Stable isotope tracing in human plasma-like medium reveals metabolic and immune modulation of the glioblastoma microenvironment
Background  In vivo stable isotope tracing is useful for natively surveying glioma metabolism but can be difficult to implement. Stable isotope tracing is tractable using in vitro glioma models, but most models lack nutrient conditions and cell populations relevant to human gliomas. This limits our ability to study glioma metabolism in the presence of an intact tumor microenvironment (TME) and immune-metabolic crosstalk.  Methods  We optimized an in vitro stable isotope tracing approach for human glioma explants and glioma stem-like cell (GSC) lines that integrates human plasma-like medium (HPLM). We performed 15N2-glutamine tracing in GSC monocultures and human IDH-wildtype glioblastoma explants and developed an analytical framework to evaluate microenvironment-dependent metabolic features that distinguish them. We also conducted spatial transcriptomics to assess transcriptional correlates to metabolic activities.  Results  Human plasma-like medium culture preserved glioma explant viability and stemness while unmasking metabolic and immune programs suppressed by conventional culture conditions. Stable isotope tracing in HPLM revealed TME-dependent and TME-independent features of tumor metabolism. Tissue explants recapitulated tumor cell-intrinsic metabolic activities, such as synthesis of immunomodulatory purines. Unlike GSC monocultures, tissue explants captured tumor cell-extrinsic activities associated with stromal cell metabolism, as exemplified by astrocytic guanosine diphosphate mannose production in heterocellular explants. Finally, glioma explants displayed tumor subtype-specific metabolic reprogramming, including robust pyrimidine degradation in mesenchymal cells.  Conclusions  We present a tractable approach to assess glioma metabolism in vitro under physiologic nutrient levels and in the presence of an intact TME. This platform opens new avenues to interrogate glioma metabolism and its interplay with the immune microenvironment. Overall design: We performed RNA sequencing on a panel of IDH mutant glioma stem-like cells, IDH WT glioma stem-like cells, and immortalized astrocytes. *************************************************************** The table below lists GEO accessions reused/reanalyzed for this study. ***************************************************************