Ventana benchmark discovery

Co-staining of CD31 and CD146 with was performed by automated multiplex IF using the Ventana Benchmark Discovery (Ventana Medical Systems Inc., Arizona, USA). In brief, following deparaffinization and heat-induced antigen retrieval with CC1 (#950-500, Ventana) for 32 min the tissue samples were incubated firstly with anti-CD31 for 32 min at 37 °C followed by detection with Red610 (#760-245, Ventana). Antibody denature step was performed using CC2 (#950-123, Ventana) for 8 min at 100 °C. Secondly, anti-CD146 was incubated for 32 min at 37 °C followed by detection with FAM (#760-243, Ventana). Slides were washed in DPBS and covered with DAPI in vectashield. Antibody information and clonality can be found in Supplementary Table 2. All slides used in this project were randomly picked before imaging, and orientation of the organoids was undetermined after processing.

Triplex staining for keratin 5, keratin7 and p63 was done by automated multiplex IF using the Ventana Benchmark Discovery (Ventana Medical Systems Inc.). In brief, following deparaffinization and heat-induced antigen retrieval with CC1 (#950-500, Ventana) for 64 min at 97 °C, the tissue samples were incubated firstly with Keratin 5 antibodies for 32 min at 37 °C followed by detection with Ultramap anti-rabbit HRP (#760–4315, Ventana) for 12 min followed by visualization with Red610 for 8 min (#760-245, Ventana). Antibody denaturing was performed using CC2 (#950-123, Ventana) for 20 min at 100 °C. Secondly, Keratin 7 antibodies were incubated for 32 min at 37 °C followed by detection with Ultramap anti-rabbit HRP (#760–4315, Ventana) followed by visualization with FAM (#760-243, Ventana) for 4 min. Antibody denaturing was performed using CC2 (#950-123, Ventana) for 8 min at 100 °C. Thirdly, P63 antibodies were incubated for 32 min at 37 °C followed by detection with Ultramap anti-mouse HRP (#760–4313, Ventana) for 12 min followed by visualization with Cy5 for 12 min (#760-238, Ventana). Slides were incubated in PBS with DAPI for 15 min and covered with anti-fading medium (DAKO, S3023).

Sections of 4µm of FFPE samples were used for immunohistochemistry analysis. To visualize M1 and M2 macrophage subsets in placental tissue, the chromogenic duplex staining was done with CD68 and CD163 by an automated staining procedure using the Ventana Benchmark Discovery (Ventana Medical Systems Inc.). In brief, following deparaffinization and heat-induced antigen retrieval with CC1 (#950-500, Ventana) for 40 minutes at 95°C, the tissue samples were firstly incubated with CD68 for 60 minutes at 37˚C, followed by detection with anti-mouse HQ (#760-4814, Ventana) for 16 minutes and subsequently anti-HQ HRP (#760-4820), and visualized with Discovery Purple (#760-229, Ventana) for 32 minutes. An antibody denature step was performed using CC2 (#950-123, Ventana) for 20 minutes at 100˚C. Secondly, CD163 was incubated for 32 minutes at 37˚C, followed by detection with anti-mouse HQ and subsequently anti-HQ HRP, and visualized with Discovery Teal (#760-247, Ventana) for 32 minutes. Finally, all slides were counterstained with hematoxylin II (#760-2208) and bluing reagents (#760-2037) for 4 minutes. Antibody information and clonality can be found in Supplementary Table S3.

All 22 tissue samples were used to validate the significantly expressed proteins by performing multiplex Immunohistochemistry (IHC). For the immune cell types, tissue sections of 5 μm were stained with CD163, CD14, PanCk antibodies and with syto13 (DNA) nuclear stating and scanned using the GeoMx DSP instrument. Additionally, all 22 samples were used for the conventional IHC to validate the expression of VISTA and IDO1 using Alkaline phosphatase, according to the manufacturer's standard protocol. The stained slides were scored and interpreted by a pathologist. VISTA was shown to be significantly highly expressed in the BM-LUAD group. In order to identify the specific cells that express VISTA in BM-LUAD samples, we performed multiplex immunofluorescence (IF) staining using 2 independent BM-LUAD samples by following the automated protocol using the Ventana Benchmark Discovery (Ventana Medical Systems Inc). The process of staining was carried out using a previously published method [31 (link)]. A summary of all antibodies is shown in Additional file 1: Table S3.

Liver tissue biopsies (n = 4) were collected during liver transplantation and fixed in 4% PFA for 24 h, followed by dehydration, paraffin embedding, and sectioning according to standard procedures. Automated multiplex immunofluorescent staining was further performed using the Ventana Benchmark Discovery (Ventana Medical Systems Inc.). For the novel series, following deparaffinization and heat-induced antigen retrieval with CC1 (#950-500, Ventana) for 64 min at 97 °C, the tissue samples were incubated with primary antibodies at 37 °C in a step-by-step manner. The antibody denature step was performed between every antibody incubation and visualization using CC2 (#950-123, Ventana) for 20 min at 100 °C. Keratin 19 (KRT19) was incubated for 32 min, detected with Universal HQ kit (#760-275, Ventana), and visualized with Red610 (#760-245, Ventana) for 4 min. TGF-β was incubated for 32 min, detected with Universal HQ kit, and visualized with R6G (#760-244, Ventana) for 4 min. N-cadherin was incubated for 32 min, detected with omnimap anti-mouse HRP, and visualized with FAM for 4 min. Slides were incubated in PBS with DAPI for 15 min and covered with an anti-fading medium (DAKO, S3023). Slides were scanned using the ZEISS Axio Imager 2.0.
Information on the antibodies used is listed in Table S3.

To determine the identity of the PD‐L1‐positive cells, triple stains were performed by automated multiplex immunofluorescence using the Ventana Benchmark Discovery (Ventana Medical Systems Inc.). In brief, following deparaffinization and heat‐induced antigen retrieval with CC1 (#950‐500, Ventana) for 64 min at 97°C, the tissue samples were incubated firstly with antibodies against either CD45, gastrin, chromogranin‐A, or somatostatin for 32 min at 37°C followed by detection with either Ultramap anti‐rabbit HRP (#760‐4315, Ventana) or Ultramap anti‐mouse HRP (#760‐4313, Ventana) for 12 min, followed by visualization with Red610 for 8 min (#760‐245, Ventana). Antibody denaturing was performed using CC2 (#950‐123, Ventana) for 20 min at 100°C. Secondly, PDL1 SP263 was incubated for 32 min at 37°C followed by detection with Ultramap anti‐rabbit HRP (#760‐4315, Ventana), followed by visualization with FAM (#760‐243, Ventana) for 4 min. Slides were incubated in PBS with DAPI for 15 min and covered with an anti‐fading medium (DAKO, S3023). Antibody information can be found in Table S1.