An Integrated Microfluidic Chip System for Single-Cell Secretion Profiling of Rare Circulating Tumor Cells

Abstract

Genetic and transcriptional profiling, as well as surface marker identification of single circulating tumor cells (CTCs) have been demonstrated. However, quantitatively profiling of functional proteins at single CTC resolution has not yet been achieved, owing to the limited purity of the isolated CTC populations and a lack of single-cell proteomic approaches to handle and analyze rare CTCs. Here, we develop an integrated microfluidic system specifically designed for streamlining isolation, purification and single-cell secretomic profiling of CTCs from whole blood. Key to this platform is the use of photocleavable ssDNA-encoded antibody conjugates to enable a highly purified CTC population with <75 ‘contaminated' blood cells. An enhanced poly-L-lysine barcode pattern is created on the single-cell barcode chip for efficient capture rare CTC cells in microchambers for subsequent secreted protein profiling. This system was extensively evaluated and optimized with EpCAM-positive HCT116 cells seeded into whole blood. Patient blood samples were employed to assess the utility of the system for isolation, purification and single-cell secretion profiling of CTCs. The CTCs present in patient blood samples exhibit highly heterogeneous secretion profile of IL-8 and VEGF. The numbers of secreting CTCs are found not in accordance with CTC enumeration based on immunostaining in the parallel experiments.

Microfluidic techniques for high throughput single cell analysis

“…Related work has introduced geometric refinements for improved isolation of CTCs from peripheral blood components[43]. Microvortex-generating microfluidic devices[44] have been demonstrated for label-free CTC separation based upon size distributions of targeted CTC and bulk blood components[45,46]. …”

Microfluidic Sample Preparation for Single Cell Analysis

“…The group used protease cleavable FRET peptides for detection. Dent et al 256 used a microfluidic platform for profiling proteins secreted by single circulating tumor cells confined in micro wells. While the proteomic approaches discussed above measured one target protein, Hughes et al 257 developed a novel microfluidic platform for multiplex single cell protein analysis.…”

Micro- and nanodevices integrated with biomolecular probes

“…Moreover, other studies improved the cell–probe functionalized surface interactions by utilizing microposts from ultra-high porosity CNT forests (Chen et al, 2012). Furthermore, herringbones on channel walls were used to disrupt streamlines of flow and, thus, enhance cell–surface interactions and capture efficiency (Deng et al, 2014; Stott et al, 2010). …”