Epithelial and mesenchymal circulating tumor cell isolation and discrimination using dual-immunopatterned device with newly-developed anti-63B6 and anti-EpCAM

We report a dual-immunopatterned microfluidic device for capturing both epithelial and mesenchymal circulating tumor cells (CTCs) simultaneously in a single device using the two antibodies of anti-epithelial cell adhesion molecule (EpCAM) antibody and newly developed anti-63B6 antibody. In addition to the conventional epithelial antibody of anti-EpCAM, our in-house produced anti-63B6 antibody targets mesenchymal stem cell-like cancer cells and intermediate cancer cells, thus overcoming limitation of the conventional single anti-EpCAM based CTC isolations. These two antibodies are immobilized to each top and bottom layer of the present device and the combination of two layers forms a circular chamber with the center inlet. The mutual complementary cell isolation through competing reaction in different velocity zones between mesenchymal and epithelial antibodies enables the precise screening and profiling of 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 captures 94.47% of cancer cells, which is substantially higher than the EpCAM-only-based method in terms of heterogeneous cancer cell isolation including mesenchymal–like cancer cells. Patient blood samples were employed to assess the clinical application of the present device for examining the patient status based on their CTC heterogeneity. The CTCs captured by both antibodies exhibit considerably varied expression profiles of epithelial protein including cytokeratin and EpCAM. The present device facilitates the isolation of heterogeneous subtypes of CTCs. These undiscovered heterogeneities would be helpful for precise analysis of CTCs to find their underlying meaning in cancer progression.