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The production of proangiogenic factors by NK cells was studied in 8 HCC patients (paired LINK and TINK) after overnight stimulation with IL-12 and IL-18 (5 ng/mL) in the presence of 10 µg/mL of brefeldin A (BFA). Surface staining with anti-CD3-BUV805, anti-CD56-BUV805, and anti-CD49a-BV510 (BD Bioscience, Franklin Lakes, NJ, USA) was performed. Cells were then fixed and permeabilized with Fixation Permeabilization Concentrate and Diluent and Permeabilization Buffer (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions and stained with Eomes-PE-Cy7 (Invitrogen), Angiopoietin 1 (ANGPT1)-Alexa Fluor750 (Bioss Antibodies, Boston, MA, USA), CXCL10/IP-10-Alexa Fluor 700 (R&D System, Minneapolis, MN, USA), Osteopontin-eFluor660 (Thermo Fisher Scientific, Waltham, MA, USA), IL-8-PerCp-eFluor710 (Thermo Fisher Scientific, Waltham, MA, USA), MMP-9-Alexa Fluor 488 (Abcam, Cambridge, UK), Placental growth Factor (PlGF)-Alexa Fluor594 (Bioss Antibodies, Boston, MA, USA), VEGF-PE (R&D system, Minneapolis, MN, USA), and Angiogenin-Alexa Fluor405 (Novus Biotechne, R&D System, Minneapolis, MN, USA). Analysis was performed on an 18-fluorescence flow cytometer (FACS Fortessa, Becton Dickinson (BD) Immunocytometry System, CA, USA). Data are expressed as the percentage of cytokine-positive cells in the CD49a+Eomes+ NK cell subset.

Freshly resected lung tissue samples were cut into small pieces and digested for 1 h at 37 °C using the tumor dissociation kit (Milteniy Biotech, Bergisch Gladbach, Germany) and the gentleMACS Dissociator (Milteniy Biotech). Digested samples were filtered through a 100 µm cell strainer, washed, and red blood cell lysis (Gibco, Waltham, MA, USA) was performed. CD45⁺ immune cells were positively sorted using CD45 (TIL) microbeads (Milteniy Biotech) according to manufacturer recommendations. Recovered cells were stained for 30 min with fluorochrome-labelled primary antibodies in PBS 1% HS, 1% FCS at 4 °C. Cell viability was ascertained by labeling with fixable viability dyes (eBioscience, San Diego, CA, USA). For intracellular staining, cells were washed in PBS and next fixed in PFA 4% for 10 min at room temperature and permeabilized with PBS 1%, FCS 1%, HS 0.1% saponin (permeabilization buffer) for 10 min. Cells were next incubated with the following antibodies VEGF-PE (R&D systems) and OPN-FITC (R&D systems, Minneapolis, MI, USA) in permeabilization buffer for 45 min at RT. Cells were next washed in PBS and proceeded to flow cytometry analysis. Stained cells were acquired using a Fortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo V10.4.2 software (TreeStar, Antwerp, Belgium).