Vacutainer plus plastic serum tube

Blood samples derived from 22 NSCLC patients were collected using BD Vacutainer Plus Plastic Serum tubes (Becton Dickinson, NJ, USA). The tubes were centrifuged for 10 min at 1800 rpm, and patients’ sera were isolated and cryopreserved (−20 °C) until use. Blood withdrawals were carried out before Nivolumab treatment (T0) and after 6 cycles of therapy (>T0) corresponding to the first clinical evaluation (12 weeks). Four non-responding patients experienced disease progression within 3 months. Their blood samples were analyzed after 2 or 3 cycles of treatment.
Fecal samples derived from 11 patients were collected at baseline and stored at −20° before analysis.

Physicians performed thoracentesis and drainage pleural effusion. We collected the pleural effusion from the sterile bottle with a gas-tight syringe (SGE Syringes, Trajan, Victoria, Australia). We transferred the fluid to a 10-mL vacutainer tube without anticoagulant (BD Vacutainer Plus Plastic Serum Tubes, Becton Dickinson, Franklin Lakes, NJ, USA) to prevent contamination. The tubes were stored in a refrigerator to keep the temperature at 4 °C before centrifugation. The collected samples were sent to the laboratory and centrifuged within three hours. The pleural fluid was centrifuged at 1500× g for 10 min by a refrigerated centrifuge at 4 °C, designed for heat-sensitive samples (Centrifuge 5702R, Eppendorf, Hamburg, Germany). The supernatant was transferred into a new vacutainer without anticoagulant and then stored at − 80 °C until further analysis. To prevent contamination by environmental air, all procedures were performed in a closed system. We placed a stir bar into a 4-mL glass vial, sealed it with a Teflon/silicone septum, and then filled it with nitrogen. The pleural fluid samples were first thawed at 4 °C. Then, we used a gas-tight syringe to inject 2 mL of pleural fluid into the sealed 4-mL glass vial (Figure S3).

Blood was collected in a closed system Vacutainer^® CPT^™ Cell Preparation Tube anti-coagulated with sodium citrate (Becton, Dickinson and Company, Franklin Lakes, New Jersey). The plasma fraction was separated from cells and frozen at -80°C using a standard operating procedure implemented across all study sites. Blood was also collected in BD Vacutainer^® Plus plastic serum tubes (Becton, Dickinson and Company, Franklin Lakes, New Jersey, separated by centrifugation and serum frozen at -80°C for GM and BG testing. All samples and clinical data were de-identified and linked to an anonymous study ID number at the AsTeC Biorepository. For proteomics assays, each plasma sample was assayed for protein content, and a few randomly selected for cysteine content estimation by amino acid analysis (for quantitative cysteine-specific fluorescence-labeling). Plasma protein profiles were determined for all samples by capillary electrophoresis. For the 2D gel electrophoresis (2DE) and peptide analyses, specimens were randomized for proteomics analysis.

A physician performed thoracentesis and the drainage of pleural fluid. The pleural fluid was collected in a sterile bottle with a gas-tight syringe (SGE Syringes, Trajan, Victoria, Australia) and transferred to a 10 mL vacutainer tube without anticoagulant (BD Vacutainer Plus Plastic Serum Tubes, Becton Dickinson, Franklin Lakes, NJ, USA) to prevent contamination. The tubes were stored in a refrigerator to maintain the samples at 4 °C before centrifugation. The collected samples were sent to the laboratory and centrifuged within three hours. The pleural fluid was centrifuged at 1500× g for 10 min by a refrigerated centrifuge designed for heat-sensitive samples to maintain the samples at 4 °C (Centrifuges 5702R, Eppendorf, Hamburg, Germany). The supernatant was transferred into new vacutainers without anticoagulant and then stored at −80 °C until further analysis. To prevent contamination by environmental air, all procedures were performed in a closed system. We placed a stir bar into a 4 mL glass vial sealed with a Teflon/silicone septum and then filled the vial with nitrogen. The pleural fluid samples were initially thawed at 4 °C. Then, we used a gas-tight syringe to inject 2 mL of pleural fluid into the sealed 4 mL glass vial. All procedures were performed in a closed system to prevent contamination by environmental air.

Sampling of blood for serum analysis was performed by a certified veterinarian and thus conducted in agreement with the provisions enforced by the Norwegian Animal Research Authority. More details about the tested animals are in Additional file 1: Table S1. Blood was collected in 10 mL collection tubes (BD Vacutainer^® Plus Plastic Serum Tubes, Becton, Dickinson and Company, Franklin Lakes), using three tubes per animal. The samples were allowed to clot at room temperature for approximately 30 to 60 min, prior to centrifugation at 2000g (10 min, 4 °C). The serum was immediately transferred into a new tube and stored at −20 °C until analysis.