==== Front bioRxiv BIORXIV bioRxiv Cold Spring Harbor Laboratory 10.1101/2023.05.29.542774 preprint 1 Article Human plasma-like medium facilitates metabolic tracing and enables upregulation of immune signaling pathways in glioblastoma explants Shami Mohamad El http://orcid.org/0000-0001-7345-4247 Savani Milan R http://orcid.org/0000-0003-1776-451X Gattie Lauren C http://orcid.org/0009-0002-9437-0610 Smith Bailey Hicks William H http://orcid.org/0000-0002-2689-3199 Rich Jeremy N Richardson Timothy E McBrayer Samuel K http://orcid.org/0000-0001-9361-675X Abdullah Kalil G http://orcid.org/0000-0002-5662-0829 31 5 2023 2023.05.29.542774http://biorxiv.org/lookup/doi/10.1101/2023.05.29.542774 nihpp-2023.05.29.542774.pdf Abstract Purpose Metabolism within the tumor microenvironment (TME) represents an increasing area of interest to understand glioma initiation and progression. Stable isotope tracing is a technique critical to the study of tumor metabolism. Cell culture models of this disease are not routinely cultured under physiologically relevant nutrient conditions and do not retain cellular heterogeneity present in the parental TME. Moreover, in vivo, stable isotope tracing in intracranial glioma xenografts, the gold standard for metabolic investigation, is time consuming and technically challenging. To provide insights into glioma metabolism in the presence of an intact TME, we performed stable isotope tracing analysis of patient-derived, heterocellular Surgically eXplanted Organoid (SXO) glioma models in human plasma-like medium (HPLM). Methods Glioma SXOs were established and cultured in conventional media or transitioned to HPLM. We evaluated SXO cytoarchitecture and histology, then performed spatial transcriptomic profiling to identify cellular populations and differential gene expression patterns. We performed stable isotope tracing with 15 N 2 -glutamine to evaluate intracellular metabolite labeling patterns. Results Glioma SXOs cultured in HPLM retain cytoarchitecture and cellular constituents. Immune cells in HPLM-cultured SXOs demonstrated increased transcription of immune-related signatures, including innate immune, adaptive immune, and cytokine signaling programs. 15 N isotope enrichment from glutamine was observed in metabolites from diverse pathways, and labeling patterns were stable over time. Conclusion To enable ex vivo, tractable investigations of whole tumor metabolism, we developed an approach to conduct stable isotope tracing in glioma SXOs cultured under physiologically relevant nutrient conditions. Under these conditions, SXOs maintained viability, composition, and metabolic activity while exhibiting increased immune-related transcriptional programs. ==== Body pmc