Abstract
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2461-4) contains supplementary material, which is available to authorized users.
Highlights
Circulating tumor cell (CTC)-filtration methods capture high numbers of Circulating tumor cells (CTCs) in non-small-cell lung cancer (NSCLC) and metastatic prostate cancer patients, and hold promise as a non-invasive technique for treatment selection and disease monitoring
Pailler et al BMC Cancer (2016) 16:477 platform (Janssen Diagnostics, LLC, Raritan, USA) enriches CTC candidates through ferromagnetic beads coated with epithelial cell adhesion molecule (EpCAM) specific antibody and defines CTCs according to the presence of cytokeratins (CK8, CK18 and CK19) and the absence of the common leukocyte antigen CD45 [5]
Using cancer cell lines spiked in normal blood and filtered by isolation by size of epithelial tumor cells (ISET), we developed a multistep process where filters were (i) treated by four-color fluorescent staining, (ii) scanned using an Ariol scanning system and automatically analyzed to select CD45− from CD45+ cells, (iii) treated by a cytomorphological dye, (iv) scanned and analyzed again using the Ariol scanning system to gather for cells of interest phenotypical and cytomorphological images, and cell characteristics (Fig. 1a)
Summary
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. We report the semi-automated microscopy method we developed to analyze filtration-enriched CTCs from NSCLC and mPCa patients. In non-small-cell lung cancer (NSCLC), CTC levels were reported to have a prognostic value, but the number of CTCs caught by the CellSearch remains low in this tumor type even in patients with metastatic advanced disease [9]. Performing molecular analysis in CTCs analyzed by the CellSearch is relatively challenging in cancers such as NSCLC due to the low numbers of CTCs captured and the processing conditions of the system
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