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

BMC Cancer

Technical Advance

Nathalie Auger¹  Benjamin Besse²  Yohann Loriot²  Maud Ngo-Camus²  Jean-Charles Soria³  Corinne Laplace-Builhé⁴  Merouan Hemanda⁵  Alexandre Galland⁵  Colin R. Lindsay⁶  Marianne Oulhen⁶  Fanny Billiot⁶  Philippe Vielh⁷  Vincent Faugeroux⁸  Françoise Farace⁸  Emma Pailler⁸ 

[1] Department of Biopathology, Gustave Roussy, Université Paris-Saclay, Villejuif, France;Department of Medicine, Gustave Roussy, Université Paris-Saclay, Villejuif, France;INSERM U981 “Identification of Molecular Predictors and new Targets for Cancer Treatment”, F-94805, Villejuif, France;Department of Medicine, Gustave Roussy, Université Paris-Saclay, Villejuif, France;Imaging and Cytometry Platform AMMICA CNRS UMS3655–INSERM US23, Gustave Roussy, Université Paris-Saclay, Villejuif, France;Pathology Imaging, Leica Biosystems, F92737, Nanterre, France;“Circulating Tumor Cells” Translational Platform AMMICA CNRS UMS3655–INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France;INSERM U981 “Identification of Molecular Predictors and new Targets for Cancer Treatment”, F-94805, Villejuif, France;“Circulating Tumor Cells” Translational Platform AMMICA CNRS UMS3655–INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France;INSERM U981 “Identification of Molecular Predictors and new Targets for Cancer Treatment”, F-94805, Villejuif, France;Department of Biopathology, Gustave Roussy, Université Paris-Saclay, Villejuif, France;“Circulating Tumor Cells” Translational Platform AMMICA CNRS UMS3655–INSERM US23, Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France;INSERM U981 “Identification of Molecular Predictors and new Targets for Cancer Treatment”, F-94805, Villejuif, France;Univ Paris Sud, Université Paris-Saclay, F-94270, Le Kremlin-Bicêtre, France;

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

DOI  :  10.1186/s12885-016-2461-4

 received in 2016-01-12, accepted in 2016-06-27,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundCirculating 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.MethodsSpiked 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.ResultsTwo 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 μm2. 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.ConclusionsThe 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.

【 授权许可】

CC BY   
© The Author(s). 2016