DOI: 10.5061/dryad.dncjsxm77
title: Metabolomic analysis of cultured TRAMP-C2 cells in the presence or absence of PD-L1 expression

description: 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.
description: 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.
description: # Metabolomic analysis of cultured TRAMP-C2 cells in the presence or
 absence of PD-L1 expression
 [https://doi.org/10.5061/dryad.dncjsxm77](https://doi.org/10.5061/dryad.dncjsxm77) Here, we analyzed the metabolome of the murine prostate cancer cell line TRAMP-C2, either in the wild-type state or with PD-L1 expression deleted via CRISPR/Cas9. Previous literature has suggested that PD-L1 can act as a metabolic regulator, in particular regulation of glycolysis. We performed this metabolomic study to determine if PD-L1 is regulating the cancer cell metabolome, thus causing differences in cell signaling and viral infection observed in our manuscript. ## Description of the data and file structure The data are provided in an Excel sheet with 2 tabs: one with the metabolite quantification data from the cell lysates, and the other from the culture media that the cells were grown in. Each row is a distinct metabolite, 186 of which were quantified in this experiment. Each column is a different sample. Both wild-type (WT) and PD-L1-deleted (KO) samples were measured, and for both types of cells, the lysate (Lys) and culture media (CM or Media) were analyzed. Different dilution factors of the metabolite samples were used to ensure that all metabolites were within the standard curves during LC/MS (UnDiluted, 1in10, 1in100). 6 biological replicates were performed for all samples (distinguished by \_01, \_02, etc. in the Excel sheet). Lastly, cell culture media that was not exposed to cells was also analyzed (MediaOnly) to determine consumption vs. production of media metabolites by the cells.  All values are in micromolar. In the "Media" tab, the data end at column AO; in the "Lysate" tab, the data end at column AL. These columns and empty cells are indicated by N/A (not applicable). ## Sharing/Access information This data is summarized in the bioRxiv preprint (link in Related Works section) and will be published in a manuscript in the Journal of Experimental Medicine.