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 fou..., 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 Metabolomi..., , # 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 ...