Fixable viability stain 780

At the end of the culture, WB samples (500 μl) were stained with an 18-color flow cytometry panel of monoclonal antibodies (mAbs) as described below. Briefly, samples were incubated with ammonium chloride for 10 min to lyse erythrocytes. After washing, they were stained with the Fixable Viability Stain 780 (BD Biosciences), then washed and stained with the combination of mAbs listed in Table 2. mAbs specific for lineage markers, HLA-DR, CD123, CD11c, CD1c, CD141, CD1a, M-DC8 (anti-slan) were used to gate on DC subsets, while the other mAbs were used to assess the immunostimulatory/regulatory phenotype of each DC subset. Staining conditions for each mAb were preliminarily determined in titration assays, as previously described (35 (link)). All operations were done at 4°C in the dark.

The cell apoptosis state of MCF‐7 cells on scaffolds was carried out by an Annexin V‐FITC/PI assay kit (Neobioscience, China). The cells were collected and stained with Annexin V‐FITC and PI in binding buffer. For evaluating HIF‐1α expression, cells were treated by 2% formaldehyde, 0.1% Triton X‐100, and anti‐HIF‐1α (Cell signaling technology, USA) for 30 min away from light. For immune cells analysis, a single cell suspension (100 µL, 5×10⁷ cells mL⁻¹) of dissociated tumor tissue was seeded on the scaffolds and cultured for 48 and 72 h. The cancer cells were harvested, rinsed with PBS, and resuspended. Then, the collected cells with Fixable viability stain 780, PerCP‐Cy5.5 Rat Antimouse CD45, FITC Hamster Antimouse CD3e, APC Rat Antimouse CD4, and PE Rat Antimouse CD8a (BD Biosciences, USA) were incubated at 4 °C for 30 min away from light. The tests were carried out on a FACS Celesta flow cytometer (BD Biosciences, USA) and calculated the data with FlowJo 10.

Epithelial organoids or fibroblasts derived from a total of 13 biopsies were prepared for FACS as described [6 (link)]. To isolate CD271 (nerve growth factor receptor)^low/mucin 1 (MUC1)^high, luminal epithelial cells, suspended cells from organoids were incubated for 30 minutes at 4 °C in the presence of CD271-APC (ME20.4, 1:50, Cedarlane Laboratories) and MUC1 (115D8, 1:50, Monosan) followed by AF488 (IgG2b, 1:500, Life Technologies). Fibroblasts were incubated with CD105-AF488 (SN6, 1:25, AbD Serotec) and CD26 (202–36, 1:200, Abcam), followed by AF647 (IgG2b, 1:500). Controls were without primary antibody. 1 μg/ml propidium iodide (Invitrogen) or Fixable Viability Stain 780 (1:1000, BD Biosciences) was added 10 minutes prior to analysis and sorting (FACSAria I and II; BD Biosciences).

Fresh mouse liver tissues were harvested, minced, and digested into single cell with mouse liver dissociation kits (Miltenyi Biotech) according to the manufacturer's instructions. Lysis buffer (BD Pharmingen, NJ, USA) was used to remove red blood cells. Fixable Viability Stain 780 (BD Pharmingen) was used to exclude the dead cells. Later, cells were blocked with the anti‐CD16/32 antibody (clone 2.4G2) for 10 min, and then incubated with fluorescently conjugated mAbs against mouse CD45, Ly6C, CD11b, and F4/80 (Biolegend, San Diego, CA) for 30 min at 4°C in the dark. Cells were detected by BD FACS Aria II and analyzed with FlowJo software. Macrophages in the liver tissue were characterized by the gating strategy CD45⁺Ly6C⁺CD11b⁺F4/80⁺.

To detect the expression of Tims in various T-cell subpopulations, PBMCs were surface stained with the following anti-human mAbs: CD3 Pacific Blue, CD4 FITC, CD8 PerCP-Cy5.5, Tim-1 PE and Tim-3 BB515 (BioLegend, San Diego, CA, USA). Dead cells were excluded from analysis by staining with the Fixable Viability Stain 780 (BD, Franklin lakes, NJ, USA) and analysed using FlowJo Software X (Tree Star, Ashland, OR, USA). The gating strategy used for T-cell subsets is shown in Figure S1.

HMNCs, PBMCs and cultured NK cells were stained with fluorescently labeled monoclonal antibodies to determine the phenotypic differences between hepatic and peripheral NK cells. The following antibodies were used: CD45 (HI30) BV510, CD3 (UCHT1) BV421 or Pacific Blue, CD56 (NCAM16.2) BV650 or APC, CD16 (3G8) PE-Cy7, CXCR6 PE-CF594. Intracellular staining was performed using FoxP3 staining buffer (00-5523-00, eBiosciences, San Diego, CA, USA) and the following antibodies were used: Eomes (WD1928) AF488 and T-bet (ebio4B10) PE (BioLegend). Dead cell exclusion was carried out using fixable viability stain 780 (BD Biosciences). Flow cytometric analysis was carried out using an LSR Fortessa (BD Biosciences) or a FACS Canto II (BD Biosciences) and data was analyzed using FlowJo (Version 7.6.5, Tree Star, Ashland, OR, USA).

For analyzing tumor-infiltrating immune cells, tumors were weighed, minced, and incubated with digestion buffer containing HBSS (Sigma-Aldrich), 5 mM CaCl₂, 40 U/ml DNase I (BIOTOOLS), 0.5 mg/ml type I collagenase (Thermo Fisher Scientific), 0.5 mg/ml type IV collagenase (Thermo Fisher Scientific), and 0.2 mg/ml hyaluronidase (MedChemExpress) for 20 min at 37 °C. The dissociated tumor cells were passed through a 100 μm cell strainer (BD) and incubated with ammonium chloride potassium (ACK) lysis buffer (MyBioSource). Dead cells were excluded using Fixable Viability Stain 780 (BD). After washing three times with PBS containing 2% inactivated FBS, cell suspensions were blocked with an anti-CD16/32 antibody (BioLegend) and stained with various antibodies to immune cell markers for 30 min on ice, followed by cell fixation and permeabilization using the Transcription Factor Buffer Set (BD) for 1 h on ice and then intracellular staining with the desired antibody for 30 min on ice. Flow cytometry analysis was performed using a Thermo Fisher Scientific Attune NxT Flow Cytometers and analyzed with FlowJo v10.8.1 software. For analyzing cell cycle profiles, DAPI-stained cells were subjected to flow cytometry with a Thermo Fisher Scientific Attune NxT Flow Cytometers together with FlowJo v8 software. For both experiments, Attune NxT software (v 4.2.0) was used for data collection.