Cell conditioning 1 cc1

Immunohistochemistry of the FFPE tumor samples was performed on a BenchMark Ultra autostainer (Ventana Medical Systems). Briefly, paraffin sections were cut at 3 µm, heated at 75°C for 28 min, and deparaffinized in the instrument with EZ prep solution (Ventana Medical Systems). Heat-induced antigen retrieval was carried out using Cell Conditioning 1 (CC1; Ventana Medical Systems) for 48 min at 95°C. PD-L1 clone 22C3 (DAKO) was detected using 1:40 dilution. Bound antibody was visualized using the OptiView DAB Detection Kit (Ventana Medical Systems). Slides were counterstained with hematoxylin II and bluing reagent (Ventana Medical Systems). After staining, slides were scanned with the P1000 (Sysmex). An experienced pathologist determined the tumor proportion score (TPS; the percentage of tumor cells with complete or partial membranous staining at any intensity) using Slide Score (https://www.slidescore.com). The TPS was classified as being <1%, 1–50%, >50%, or not evaluable (due to pigmentation or little to no tumor cells).

Hematoxylin and eosin were used for staining the tumor tissue section for morphological visualization. Furthermore tissues were fixed in 10% formalin. Deparaffinization was done with with EZ Prep (Ventana, Arizona, USA) at 70 °C, heat pretreated in Cell Conditioning 1 (CC1; Ventana, Arizona, USA) using “standard cell conditioning” for antigen reclamation at 90 °C. Overnight incubation with antibodies against β-catenin, cyclinA and cleaved caspase3 (diluted 1:75). Then incubation with ultraview universal HRP multimer (secondary antibody), diamminnobenzidine/ DAB (DAKO, CA) staining and counter staining with hematoxylin. After mounting in DPX (distyrene, a plasticizer and xylene), the sections were covered with cover slips. Immunostained tissue sections were assessed at magnification of 100× and 200× (Olympus BX51 light Microscope and DP72 Olympus Digital Camera, Olympus America Inc., Center Valley, PA, USA).

Immunohistochemical staining for FOLR1 distribution in cynomolgus monkey or human formalin-fixed, paraffin-embedded tissues was carried out on a Discovery XT automated slide stainer using a mouse anti-human monoclonal antibody for FOLR1 (Novocastra Clone BN3.2; Leica Biosystems, Wetzlar, Germany) at 15 µg/mL after antigen retrieval with Cell Conditioning 1 (CC1; Ventana Medical Systems Inc). As secondary Antibody was used a donkey anti-mouse biotinylated polyclonal IgG (Jackson Immunoresearch Lab, cat: 715-065-151) at 6 µg/mL. DAB Map Kit (Ventana 760–124) was used as a detection system. Xenograft tumors from FOLR1-expressing HeLa cells were used as a positive control.

We measured p53 levels by IHC using mouse monoclonal anti-human p53 protein antibody (DO7; Nichirei Biosciences Inc., Tokyo, Japan) and rabbit monoclonal anti-human PD-L1 protein antibody (SP263; Ventana Medical Systems, Inc., Tucson, AZ, USA). Five-micron-thick sections were obtained from formalin-fixed, paraffin-embedded tissues and set aside for p53 antibody (DO7) and PD-L1 (SP263) assay using a VENTANA OptiView DAB universal kit (Roche, Bazel, Switzerland) and VENTANA BenchMark ULTRA automated slide stainer (Roche, Bazel, Switzerland). Heat-induced antigen retrieval was performed using Cell Conditioning 1 (CC1; Ventana Medical Systems) for 32 min at 100 °C, followed by application of the primary antibody against p53 for 16 min at 36 °C, that of CC1 for 64 min at 100 °C, and of the primary antibody against PD-L1 for 16 min at 36 °C.
The IHC results were scored based on the percent positivity of staining. Protein expression of p53 and PD-L1 was evaluated by two pathologists as the percentage of staining area of all tumour cells. p53 status was determined by the percentage range of stained tumour cell nuclei. PD-L1 status was determined by the percentage of tumour cells with membrane staining above background.

Tissue stainings were performed using the Ventana Discovery platform. Sectioned paraffin embedded tissue samples were deparaffinized in EZ prep solution (Ventana) and stained with V5-tagged rVAR2 or anti-AR (N-20, Santa Cruz) antibody. In brief, tissue sections were incubated in Cell Conditioning 1 (CC1) or Cell Conditioning 2 (CC2) solution (Ventana) to retrieve antigen, followed by incubation with primary staining molecules. Bound rVAR2 was detected with an anti-V5 antibody, and an anti-mouse-HRP detection step. Bound antibodies were incubated with universal secondary antibody and visualized using Streptavidin-biotin peroxidase detection system and 3.3′-diaminobenzidine as chromogen.