Abstract 3645: Quantifying cell populations in CTC enriched samples with the open-source imaging program ACCEPT

Abstract

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.