Anti egfr

Cells were kept in culture for 72 hours. Cells collected for flow cytometry were washed with 1x PBS, trypsinised, and resuspended in PBS-2% BSA-0.05% Tween® 20. One million (1 x 10⁶) cells were used for each labelling. The primary antibodies used were: anti-CD49f-PE (BD, Pharmigen, Franklin Lakes, NJ, USA); anti-EpCAM, anti-MUC1, anti-EGFR, anti-N-cadherin, anti-E-cadherin, anti-CD66a/c/e (Biolegend, San Diego, CA, USA); anti-EpHB4 and anti-claudin-3 (R&D Systems, Inc., MN, USA); anti-vimentin, anti-claudin-4, anti-ALDH1, anti-FGFR; secondary antibodies were Alexa488 and Alexa647 (Abcam, Cambridge, UK). Unlabelled cells and with only secondary antibody labelling were included in each independent assay and considered autofluorescent. Dead cells and debris were excluded from the analysis. Cells were analyzed in a FACs-Aria flow cytometer (BD Bioscience).

Buffy coats obtained from AccuCyte diluted in 1 ml of media were stained with a cocktail of PE-CF594 conjugated antibodies against IM and a cocktail of AlexaFluor647 conjugated antibodies against CSMs for 30 min at 37 °C. The IM antibody cocktail included anti-CD45, anti-CD14 and anti-CD16 antibodies (BD Biosciences) at a dilution of 1 in 20. CSM antibody cocktail in spike in experiments is anti-EpCAM (Cell Signaling) at a dilution of 1 in 100, whereas in patients’ samples anti-HER2 (Biolegend) and anti-EGFR (Biolegend) antibodies were added to the cocktail. Cells were then transferred to 50 ml Falcon tubes and washed in 50 ml of CTC media. Tubes were centrifuged for 20 min at 200 g. After centrifugation, 25 ml of the supernatant was discarded carefully from each tube without disturbing cell pellets. An additional 25 ml of fresh CTC media was added, and tubes were centrifuged for an additional 5 min. All supernatant was removed except 24 ml of media at the bottom of each tube. Cells were pipetted up and down using 25 ml serological pipettes to obtain single cell suspensions. Cells were then plated on 4 two-well cyteslides (Rarecyte) at 3 ml/well and incubated for 30 min at 37 °C. After incubation, slides were scanned for IM⁻/CSM⁺ cells (CTCs).

Primary antibodies used in this study were anti-ADAM10 (abcam #ab1997), PE conjugated anti-ADAM10 (Biolegend #352704), anti-v-Src (Calbiochem #OP07), anti-Src family pY416 (CST #2101), anti-MAPK14 (CST #9212, anti-MAPK14 pT180/pY182 (CST #9211), anti-FAK (BD #610088), anti-FAK pY397 (Biosource #44-625G), anti-FAK pY576 (Santa Cruz #sc-16563), anti-FAK pS910 (Biosource #44-596G), anti-MAPK3/1 (CST #9102), anti-MAPK3/1 pT202/pY204 (CST #9106), anti-PAK2 (CST #2608), anti-PAK1/2 pS144/pS141 (CST #2606), anti-BCAR1 (Santa Cruz #sc-860), anti-BCAR1 pY249 (CST #4014), anti-Paxillin (Santa Cruz #sc-5574), anti-Paxillin pY118 (CST #2541), anti-Shc pY239/pY240 (CST # 2434), anti-Gab1 pY627 (CST #3231), PE-conjugated anti-E-cadherin (BioLegend #324106), anti-EGFR (Biolegend #352904), anti-Vinculin (Life Technologies #700062).
Protein extracts were separated by one-dimensional SDS gel electrophoresis using 4–15% TGX gradient gels (Biorad) and subsequently transferred on a PVDF membrane (Merck-Millipore). Membranes were blocked for 1.5 h in 5% (w/v) dried milk in PBS-0.05% (v/v) Tween-20 and probed with various phospho-site specific primary antibodies and their corresponding pan antibodies overnight at 4°C. Detection was performed using either IRDye680RD or IRDye800CW secondary antibodies (LI-COR) and the Odyssey infrared imaging system (LI-COR).