Profiling protein expression in circulating tumour cells using microfluidic western blotting

Sinkala, Elly; Sollier‐Christen, Elodie; Renier, Corinne; Rosàs-Canyelles, Elisabet; Che, James; Heirich, Kyra; Duncombe, Todd A.; Vlassakis, Julea; Yamauchi, Kevin A.; Huang, Haiyan; Jeffrey, Stefanie S.; Herr, Amy E.

doi:10.1038/ncomms14622

Cited by 213 publications

(212 citation statements)

References 69 publications

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“…Increasing the assay duration presents trade-offs, as longer durations may improve separation performance but concomitantly increase diffusive losses of the cell lysate. 41,71 We thus increased the EF duration modestly, from 6 min to 8 min, and immunoprobed for lamin A/C and β-tubulin (Fig. 6A and S11†).…”

Section: Resultsmentioning

confidence: 99%

Electrophoretic cytometry of adherent cells

Herr

2017

Lab Chip

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Cell-matrix and cell-cell interactions influence intracellular signalling and play an important role in physiologic and pathologic processes. Detachment of cells from the surrounding microenvironment alters intracellular signalling. Here, we demonstrate and characterise an integrated microfluidic device to culture single and clustered cells in tuneable microenvironments and then directly analyse the lysate of each cell in situ, thereby eliminating the need to detach cells prior to analysis. First, we utilise microcontact printing to pattern cells in confined geometries. We then utilise a microscale isoelectric focusing (IEF) module to separate, detect, and analyse lamin A/C from substrate-adhered cells seeded and cultured at varying (500, 2000, and 9000 cells per cm2) densities. We report separation performance (minimum resolvable pI difference of 0.11) that is on par with capillary IEF and independent of cell density. Moreover, we map lamin A/C and β-tubulin protein expression to morphometric information (cell area, circumference, eccentricity, form factor, and cell area factor) of single cells and observe poor correlation with each of these parameters. By eliminating the need for cell detachment from substrates, we enhance detection of cell receptor proteins (CD44 and β-integrin) and dynamic phosphorylation events (pMLCS19) that are rendered undetectable or disrupted by enzymatic treatments. Finally, we optimise protein solubilisation and separation performance by tuning lysis and electrofocusing (EF) durations. We observe enhanced separation performance (decreased peak width) with longer EF durations by 25.1% and improved protein solubilisation with longer lysis durations. Overall, the combination of morphometric analyses of substrate-adhered cells, with minimised handling, will yield important insights into our understanding of adhesion-mediated signalling processes.

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Section: Resultsmentioning

confidence: 99%

Electrophoretic cytometry of adherent cells

Herr

2017

Lab Chip

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“…Here, CTC capture is based on cell size, cell shape, and deformability and is the agnostic of tumor biomarkers (Fig. Numerous workflows have been optimized for various CTC research applications, such as immunofluorescence staining and CTC enumeration (23)(24)(25)(26)29), cytopathology and cytogenetics (24), Sanger sequencing (26), next-generation sequencing (27,29), and even Western-blotting on single cells (28) (Fig. The Vortex chip has been characterized and validated for CTC isolation from blood samples of metastatic cancer patients with breast, colon, lung and prostate cancer (23)(24)(25)(26)(27)(28)(29).…”

Section: Vortex Technology For Label-free Ctc Isolationmentioning

confidence: 99%

VTX‐1 Liquid Biopsy System for Fully‐Automated and Label‐Free Isolation of Circulating Tumor Cells with Automated Enumeration by BioView Platform

Sollier‐Christen¹,

Renier²,

Kaplan³

et al. 2018

Cytometry Pt A

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Clinicians continue to rely on invasive tissue biopsies as a mean to assess a patient's disease and prescribe appropriate treatment regimens. Biopsies not only are risky and expensive but also limit the understanding of disease. Circulating tumor cells (CTCs) can be isolated from a simple blood draw and offer a promising potential to both diagnose and monitor cancer progression. The VTX-1 Liquid Biopsy System automates the isolation of clinically relevant CTC populations, while simplifying their collection for easy analysis, ultimately expanding the clinical possibilities for CTCs. We present here the key features and performance of this automated system for isolating CTCs directly from whole blood, both with cell spiking experiments and patient samples. As a first step toward the characterization of CTCs for research applications and transfer to clinical practice, we present workflows for both molecular analyses and automated cell enumeration and biomarker quantification with the BioView imaging platform.

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“…Similarly, using RNA probes that are conjugated to the magnetic nanoparticles, a quantification analysis of interested gene expression in CTCs could be achieved with high sensitivity and efficacy (Labib et al, 2018). In addition, novel technologies for analyzing the proteomics of CTCs, especially for the phosphorylation and proteolytic processing, were also under development (Sinkala et al, 2017). These techniques hold the potentials to reveal the dynamic phenotypes of CTCs that are required for the metastasis-seeding.…”

Section: Epithelial-to-mesenchymal Transition and Metastasismentioning

confidence: 99%

Metastatic tumor cells – genotypes and phenotypes

Gao

Mittal

et al. 2018

Front. Biol.

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BACKGROUND: Metastasis is the primary cause of mortality in cancer patients. Therefore, elucidating the genetics and epigenetics of metastatic tumor cells and the mechanisms by which tumor cells acquire metastatic properties constitute significant challenges in cancer research. OBJECTIVE: To summarize the current understandings of the specific genotype and phenotype of the metastatic tumor cells. METHOD and RESULT: In-depth genetic analysis of tumor cells, especially with advances in the next-generation sequencing, have revealed insights of the genotypes of metastatic tumor cells. Also, studies have shown that the cancer stem cell (CSC) and epithelial to mesenchymal transition (EMT) phenotypes are associated with the metastatic cascade. CONCLUSION: In this review, we will discuss recent advances in the field by focusing on the genomic instability and phenotypic dynamics of metastatic tumor cells.

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Profiling protein expression in circulating tumour cells using microfluidic western blotting

Cited by 213 publications

References 69 publications

Electrophoretic cytometry of adherent cells

Electrophoretic cytometry of adherent cells

VTX‐1 Liquid Biopsy System for Fully‐Automated and Label‐Free Isolation of Circulating Tumor Cells with Automated Enumeration by BioView Platform

Metastatic tumor cells – genotypes and phenotypes

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