Abstract 5108: Capture and enumeration of mesenchymal CTCs on the OnQChip platform.
Authors: Merdek Keith D. , Sprott Kam , Jiang Chunsheng , Stocum Michael , Bubley Glenn , Carney Walter 
Published: August 2014

Circulating tumor cells (CTCs) are cancer cells that disseminate from the primary tumor and enter the blood. While cells in the primary tumor are generally epithelial in nature, as they become migratory and invasive, changes such as loss of epithelial markers (ie: EpCAM and cytokeratin (CK)) and a switch to a mesenchymal phenotype (EMT) often occur. CTC platforms must be flexible in capturing and identifying cells, and not limited to the use of only epithelial markers. Dual modal capture on the OnQChip provides this flexibility, allowing for CTC isolation based on size and independent of surface marker expression in addition to specific antibody mediated capture targeting markers such as EpCAM. For cell lines ranging in EpCAM expression, the OnQChip captures 90% or greater of mid to high EpCAM expressing cells, and 79% or greater of very low EpCAM expressing cells. Due to the OnQChip's gradient design, distinct capture patterns related to EpCAM levels are observed, allowing for capture location to be informative about EpCAM expression. When Hs578T cells, a mesenchymal model with very little EpCAM and CK, are prestained around 79% of spiked cells are enumerated on the OnQChip, but when the same cells are not prestained and only CK staining is used, 0-2% of spiked cells are counted. When two new mesenchymal staining markers are incorporated alongside CK, 60-80% of spiked Hs578T cells are now enumerated, translating to a 75-100% staining efficiency of captured cells. Although very few cells are visualized with CK alone, pooling CK staining with mesenchymal staining shows increased staining intensity compared to mesenchymal staining alone, highlighting benefits of a multi-marker staining panel for visualizing cells with low expression of several different markers, likely the case for subpopulations of cells at different stages of the EMT. On a population of 44 prostate cancer samples, an increase of 28.5, 8.2, 12.7, and 7.6 in the mean number of total, intact, irregular, and fragmented CTCs, respectively, was observed with staining for CK plus the mesenchymal markers as compared to CK staining alone. Specifically, 70.5%, 18.2%, 50%, and 38.6% of patients showed an increase of 5 or more, and 40.9%, 9.1%, 31.8%, and 25% showed an increase of 10 or more, total, intact, irregular, and fragmented CTCs, respectively, when mesenchymal staining was included. Additionally, a few case studies from this population for which serial draws during treatment were obtained indicate changes in the ratio of CTCs enumerated with staining for CK plus mesenchymal markers compared to CK only, suggesting the ratio of mesenchymal to epithelial like CTCs may reflect a clinical outcome. Our data suggests that the OnQChip's ability to capture CTCs based on size independent of surface marker expression and inclusion of two new stains, allow for capture and enumeration of mesenchymal CTCs that would be missed by platforms incorporating only epithelial markers such as EpCAM and CK.

Citation Format: Keith D. Merdek, Kam Sprott, Chunsheng Jiang, Michael Stocum, Glenn Bubley, Walter Carney. Capture and enumeration of mesenchymal CTCs on the OnQChip platform. [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 5108. doi:10.1158/1538-7445.AM2013-5108

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.