Nbp1 76769

Tumor tissues or cells were lysed in RIPA buffer with a protease inhibitor cocktail. Protein was separated by SDS-PAGE gel and transferred to PVDF membrane (Millipore) for 90 min. The membrane was blocked for 60 min in 5% skim milk at room temperature. The membrane was briefly rinsed with 1xTBST and incubated with the respective primary antibodies at 4 °C overnight. Primary antibody of PD-L1 was purchased from NOVUS (NBP1-76769), and p-STAT3 (#9145), STAT3 (#9139), p-NF-κB (#3033), NF-κB (#8242), β-actin (#3700) and GAPDH (#5174) were purchased from CST. After incubation with the secondary antibodies, the protein bands were developed with the chemiluminescent reagents.
Media from adipocytes were collected and TNF-α and IL-6 levels were measured by using ELISA kits (Multi Sciences, EK282, EK260).

The following primary antibodies were used in the study including anti-CXCR4 (ab124824, AbCam), anti-TRAIL (AF1121, R&D), anti-c-Kit (AF1356, Novus Biological), anti-CD47 (ab175388, Abcam), anti-Fasl (ab15285, Abcam), anti-PDL1 (NBP1-76769, Novus Biological), anti-CD4 (ab183685, Abcam), anti-SSEA1 (ab16285, Abcam), anti-Oct4 (ab184665, Abcam), anti-Sox2 (MAB2018R-100, R&D), anti-Nanos3 (ab70001, Abcam), anti-Ifitm3 (ab109429, Abcam), anti-Stellar (Invitrogen, PA5-34601), anti-PRDM14 (ab187881, Abcam), anti-Vasa (ab27591, Abcam), anti-DAZL (NB100-2437, Novus biologicals), anti-SMAD2 (ab33875, Abcam).

The immunofluorescence staining was processed using a previously published method [28 (link)]. The organoids were fixed by adding 3.7% paraformaldehyde, 200 μL/well for 20 min at room temperature. huTGOs were then washed with 200 μL/well DPBS (Fisher Scientific) for 5 min at room temperature (RT). The organoids were permeabilized for 20 min at RT with 200 μL/well 0.5% Triton X-100 in PBS (PBST). The organoids were washed with 200 μL/well 0.01% PBST for 5 min at RT, followed by blocking for 1 h at RT with 2% normal donkey serum (Jackson Immuno Research, West Grove, PA, USA) diluted in 0.01% PBST, and the incubation with specific primary antibodies, diluted in 0.01% PBST, was at 4 °C for 16–18 h. The organoids were washed for 5 min at RT with 0.01% PBST and incubated under dark conditions for 1 h at RT with the secondary antibody and Hoechst (diluted 1:1000 in 0.01% PBST), diluted in 0.01% PBST. The organoids were then washed for 5 min at RT with 0.01% PBST and stored in DPBS. The primary antibodies used were rabbit anti-PD-L1 (Novus Biologicals, Littleton, CO, USA, NBP1-76769) reactive to human and mouse, and human-specific mouse anti-HER2 (Novus Biologicals, NBP2-01152) with a 1:100 dilution for both.

Sections of formalin‐fixed paraffin‐embedded tissues were prepared and stained with H&E. Immunohistochemistry (IHC) was performed on 4 μm tissue sections using a Ventana Bench Mark XT Autostainer (Ventana Medical Systems, Tucson, USA). PD‐L1 expression on tumor cells was analyzed by applying a modification of a previously used approach.15 Primary antibody against PD‐L1 (dilution 1:100; NBP1‐76769; Novus Biologicals, Littleton, USA) was used. PD‐L1 staining was evaluated by 2 independent pathologists with frequency (from 0% to 100%) and the intensity (negative or trace, weak, moderate, intense) per slide. PD‐L1 positivity was defined as PD‐L1 expression in at least 5% of tumor cells ranging from moderate to intense based on IHC staining by examining 5 representative high‐power fields under 200× magnifications.

HNSCC lines cells were harvested and lysed in CelLytic M Cell Lysis reagent (Sigma‐Aldrich, St. Louis, MO, USA) with protease and phosphatase inhibitor cocktails (Pierce Biotechnology, Rockford, USA). Protein concentrations were determined (Bio‐Rad, Munich, Germany). Standard Western blotting (WB) assay was used to analyze protein expression, as described previously. Briefly, immunostaining was detected with primary antibody to PD‐L1 (rabbit polyclonal, dilution 1:1000; NBP1‐76769; Novus Biologicals, Littleton, USA), anti‐β‐tubulin (mouse monoclonal, 1:1000; Abcam, Cambridge, UK) and anti‐rabbit IgG (1:10 000; Sigma‐Aldrich) or mouse IgG (1:10 000; Dako, Glostrup, Denmark) antibodies The immunoreactive signals were visualized by scanning densitometry with ChemiDoc^™ Touch Imaging System.

For immunohistochemistry analysis, 3 μm thick cross‐sections were deparaffinized and rehydrated, and then incubated at 4°C overnight with the following primary antibodies: anti‐CD3 (1:800; ab33429; Abcam), anti‐CD4 (1:800; ab203034; Abcam), anti‐CD8 (1:1000; ab217344; Abcam), anti‐CD68 (1:600; ab125212; Abcam), and anti‐FOXP3 (1:800; ab215206; Abcam). Then, the samples were stained with goat anti‐rabbit IgG/HRP (1:100; bs‐0295G‐HRP; Bioss) for 1 h at 37°C. Semi‐quantitative analysis of the inflammation characteristics in samples was performed using Image‐Pro Plus (IPP) 6.0 imaging software (Media Cybernetics). Immunofluorescence was used to detect the expression of PD‐L1 (20 μg/ml; NBP1‐76769; Novus Biologicals) in the grafts and the samples were examined using confocal laser scanning microscope (Zeiss LSM880).