Anti β4 integrin

For stem and EMT features evaluation, 4-μm tissue sections were stained according to a well-established protocol in a Bond-RXm immunostainer (Leica, Munich, Germany). The following panels of primary antibodies were used: (1) Anti-panCK (1:10, clone AE1/AE3, Roche, Indianapolis, IN, USA), Anti-EpCam (1:500, clone E144, Abcam, Cambridge, UK), Anti-E-cadherin (1:10, clone EP700Y, CellMarque, USA), Anti-N-Cadherin (1:500, clone EPR1791-4, Abcam, UK), Anti-β4 integrin (1:400, clone JM11-06, Thermo, Waltham, MA, USA); (2) Anti-panCK (1:10, clone AE1/AE3, Roche, Indianapolis, IN, USA), Anti-CD24 (1:100, clone SN3b, Thermo), Anti-CD44 (1:100, clone 156-3C11, Thermo, USA), Anti-ALDH1 (1:500, polyclonal, Abcam, UK), Anti-β4 integrin (1:400, clone JM11-06, Thermo, USA). A 7-color Opal Kit (NEL797B001KT; PerkinElmer, Waltham, MA, USA) containing Opal 520, Opal 540, Opal 570, Opal 620, Opal 650, and Opal 690 fluorophores was used. The obtained slides were embedded in Mounting Medium with DAPI (VECTOR Laboratories Inc., Newark, CA, USA). The slides were scanned using Vectra 3.0 system (PerkinElmer, USA). Images were processed using inForm 2.2.1 software (PerkinElmer, USA). The frequency and the number of tumor cells in different populations as a percent of all tumor cells were assessed.