Cellbrowser software

Peripheral blood (7.5 mL) was collected into CellSave^® tubes and EpCAM-positive CTCs were captured and enumerated by the FDA-cleared CellSearch^TM platform (Menarini Silicon Biosystems Inc., Huntingdon Valley, PA, USA) following the manufacturer guidelines, according to the CTC definition by CellSearch (EpCAM+/CK+/DAPI+/CD45− cells). CTC enumeration of blood from NSG mice was performed using 7.0 mL of blood from healthy donors spiked with 500 µL of mouse blood using the CellSearch Circulating Epithelial Cell Kit (Menarini Silicon Biosystems, Inc.). Fluorescent CTCs were imaged and enumerated by the automated CellBrowser software (Menarini Silicon Biosystems, Inc.) according to the manufacturer guidelines.

Peripheral blood mononuclear cells (PBMCs) were isolated using red blood cell lysis buffer (154 mm NH₄Cl, 10 mm KHCO₃, and 0.1 mm EDTA) either from patient blood (8 mL), CDX blood (500‐800 μL), or BM (Vishnoi et al., 2018). PBMCs were stained with antibodies for respective markers and loaded to FACSAria III flow cytometer (BD Biosciences, San Jose, CA, USA) for multiparametric selection. All sorted‐cell population were collected in 10% Dulbecco's modified Eagle's medium‐F12 media + 90% 1× PBS (both from Gibco, Dún Laoghaire, Dublin, Ireland) for downstream interrogation. FACS data were analyzed by software flowjo ver 10 (Ashland, OR, USA), LLC. In addition, CTC enumeration by CellSearch™ (Menarini Silicon Biosystems) was performed using mice blood (500 μL) spiked with blood (7 mL) from healthy donors, according to the user‐defined CellSearch™ platform and CellSearch™ Circulating Epithelial Cell Kit [Menarini Silicon Biosystems (Huntingdon Valley, PA, USA); the definition of CTC by Federal Drug Administration (FDA)‐cleared CellSearch™ platform is epithelial cell adhesion molecule (EpCAM)+/cytokeratin (CK)+/DAPI+ but CD45‐ cell]. Fluorescent CTC images along with DAPI were scanned and enumerated by an automated CellBrowser™ software (Menarini Silicon Biosystems), following the manufacturer's guidelines.

Circulating cell subgroups created during the DEPArray analysis were: Epithelial cells (E) characterized by nuclear positivity in blue (HOECHST 33342+) and a green signal (FITC+) specific for epithelial markers; Mesenchymal cells (M) characterized by nuclear positivity in blue (HOECHST 33342+) and by a red signal (PE+) specific for mesenchymal markers; Epithelial–Mesenchymal Cells (EM) characterized by blue nuclear positivity (HOECHST 33342+) and the simultaneous presence of a red signal (PE+) for mesenchymal markers and a green one (FITC+) for the epithelial ones; Lymphocytes (L) characterized by nuclear positivity (HOECHST 33342+) in blue and a blue signal (APC+) specific for CD45, sometimes by a mesenchymal red signal (PE+) and Negative cells (N) characterized by only the nuclear positivity in blue.
Cells of interest were selected using the CellBrowser Software (Menarini Silicon Biosystems), and sorted individually. Parameters provided by CellBrowser were morphological features such as: such as diameter, circularity, OV circularity, perimeter and fluorescence intensities for each channel (mean fluorescence intensity, max intensity, mean intensity without background) of each single cell found. All raw data were exported from the instrument and elaborated through bioinformatic tools.