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BMC Cancer. 2016 Jul 14;16:477. doi: 10.1186/s12885-016-2461-4.

Method for semi-automated microscopy of filtration-enriched circulating tumor cells.

Author information

1
"Circulating Tumor Cells" Translational Platform AMMICA CNRS UMS3655-INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France.
2
INSERM U981 "Identification of Molecular Predictors and new Targets for Cancer Treatment", F-94805, Villejuif, France.
3
Univ Paris Sud, Université Paris-Saclay, F-94270, Le Kremlin-Bicêtre, France.
4
Pathology Imaging, Leica Biosystems, F92737, Nanterre, France.
5
Department of Biopathology, Gustave Roussy, Université Paris-Saclay, Villejuif, France.
6
Imaging and Cytometry Platform AMMICA CNRS UMS3655-INSERM US23, Gustave Roussy, Université Paris-Saclay, Villejuif, France.
7
Department of Medicine, Gustave Roussy, Université Paris-Saclay, Villejuif, France.
8
"Circulating Tumor Cells" Translational Platform AMMICA CNRS UMS3655-INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France. francoise.farace@gustaveroussy.fr.
9
INSERM U981 "Identification of Molecular Predictors and new Targets for Cancer Treatment", F-94805, Villejuif, France. francoise.farace@gustaveroussy.fr.
10
Univ Paris Sud, Université Paris-Saclay, F-94270, Le Kremlin-Bicêtre, France. francoise.farace@gustaveroussy.fr.

Abstract

BACKGROUND:

Circulating tumor cell (CTC)-filtration methods capture high numbers of CTCs in non-small-cell lung cancer (NSCLC) and metastatic prostate cancer (mPCa) patients, and hold promise as a non-invasive technique for treatment selection and disease monitoring. However filters have drawbacks that make the automation of microscopy challenging. We report the semi-automated microscopy method we developed to analyze filtration-enriched CTCs from NSCLC and mPCa patients.

METHODS:

Spiked cell lines in normal blood and CTCs were enriched by ISET (isolation by size of epithelial tumor cells). Fluorescent staining was carried out using epithelial (pan-cytokeratins, EpCAM), mesenchymal (vimentin, N-cadherin), leukocyte (CD45) markers and DAPI. Cytomorphological staining was carried out with Mayer-Hemalun or Diff-Quik. ALK-, ROS1-, ERG-rearrangement were detected by filter-adapted-FISH (FA-FISH). Microscopy was carried out using an Ariol scanner.

RESULTS:

Two combined assays were developed. The first assay sequentially combined four-color fluorescent staining, scanning, automated selection of CD45(-) cells, cytomorphological staining, then scanning and analysis of CD45(-) cell phenotypical and cytomorphological characteristics. CD45(-) cell selection was based on DAPI and CD45 intensity, and a nuclear area >55 μm(2). The second assay sequentially combined fluorescent staining, automated selection of CD45(-) cells, FISH scanning on CD45(-) cells, then analysis of CD45(-) cell FISH signals. Specific scanning parameters were developed to deal with the uneven surface of filters and CTC characteristics. Thirty z-stacks spaced 0.6 μm apart were defined as the optimal setting, scanning 82 %, 91 %, and 95 % of CTCs in ALK-, ROS1-, and ERG-rearranged patients respectively. A multi-exposure protocol consisting of three separate exposure times for green and red fluorochromes was optimized to analyze the intensity, size and thickness of FISH signals.

CONCLUSIONS:

The semi-automated microscopy method reported here increases the feasibility and reliability of filtration-enriched CTC assays and can help progress towards their validation and translation to the clinic.

KEYWORDS:

Circulating tumor cells; FA-FISH; Filtration enrichment; Fluorescent staining; Predictive biomarkers

PMID:
27417942
PMCID:
PMC4946105
DOI:
10.1186/s12885-016-2461-4
[Indexed for MEDLINE]
Free PMC Article

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