Isolation of circulating tumor cells using CellCelector post CellSearch enables characterization of single cells

Context.— As circulating tumor cell (CTC) assays gain clinical relevance, it is essential to address preanalytic variability and to develop standard operating procedures for sample handling in order to successfully implement genomically informed, precision health care. Objective.— To evaluate the effects of blood collection tube (BCT) type and time-to-assay (TTA) on the enumeration and high-content characterization of CTCs by using the high-definition single-cell assay (HD-SCA). Design.— Blood samples of patients with early- and advanced-stage breast cancer were collected into cell-free DNA (CfDNA), EDTA, acid-citrate-dextrose solution, and heparin BCTs. Time-to-assay was evaluated at 24 and 72 hours, representing the fastest possible and more routine domestic shipping intervals, respectively. Results.— We detected the highest CTC levels and the lowest levels of negative events in CfDNA BCT at 24 hours. At 72 hours in this BCT, all CTC subpopulations were decreased with the larger effect observed in high-definition CTCs and cytokeratin-positive cells smaller than white blood cells. Overall cell retention was also optimal in CfDNA BCT at 24 hours. Whole-genome copy number variation profiles were generated from single cells isolated from all BCT types and TTAs. Cells from CfDNA BCT at 24-hour TTA exhibited the least noise. Conclusions.— Circulating tumor cells can be identified and characterized under a variety of collection, handling, and processing conditions, but the highest quality can be achieved with optimized conditions. We quantified performance differences of the HD-SCA for specific preanalytic variables that may be used as a guide to develop best practices for implementation into patient care and/or research biorepository processes.

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Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion

PLoS ONE ◽

10.1371/journal.pone.0169427 ◽

2017 ◽

Vol 12 (1) ◽

pp. e0169427 ◽

Cited By ~ 34

Author(s):

Sophie Laget ◽

Lucile Broncy ◽

Katia Hormigos ◽

Dalia M. Dhingra ◽

Fatima BenMohamed ◽

...

Keyword(s):

Early Detection ◽

Tumor Cells ◽

Molecular Characterization ◽

Circulating Tumor Cells ◽

Tumor Invasion ◽

Highly Sensitive

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Detection and Characterization of Circulating Tumor Cells by Quantitative Real-Time PCR

Methods in Molecular Biology - Quantitative Real-Time PCR ◽

10.1007/978-1-4939-9833-3_11 ◽

2019 ◽

pp. 139-151 ◽

Cited By ~ 1

Author(s):

Francesca Salvianti ◽

Filomena Costanza ◽

Gemma Sonnati ◽

Pamela Pinzani

Keyword(s):

Real Time ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Real Time Pcr ◽

Quantitative Real Time Pcr

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Molecular Detection and Characterization of Circulating Tumor Cells and Micrometastases in Solid Tumors

Surrogate Tissue Analysis ◽

10.1201/9781420038095.ch13 ◽

2005 ◽

pp. 203-228

Author(s):

Satyajit Bhattacharya ◽

Ronald Ghossein ◽

Hikmat Al-Ahmadie

Keyword(s):

Solid Tumors ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Molecular Detection

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A small molecule screen identified N-Acetyl-L-cysteine for promoting ex vivo growth of single circulating tumor cells

10.1101/2020.08.19.257378 ◽

2020 ◽

Author(s):

Teng Teng ◽

Mohamed Kamal ◽

Oihana Iriondo ◽

Yonatan Amzaleg ◽

Chunqiao Luo ◽

...

Keyword(s):

Tumor Cells ◽

Circulating Tumor Cells ◽

Small Molecule ◽

Ex Vivo ◽

Single Cells ◽

Small Subset ◽

Blood Draw ◽

Small Molecule Screen ◽

Functional Analyses ◽

Single Ctcs

AbstractCirculating tumor cells (CTCs) can be isolated via a minimally invasive blood draw and are considered a “liquid biopsy” of their originating solid tumors. CTCs contain a small subset of metastatic precursors that can form metastases in secondary organs, and provide a resource to identify mechanisms underlying metastasis-initiating properties. Despite technological advancements that allow for highly sensitive approaches of detection and isolation, CTCs are very rare and often present as single cells, posing an extreme challenge for ex vivo expansion after isolation. Here, using previously established patient-derived CTC lines, we performed a small molecule drug screening to identify compounds that can improve ex vivo culture efficiency for single CTCs. We found that N-acetylcysteine (NAC) and other antioxidants can promote ex vivo expansion of single CTCs, by reducing oxidative and other stress particularly at the initial stage of single cell expansion. RNA-seq analysis of growing clones and non-growing clones confirmed the effect by NAC, but also indicate that NAC-induced decrease in oxidative stress is insufficient for promoting proliferation of a subset of cells with heterogeneous quiescent and senescent features. Despite the challenge in expanding all CTCs, NAC treatment lead to establishment of single CTC clones that have similar tumorigenic features, which will facilitate future functional analyses.

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