Multiplex immunofluorescence was used for the evaluation of epithelial and mesenchymal markers in tumour cell in primary lesions. Multiplex IF was performed by using the Bond RXm staining system (Leica Microsystems, GmbH, Wetzlar, Germany). The following antibody-Opal pair and dilutions were used: Anti-EpCAM-Opal 520 (1:1000, clone E144, Abcam, Cambridge, UK), Anti-N-cadherin-Opal 650 (1:500, clone EPR1791-4, Abcam, Cambridge, UK), and Anti-CK7-Opal 690 (1:10, OV-TL 12/30, Agilent, Santa-Clara, CA, USA). The slides were counterstained with DAPI for 5 min and mounted with VECTASHIELD Hard Set (Vector Labs, Burlingame, CA, USA). All primary antibodies were optimized using control tissues. Slides were scanned using the PerkinElmer Vectra (Vectra 3.0.3; PerkinElmer, Waltham, MA, USA). Tissue imaging and analysis were performed using inForm Advanced Image Analysis software (inForm 2.2.1; PerkinElmer, Waltham, MA, USA) according to the recommendations [24 (link)].
The frequency and count of the following cell populations were scored: EpCAM+CK7–N-cadherin–; EpCAM+CK7–N-cadherin+; and EpCAM+CK7+N-cadherin–; EpCAM+CK7+N-cadherin+; and EpCAM–CK7+N-cadherin–; EpCAM–CK7+N-cadherin+.
The frequency and count of the following cell populations were scored: EpCAM+CK7–N-cadherin–; EpCAM+CK7–N-cadherin+; and EpCAM+CK7+N-cadherin–; EpCAM+CK7+N-cadherin+; and EpCAM–CK7+N-cadherin–; EpCAM–CK7+N-cadherin+.