Simple SummaryCardiolipin is an important mitochondrial lipid for organelle and cellular energy production. Cardiolipin has been found in altered abundance and diversity in glioma xenograft models. Here, we utilize mass spectrometry imaging to map cardiolipin alterations in human normal and astrocytoma tumors in the histologically diverse tumor microenvironment. Longer chain cardiolipin species were detected at significantly lower relative abundance in tumor tissues versus normal cortex. Cardiolipin diversity correlated with histological trends in the tumor microenvironment including tumor cell invasion and tumor viability. The expression level of proteins involved in mitochondrial energy production was found to decrease with increasing tumor grade among human glioma tumors, though the major enzyme involved in cardiolipin synthesis was not found to be differentially expressed. This work provides confirmation of cardiolipin alterations in human astrocytomas and provides rationale for a spatially aware approach when considering cardiolipin diversity in the human tumor microenvironment.AbstractCardiolipin (CL) is a mitochondrial lipid with diverse roles in cellular respiration, signaling, and organelle membrane structure. CL content and composition are essential for proper mitochondrial function. Deranged mitochondrial energy production and signaling are key components of glial cell cancers and altered CL molecular species have been observed in mouse brain glial cell xenograft tumors. The objective of this study was to describe CL structural diversity trends in human astrocytoma tumors of varying grades and correlate these trends with histological regions within the heterogeneous astrocytoma microenvironment. To this aim, we applied desorption electrospray ionization coupled with high field asymmetric ion mobility mass spectrometry (DESI-FAIMS-MS) to map CL molecular species in human normal cortex (N = 29), lower-grade astrocytoma (N = 19), and glioblastoma (N = 28) tissues. With this platform, we detected 46 CL species and 12 monolysocardiolipin species from normal cortex samples. CL profiles detected from glioblastoma tissues lacked diversity and abundance of longer chain polyunsaturated fatty acid containing CL species when compared to CL detected from normal and lower-grade tumors. CL profiles correlated with trends in tumor viability and tumor infiltration. Structural characterization of the CL species by tandem MS experiments revealed differences in fatty acid and double bond isomer composition among astrocytoma tissues compared with normal cortex and glioblastoma tissues. The GlioVis platform was used to analyze astrocytoma gene expression data from the CGGA dataset. Decreased expression of several mitochondrial respiratory enzyme encoding-genes was observed for higher-grade versus lower-grade tumors, however no significant difference was observed for cardiolipin synthesis enzyme CRLS1.