Ventana automated staining system

Organoids were fixed in 4% paraformaldehyde (20 min at room temperature) before embedding (first in a 2% agar solution, and then in paraffin). Organoid sections (5-µm width), as well as sections from matching primary BC tissues, were prepared and stained for ER (#13258, Cell Signaling Technology), PR (#8757, Cell Signaling Technology), HER2 (#2165, Cell Signaling Technology) and Ki67 (#MA5-14520, Thermo Fisher Scientific) with a VENTANA automated staining system (Roche) at CUSL. Image acquisition was performed using an AxioImager Z1 microscope equipped with an Apotome1 (Zeiss).

TMA were generated from primary tumors of all patients in both cohorts, using an automated tissue microarrayer (VTA-100, Veridiam, Oceanside, CA, USA). Representative tumor-rich areas, previously annotated by a certified pathologist, were selected from the formalin-fixed paraffin-embedded (FFPE) patient tissue blocks. Each TMA consisted of cores with a diameter of 1 mm and included duplicate cores per tumor/patient. Tissue sections (thickness: 4 μm) were prepared from the TMA in cohort 1 for staining with primary monoclonal SP142 and SP263 PD-L1 antibody clones (Ventana, Roche, Basel, Switzerland) using Ventana automated staining system (Roche, Basel, Switzerland) according to the manufacturer’s protocol. Reactive lymphoid tissue of the tonsil was used as a control. PD-L1 was evaluated in both tumor and immune cells as previously described [12 (link)]. A core was considered as PD-L1-positive (PD-L1+) when at least one cell with membranous immunostaining was detected in at least one TMA core. TMA cores with poor tissue quality were excluded from the analysis.