Sensitive and easy screening for circulating tumor cells by flow cytometry
Alexia Lopresti, Fabrice Malergue, François Bertucci, Maria Lucia Liberatoscioli, Severine Garnier, Quentin DaCosta, Pascal Finetti, Marine Gilabert, Jean Luc Raoul, Daniel Birnbaum, Claire Acquaviva, Emilie Mamessier
Alexia Lopresti, Fabrice Malergue, François Bertucci, Maria Lucia Liberatoscioli, Severine Garnier, Quentin DaCosta, Pascal Finetti, Marine Gilabert, Jean Luc Raoul, Daniel Birnbaum, Claire Acquaviva, Emilie Mamessier
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Resource and Technical Advance Oncology

Sensitive and easy screening for circulating tumor cells by flow cytometry

Abstract

Circulating tumor cells (CTCs) provide easy, repeatable, and representative access to information regarding solid tumors. However, their detection remains difficult because of their paucity, their short half-life, and the lack of reliable surface biomarkers. Flow cytometry (FC) is a fast, sensitive, and affordable technique, ideal for rare-cell detection. Adapted to CTC detection (i.e., extremely rare cells), most FC-based techniques require a time-consuming pre-enrichment step, followed by a 2-hour staining procedure, impeding the efficiency of CTC detection. We overcame these caveats and reduced the procedure to less than 1 hour, with minimal manipulation. First, cells were simultaneously fixed, permeabilized, and then stained. Second, using low-speed FC acquisition conditions and 2 discriminators (cell size and pan-cytokeratin expression), we suppressed the pre-enrichment step. Applied to blood from donors with or without known malignant diseases, this protocol ensures a high recovery of the cells of interest independently of their epithelial-mesenchymal plasticity and can predict which samples are derived from cancer donors. This proof-of-concept study lays the bases of a sensitive tool to detect CTCs from a small amount of blood upstream of in-depth analyses.

Authors

Alexia Lopresti, Fabrice Malergue, François Bertucci, Maria Lucia Liberatoscioli, Severine Garnier, Quentin DaCosta, Pascal Finetti, Marine Gilabert, Jean Luc Raoul, Daniel Birnbaum, Claire Acquaviva, Emilie Mamessier

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Figure 2

Principle of our gating strategy to select rare cells of epithelial origin from blood.

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Principle of our gating strategy to select rare cells of epithelial orig...
The strategy was applied using the MDA-MB-231 cell line that expresses both epithelial and mesenchymal markers. This experiment was reproduced 3 times. After applying a double threshold based on cell size and pan-cytokeratin (pan-KRT) staining, gates were defined to progressively refine the selection. (A) A first gate was used to select for events with a size larger than most monocytes and granulocytes. These events were plotted against DAPI to select for nucleated cells only. Debris was removed with the “Cleaning zone 1.” (B) As cells can form small clusters, including with CTCs, a wide range of DAPI intensities was used. (C) Remaining contaminating CD45+ cells were excluded (Cleaning zone 2). The pan-KRT+ population of interest was then plotted according to VIM-PE and APC Alexa 750 (APC AF750) values. (D) All events that fluoresce in this empty channel should be considered contamination (Cleaning zone 3), as these events displayed nonspecific staining. (E) Cells negative to positive for VIM were selected, then analyzed for their respective expression of VIM and EPCAM (EPCAM+VIMlo/dim and EPCAM–/loVIMdim/hi). Double-negative residual events were excluded as potential false-positive events (Cleaning zone 4). We have indicated the potential CTCs in 2 gates which allow the identification of EPCAM+VIMlo/hi population and EPCAM–/dimVIMhi. (F) Finally, a last readjustment based on the FSC parameter, which was already used to preselect the cells in the beginning, was made to eliminate cells of small size and with low granularity (Cleaning zone 5).