TY - JOUR
T1 - Method for semi-automated microscopy of filtration-enriched circulating tumor cells
AU - Pailler, Emma
AU - Oulhen, Marianne
AU - Billiot, Fanny
AU - Galland, Alexandre
AU - Auger, Nathalie
AU - Faugeroux, Vincent
AU - Laplace-Builhé, Corinne
AU - Besse, Benjamin
AU - Loriot, Yohann
AU - Ngo-Camus, Maud
AU - Hemanda, Merouan
AU - Lindsay, Colin R.
AU - Soria, Jean Charles
AU - Vielh, Philippe
AU - Farace, Françoise
N1 - Publisher Copyright:
© 2016 The Author(s).
PY - 2016/7/14
Y1 - 2016/7/14
N2 - 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 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. 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.
AB - 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 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. 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.
KW - Circulating tumor cells
KW - FA-FISH
KW - Filtration enrichment
KW - Fluorescent staining
KW - Predictive biomarkers
UR - http://www.scopus.com/inward/record.url?scp=84987700978&partnerID=8YFLogxK
U2 - 10.1186/s12885-016-2461-4
DO - 10.1186/s12885-016-2461-4
M3 - Article
C2 - 27417942
AN - SCOPUS:84987700978
SN - 1471-2407
VL - 16
JO - BMC Cancer
JF - BMC Cancer
IS - 1
M1 - 477
ER -