Chip-Assisted Single-Cell Biomarker Profiling of Heterogeneous Circulating Tumor Cells Using Multifunctional Nanospheres

Abstract

Profiling the heterogeneous phenotypes of individual circulating tumor cells (CTCs) from patients is a very challenging task, but it paves new ways for cancer management, especially personalized anticancer therapy. Herein, we propose a chip-assisted multifunctional-nanosphere system for efficient and reliable biomarker phenotype analysis of individual heterogeneous CTCs. Red fluorescent magnetic biotargeting multifunctional nanospheres and green fluorescent biotargeting nanospheres targeting to two kinds of CTC biomarkers are used for convenient dual-fluorescence labeling of CTCs along with magnetic tags. By integrating magnetic enrichment with a size-selective single-cell-trapping microfluidic chip (SCT-chip), over 90% of CTCs, even when the concentrations is as low as 10 CTCs per milliliter of blood, can be individually trapped at highly ordered micropillars, spatially separated from the minimal residual blood cells. Such single CTCs offer easy-readout fluorescence signals, facilitating efficient identification and reliable phenotype analysis in accordance with their biomarker expressions. Therefore, the phenotypes of breast tumor cells in terms of the expression level of human epidermal-growth-factor receptor 2, an important target of clinical anticancer drugs, are accurately assessed, and over 82% of them can be classified into corresponding cell subpopulations. Furthermore, this system demonstrates successful detection and subpopulation analysis of heterogeneous CTCs from seven breast cancer patients, which provides a promising new means for single-cell profiling of CTC-biomarker phenotypes and guiding of personalized anticancer therapy.

Sarcoma treatment in the era of molecular medicine

“…The biological value of CTCs was assessed by comparing the molecular profiles of CTCs and primary tumors (Keller & Pantel, 2019). Controversial conclusions showed that CTCs only partly reflect the spectrum of mutations in the primary and metastatic tumors (Paoletti et al , 2018; Wu et al , 2018; Brown et al , 2019; Keller & Pantel, 2019). CTCs may be considered a snapshot of tumor tissue heterogeneity at a given time and could have strong implications for longitudinal patient monitoring (Brown et al , 2019; Tellez‐Gabriel et al , 2019).…”

Beyond Capture: Circulating Tumor Cell Release and Single‐Cell Analysis

“…However, when it comes to the analysis of rare CTCs, FACS suffers from low sensitivity and sample losses . Alternatively, microfluidic technology allowing capture and in situ identification of CTCs is well suitable to conduct phenotypic‐relevant imaging . For example, Pang's group combined micropillar‐array chip and multifunctional nanospheres for capture and analysis of biomarker phenotypes of individual CTCs ( Figure a) .…”

“…Alternatively, microfluidic technology allowing capture and in situ identification of CTCs is well suitable to conduct phenotypic‐relevant imaging . For example, Pang's group combined micropillar‐array chip and multifunctional nanospheres for capture and analysis of biomarker phenotypes of individual CTCs ( Figure a) . Red fluorescent magnetic nanospheres modified anti‐EpCAM Ab (IRMNs) and green fluorescent nanospheres modified anti‐HER2 Ab (IGNs) were targeted to two kinds of CTC biomarkers, endowing simultaneous magnetic labeling and fluorescence measurement.…”

“…a) Chip‐assisted multifunctional nanosphere system for single‐cell biomarker analysis. Reproduced with permission . Copyright 2018, American Chemical Society.…”

Negative depletion mediated brightfield circulating tumour cell identification strategy on microparticle-based microfluidic chip

“…On the other side, the cell morphology or phenotype characteristics are destroyed after traditional immunofluorescence identification. Fluorescence photobleaching and spectral overlap also limit long term and multi-biomarkers cell identification [15].…”