Abstract 3645: Quantifying cell populations in CTC enriched samples with the open-source imaging program ACCEPT
Authors: Wit Sanne De , Zeune Leonie L. , Hiltermann T. Jeroen N. , Groen Harry J. , Terstappen Leon W.M. 
Published: August 2018

Abstract
Introduction.
Composition of cell suspensions enriched for Circulating Tumor Cells (CTC) by the CellSearch system has not yet been explored as the system only presents DAPI+/Cytokeratin+ objects to the operator to identify CTC. We used ACCEPT (http://github.com/LeonieZ/ACCEPT) to identify and characterize all DAPI+ nuclei in EpCAM enriched samples and explored avenues to increase the number of nuclei that can be assigned to a cell lineage. Addition of CD16, expressed on granulocytes and NK-cells, the detection of a cellular plasma membrane and the use of LED light, as opposed to the mercury arc lamp, to improve the excitation of CD45-APC were tested.
Methods.
In 127 controls and 300 samples from 192 metastatic lung cancer patients, CD16-PerCP was added to the CellSearch CTC identification antibody cocktail. A CellTracks Analyzer equipped with six filter cubes was used to acquire the images. For exploration, after the CellTracks image acquisition 5 cartridges were scanned again on a microscope that contains a LED light source to determine if this would improve the detection of CD45-APC. Also, wheat germ agglutinin (wga) conjugated to AlexaFluor488 stains the cellular plasma membrane and was added to the CellSearch antibody cocktail to discriminate between bare nuclei and unstained cells in 4 samples. The stack of fluorescence images was analyzed with the open source imaging program ACCEPT to define and identify various cell populations.
Results.
In 427 samples 26,285 ±26,590 [603-156,519] (mean, SD, [range]) nuclei were identified with ACCEPT in the CellSearch images of which only 14% ±15 were identified as leukocytes or CTC, remaining 86% ±15 cells unidentified. Adding CD16 increased the leukocyte population by 47% ±17, thereby reducing the unidentified population to 39% ±19 of the total number of nuclei. CD16-staining was found on 13% of the CTC. Using a LED-light source improved the identification of APC+ cells, and reduced the amount of unidentified nuclei in these samples from 29% (±5) to 14% (±10). Exploration with wga indicated that 43% (±11) (mean 987 cells, range 331-2,638 cells) of the unstained cells contained a cellular membrane.
Conclusion.
In 427 blood samples run on CellSearch, only 14% of the nuclei obtained after EpCAM enrichment could be assigned as a leukocyte or a CTC. By adding CD16 to the antibody cocktail, the unidentified population is reduced from 86% to 39%. Also, 13% of nuclei scored as CTC express CD16, suggesting these are granulocytes that are incorrectly designated as tumor cells. Using a LED light source improves the identification of dimly stained CD45+ cells. Exploration with wga indicates that 57% of the unidentified nuclei are simply bare nuclei and the origin of 43% still remains unknown. Further studies are needed to determine whether EpCAM+/Cytokeratin- CTC are present among the nucleated cells of which the origin is not yet known.
Citation Format: Sanne De Wit, Leonie L. Zeune, T. Jeroen N. Hiltermann, Harry J. Groen, Leon W.M. Terstappen. Quantifying cell populations in CTC enriched samples with the open-source imaging program ACCEPT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3645.