Epithelial and Mesenchymal Circulating Tumor Cell Isolation and Discrimination using Dual-immunopatterned Device with Newly-developed Anti-63B6 and Anti-EpCAM

Abstract

We report a dual-immunopatterned microfluidic device for capturing both epithelial and mesenchymalcirculating tumor cells (CTCs) simultaneously in a single device using the two antibodies of anti-epithelialcell adhesion molecule (EpCAM) antibody and newly developed anti-63B6 antibody. In addition to theconventional epithelial antibody of anti-EpCAM, our in-house produced anti-63B6 antibody targets mes-enchymal stem cell-like cancer cells and intermediate cancer cells, thus overcoming limitation of theconventional single anti-EpCAM based CTC isolations. These two antibodies are immobilized to each topand bottom layer of the present device and the combination of two layers forms a circular chamber withthe center inlet. The mutual complementary cell isolation through competing reaction in different veloc-ity zones between mesenchymal and epithelial antibodies enables the precise screening and profilingof CTCs depending on their positivity and degree of epithelial/mesenchymal surface antigen expression,which may differ in compliance with disease status.From the experiments using epithelial and mesenchymal-like cancer cells, the present device captures94.47% of cancer cells, which is substantially higher than the EpCAM-only-based method in terms ofheterogeneous cancer cell isolation including mesenchymal–like cancer cells. Patient blood samples wereemployed to assess the clinical application of the present device for examining the patient status basedon their CTC heterogeneity. The CTCs captured by both antibodies exhibit considerably varied expressionprofiles of epithelial protein including cytokeratin and EpCAM. The present device facilitates the isolationof heterogeneous subtypes of CTCs. These undiscovered heterogeneities would be helpful for preciseanalysis of CTCs to find their underlying meaning in cancer progression.