiac puncture and Ficoll-centrifuged to remove red blood cells. The PBMC layer was resuspended in PBS and passed through the slot microfilter. Captured GFP+ CTC were identified under fluorescence microscopy and transferred into culture media (DMEM/10% FBS). Cultured CTC and parental cancer cells were analyzed by qPCR for gene expression. Results: Subcutaneous tumors and lung metastases (GFP+) formed 4–6 weeks after inoculation in 4 of 4 mice as confirmed by fluorescence microscopy. Using the slot microfilter, GFP+ live CTC were successfully captured from the blood of 2 of the 4 mice. Notably, the captured CTC expanded to form spheres in culture, in contrast to parental cells which formed an adherent monolayer under identical culture conditions. Because sphere formation and cancer dissemination have been associated with epithelial-mesenchymal transition (EMT), we quantified the expression of EMT-associated genes in the cultured CTC compared to parental cells and found a marked elevation in N-cadherin (468x), Vimentin (307x), Foxc2 (356x) and Slug (64x). Conclusions: We have developed a new mouse model for the study of cancer dissemination which – for the first time – enables capture, manipulation, expansion, and analysis of live CTC in a manner that is independent of cell surface marker expression. As proof of principle, we used this approach to compare the morphologic and gene expression profiles of captured CTC to those of parental cancer cells, and we observed phenotypic changes suggestive of EMT. Our findings illustrate the considerable strength and versatility of this model, which allows for primary inoculation of cancer cells with pre-specified properties and subsequent capture and direct characterization of the disseminating cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-34. doi:10.1158/1538-7445.AM2011-LB-34