Anti epcam pe

Cells were exfoliated from culture dish by accutase (Innovative Cell Technology) and filtered through 40μm cell strainers (Greiner) to obtain single cells. The ALDH1^high cells were isolated from MDA-MB157 and MDA-MB468 cells by ALDEFLUOR assay kit (Stem Cell Technology) or AldeRed ALDH detection assay kit (MERCK) according to the manufacturer's instructions. Briefly, cells (2×10⁶) were incubated with the substrate for ALDH1 (5μL substrate/mL medium) for 30 min at 37°C. As a negative control for the ALDEFLUOR assay and AldeRed assay, cells were incubated with ALDH1 inhibitor, diethylaminobenzaldehyde (DEAB). The ALDH1^high cells were sorted by cell sorter (FACS AriaII, BD Bioscience) by taking the negative control into consideration. The analysis of CD10/EpCAM positive cells from MDA-MB157 and MDA-MB468 cells. Suspended MDA-MB468 cells (1×10⁶) were incubated with anti-CD10 (APC) (BD Bioscience) and anti-EpCAM (PE) (BD Bioscience) for 1hr on ice, after which the sample was washed with fresh FACS buffer (2%FBS in 1×PBS (-)). For this experiments, cells were analyzed using a FACS Calibur (BD Bioscience).

Single cells from patient P1 were stained with Sytox Blue viability dye (S34857, Life Technologies) and processed on a FACS Aria I instrument. Cells from P2–P16 were stained with anti-EPCAM-PE (347198, BD Biosciences; 1:50 dilution in ice-cold PBS containing 2% FBS) for 30 min with gentle rotation at 4 °C. Mouse lung single cells were similarly stained but with a cocktail of antibodies (1:250 each) against CD45-PE/Cy7 (103114, BioLegend), ICAM2-A647 (A15452, Life Technologies), EPCAM-BV421 (118225, BioLegend) and ECAD-A488 (53-3249-80, eBioscience). Stained cells were then washed, filtered using 40 μm filters, stained with Sytox Blue (human) or Sytox Green (mouse) and processed on a FACS Aria I instrument (gating strategies for epithelial cell sorting are shown in Supplementary Figs. 1 and 4 for human and mouse cells, respectively). Doublets and dead cells were eliminated, and viable (Sytox-negative) epithelial singlets were collected in PBS containing 2% FBS. Cells were washed again to eliminate ambient RNA, and a sample was taken for counting by Trypan Blue exclusion before loading on 10X Genomics Chromium microfluidic chips.

Cells were analyzed with a BD FACS CantoII
(BD Bioscience, San Jose, CA) or imaged by the Amnis ImageStream
Mk II image flow cytometer (Millipore, Burlington, MA) and analyzed
by Ideas software. For CTC identification and WBC exclusion, acoustophoresis
isolated cells were stained with a fluorescent antibody cocktail;
anti-EpCAM-PE (BD biosciences) and anti-panCytokeratin (CK)-Alexa
Fluor 488 (Thermo Fisher, Gothenburg Sweden), identifying CTCs, and
anti-CD45-APC (BD biosciences) and anti-CD66b-Alexa Fluor 647 (BD
biosciences) to distinguish WBCs. DAPI (Sigma-Aldrich) identified
intact cells.

Cells were analyzed with a BD FACS CantoII or imaged by the image flow cytometer Amnis ImageStream Mk II (Millipore, Burlington, MA) and analyzed by the Ideas software. For CTC identification and WBC exclusion, acoustophoresis isolated cells were stained with a fluorescent antibody cocktail; anti-EpCAM-PE (BD biosciences) and anti-panCytokeratin (CK)-Alexa Fluor 488 (Thermo Fisher, Gothenburg Sweden), identifying CTCs, and anti-CD45-APC (BD biosciences) and antiCD66b-Alexa Fluor 647 (BD biosciences) to distinguish WBCs. DAPI (Sigma-Aldrich) identified intact cells.