Tryptophan metabolism is inversely regulated in the tumor and blood of patients with glioblastoma.

Tryptophan (Trp)-catabolic enzymes (TCEs) produce metabolites that activate the aryl hydrocarbon receptor (AHR) and promote tumor progression and immunosuppression in glioblastoma. As therapies targeting TCEs or AHR become available, a better understanding of Trp metabolism is required. Methods: The combination of LC-MS/MS with chemical isobaric labeling enabled the simultaneous quantitative comparison of Trp and its amino group-bearing metabolites in multiple samples. We applied this method to the sera of a cohort of 43 recurrent glioblastoma patients and 43 age- and sex-matched healthy controls. Tumor volumes were measured in MRI data using an artificial neural network-based approach. MALDI MSI visualized Trp and its direct metabolite N-formylkynurenine (FK) in glioblastoma tissue. Analysis of scRNA-seq data was used to detect the presence of Trp metabolism and AHR activity in different cell types in glioblastoma. Results: Compared to healthy controls, glioblastoma patients showed decreased serum Trp levels. Surprisingly, the levels of Trp metabolites were also reduced. The decrease became smaller with more enzymatic steps between Trp and its metabolites, suggesting that Trp availability controls the levels of its systemic metabolites. High tumor volume associated with low systemic metabolite levels and low systemic kynurenine levels associated with worse overall survival. MALDI MSI demonstrated heterogeneity of Trp catabolism across glioblastoma tissues. Analysis of scRNA-seq data revealed that genes involved in Trp metabolism were expressed in almost all the cell types in glioblastoma and that most cell types, in particular macrophages and T cells, exhibited AHR activation. Moreover, high AHR activity associated with reduced overall survival in the glioblastoma TCGA dataset. Conclusion: The novel techniques we developed could support the identification of patients that may benefit from therapies targeting TCEs or AHR activation.

New Derivatization Reagent for Detection of free Thiol-groups in Metabolites and Proteins in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Several diseases are associated with disturbed redox signaling and altered metabolism of sulfur-containing metabolites and proteins. Importantly, oxidative degradation of fresh-frozen tissues begins within the normal time scale of MALDI MSI sample preparation. As a result, analytical methods that preserve the redox state of the tissue are urgently needed for refined studies of the underlying mechanisms. Nevertheless, no derivatization strategy for free sulfhydryl groups in tissue is known for MALDI MSI. Here, we report the first derivatization reagent, (E)-2-cyano-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-(4-hydroxyphenyl)acrylamide (CHC-Mal), for selective detection of free thiols using MALDI MSI. We performed in situ derivatization of free thiol groups from thiol-containing metabolites such as glutathione and cysteine and reduced proteins such as insulin and imaged their spatial distribution in porcine and mouse xenograft tissue. Derivatization of thiol-containing metabolites with CHC-Mal for MALDI MSI was also possible when using aged tissue in the presence of excess reducing agents. Importantly, CHC-Mal-derivatized low mass-metabolites could be detected without the use of a conventional MALDI matrix.