Serpina1

Immunohistochemical (IHC) staining was performed incubating 4 µm FFPE sections in 10 mmol/L citrate buffer (pH 6.0) at 120°C for 5 minutes for antigen retrieval. Endogenous peroxidase was neutralized using EnVision FLEX Peroxidase‐Blocking Reagent (Dako) for 10 minutes. Tissue sections were blocked with 3% bovine serum albumin and incubated with the primary antibodies overnight at 4°C. Primary antibodies used were FRMD6 (Sigma‐Aldrich), ZEB1 (Sigma‐Aldrich), HTR2B (Sigma‐Aldrich), AE1AE3 (Thermo Scientific), CDX2 (Novus Biologicals), SRSF3 (Abcam) and SERPINA1 (Sigma‐Aldrich). After incubation with the EnVision FLEX + mouse or rabbit linker (Dako), EnVision FLEX/HRP (Dako) was used as the secondary antibody for 1 hour at room temperature, followed by 3,3'‐diaminobenzidine (DAB) staining (Dako). CMS molecular classification was performed according to Thrin et al,14 analysing the intensity and content of FRMD6, ZEB1, HTR2B, AE1AE3 and CDX2. SRSF3 and SERPINA1 tumoral epithelial expression was categorized as high (intense staining) and low expression (moderate and negative staining). Individual cores were scored by trained pathologists (CVP and SGL).

Proteins (20 μg) were separated on a Criterion^™ TGX Stain‐Free 4%‐20% acrylamide gel in the Bio‐Rad Criterion System and transferred to PVDF membranes (Bio‐Rad Laboratories). After blocking with 5% non‐fat milk, membranes were incubated with the specific primary antibodies followed by incubation with secondary antibody conjugated with horseradish peroxidase and detection was performed with chemiluminescent reaction with the Clarity Western ECL Substrate (Bio‐Rad). Images were captured on a ChemiDoc XRS Imaging System (Bio‐Rad). Stain‐free technology was used as protein loading control, and densitometric analysis was performed with Image Lab software (Bio‐Rad). Antibodies used were as follows: SPP1 (OPN), HDAC2, SERPINA1 and RPS27L from Sigma‐Aldrich, and SFRS3 from Abcam. Secondary antibodies conjugated with HRP were from Santa Cruz Biotechnology.