Precision xceed system

Plasma interleukin-6 levels were analyzed using a mouse cytometric bead array inflammation kit (BD Biosciences, San Diego, CA, USA) on a FACSCalibur flow cytometer affixed with a 488-nm laser (BD Immunocytometry Systems, San Jose, CA, USA), according to the manufacturer’s protocol. Plasma PGE₂ levels were determined using commercially available purification kits and enzyme-linked immunosorbant assay kits (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer’s protocols. Blood β-hydroxybutyrate (βHB) levels were measured enzymatically on day 21 using the Precision Xceed system (Abbott, Tokyo, Japan) to evaluate the production of ketone bodies and to investigate the correlation between ketone bodies and anti-tumor efficacy.

The in vivo experiments were performed in accordance with the Salzburg Animal Care and Use Committee (Study approval no. 20901-TVG/112/6-2018). Animals were maintained under specific pathogen-free conditions and care conformed to the Austria Act on Animal Experimentation. Mice had ad libitum access to water and chow. A total of 1 × 107 cells in 200 µL of a 1:1 mixture of matrigel (Corning, New York, NY, USA) and serum-free medium were subcutaneously injected into the right flank of 5- to 7-week-old female CD-1 nude mice (n = 10–13) (Charles River, Sulzfeld, Germany). Control animals (n = 9) were kept tumor-free. Blood glucose and beta-hydroxybutyrate levels were monitored over time (Precision Xceed System, Abbott, Chicago, IL, USA). For metabolomics analysis, blood was taken by cardiac puncture and collected in MiniCollect^® Lithium Heparin tubes (Greiner Bio-One, Kremsmünster, Austria) and centrifuged for 3 min at 2000 g. Plasma samples were stored at −80 °C until metabolomics analysis. Tumor size at the day of collection is shown in Figure S16. When tumors were harvested, one half of the tumor was snap frozen in liquid nitrogen for metabolomics analysis while the other half was formalin-fixed and paraffin-embedded (FFPE) for histological analysis.