Autostainer system

Tissue microarrays (TMAs) were constructed using duplicate tumor cores from primary tumors and an automated tissue microarrayer (VTA-100, Veridiam, San Diego, CA, USA). Tissue sections from the TMAs were used for PD-L1 immunohistochemistry (IHC) using the Ventana autostainer system according to manufacturer’s protocols. Positivity was defined by the presence of any single cell with membranous expression of PD-L1 either in tumor or in immune cells (total cells). In addition, whole tissue sections (4 μm) were prepared for a subset of patients in this cohort based on the expression of PD-L1 in tumor cells (positive and randomly selected negative cases) and stained using an anti-phosphorylated STAT3 (pSTAT3) antibody (Y705). At least 300 tumor cells were counted in five different high power fields in order to calculate the percentage of pSTAT3 positive cells. The median percentage of expression was used as a cut-off for dividing patient tumors in pSTAT3-high and pSTAT3-low expressing ones. Furthermore, cell pellets from cell lines were collected, fixed in formalin and embedded in paraffin to prepare cell blocks. Subsequently, IHC was performed using anti-pSTAT3 and anti-PD-L1 antibodies, as previously described [41 (link)]. The antibodies used are listed in Table S2. Moreover, Ki67 immunohistochemical staining and evaluation method have been previously described [42 (link)].

Immunocytochemistry (ICC) analyses using different antibodies (listed in Table 5) were performed on the Dako Autostainer system or the Ventana Benchmark automated staining system according to the manufacturers’ recommendations. A polymer linked to a peroxidase system with DAB was used to amplify and visualize the signal. ICC results were classified as being either not contributing for the diagnosis, eliminating metastases of secondary tumours, differentiating Benign/LG-M from HG-M or providing the definitive diagnosis.