Exoquick serum exosome precipitation solution

Exosomes were isolated from serum samples using differential centrifugation and two commercial kits: ExoQuick Serum Exosome Precipitation Solution (System Biosciences, Palo Alto, CA, USA) and exoEasy kit (Qiagen GmbH, Hilden, Germany). Serum samples were centrifuged at 2,000×g for 10 min and 10,000×g for 30 min at 4 °C to thoroughly remove cellular debris. Next, supernatants were filtered through a 0.2 μm pore size filter to exclude particles > 200 nm in diameter. For exosome isolation by differential ultracentrifugation, the filtered supernatant was collected into ultracentrifuge tubes and centrifuged in an Optima MAX-XP ultracentrifuge with an MLA-130 rotor (Beckman Coulter, Jersey City, NJ, USA) at 100,000 g for 1 h at 4 °C. The pellets were washed with PBS, ultracentrifuged again, and resuspended in PBS. For exosome isolation by ExoQuick and exoEasy, the two commercial kits were used according to the manufacturers’ instructions. Exosome preparations were conserved at − 80 °C for later use.

H&E stains were prepared on 4 µm sections from the FFPE tissue blocks. Pathology review was undertaken to identify tumor and benign regions of interest (ROIs). Coring tools of 0.6 mm diameter were used to punch the ROI for subsequent RNA isolation. We implemented the Qiagen AllPrep DNA/RNA FFPE Kit (Qiagen, USA—cat# 80234) following the manual’s instructions for RNA extraction. TRIzol Reagent (Life Technologies, Inc., USA—cat# 15596-026) was utilized in the isolation of RNA from the matched plasma specimens, as per the manufacturer’s instructions. Exosomes were isolated with the ExoQuick serum exosome precipitation solution (Systems Biosciences, Inc., USA—cat# EXOQ5A-1) with the addition of pacific hemostasis thromboplastin D to remove coagulating material, as per the manufacturer’s instructions. The exosome solution was then subjected to TRIzol RNA isolation, as described for plasma. RNA isolates were quantified utilizing the Quant-iT RiboGreen assay (Life Technologies, Inc., USA—cat# R11490) over the concentration range 1–100 ng/µl.