Metabolomic analysis of cultured TRAMP-C2 cells in the presence or absence of PD-L1 expression

The interaction between the immune inhibitory receptor PD-1 and its ligand PD-L1 is a critical mechanism for altering immune responses, especially during chronic antigen exposures such as cancer. While much research has focused on the PD-1 receptor, recent evidence suggests that PD-L1 can have cell-intrinsic effects in cancer and immune cells. These functions are distinct from its ability to bind and trigger PD-1 activity and are notable given that PD-L1 is widely expressed in mammals. One such cell-intrinsic function is the modulation of cellular metabolism, including regulation of mTOR activity and glycolysis. As part of our investigation into PD-L1 function, we analyzed the metabolome of cultured mouse prostate cancer cells (TRAMP-C2) expressing PD-L1 or with PD-L1 deleted via CRISPR/Cas9. We quantified 186 water-soluble metabolites from TRAMP-C2 cells expressing PD-L1 or not to better understand what metabolic pathways and processes are regulated by PD-L1 expression/activity. We found a broad range of differentially abundant metabolites, most notably a decreased abundance of glycolytic metabolites when PD-L1 expression is knocked out. In our manuscript, we show that this has a functional outcome on viral infection and cytokine signaling.

Methods
TRAMP-C2 cells were cultured in standard conditions. Cells were placed on ice and the culture media was removed. Cells were washed twice with ice-cold PBS and scraped into chilled 2 mL tubes, and frozen until metabolite extraction. Samples were mixed with 230 uL of 1:1 methanol:water, along with 6 washed 1.4 mm ceramic beads. Samples were vortexed for 10 s and cell lysis was done by beating for 60 s at 2000 rpm (bead beating was done twice) after adding 220 µL of acetonitrile. Samples were then incubated with a 2:1 dichloromethane:water solution on ice for 10 minutes. The polar and non-polar phases were separated by centrifugation at 4000g for 10 minutes at 1°C. The upper polar phase was dried using a refrigerated CentriVap Vacuum Concentrator at -4°C. Samples were resuspended in water.

Samples were resuspended in water and run on an Agilent 6470A tandem quadruple mass spectrometer equipped with a 1290 Infinity II ultra-high performance LC (Agilent Technologies) utilizing the Metabolomics Dynamic MRM Database and Method (Agilent), which uses an ion-pairing reverse phase chromatography (Reference: ‘The Agilent Metabolomics Dynamic MRM Database and Method’). This method was further optimized for phosphate-containing metabolites with the addition of a 5 µM InfinityLab deactivator (Agilent) to mobile phases A and B, which requires decreasing the backflush acetonitrile to 90%. Multiple reaction monitoring (MRM) transitions were optimized using authentic standards and quality control samples. Metabolites were quantified by integrating the area under the curve of each compound using external standard calibration curves with Mass Hunter Quant (Agilent). No corrections for ion suppression or enhancement were performed, as such, uncorrected metabolite concentrations are presented.