Background: Malignant ascites is a common complication of advanced ovarian and gastrointestinal cancer, significantly affecting metastasis, patient quality of life, and survival. In here, increased intestinal permeability cannot only result in blood or lymph infiltration but also microbial translocation from the gastrointestinal or uterine tract. This study aimed to discover microbiota-derived metabolites in ascites of ovarian cancer stages II-III, IV, and gastrointestinal cancer and to assess and discuss their potential roles in tumor progression and aggressiveness. Methods: In an explorative approach, this study analyzed malignant ascites samples from a total of 18 ovarian and gastrointestinal cancer patients. Using reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) coupled to trapped ion mobility time of flight mass spectrometry (timsTOF), we performed a fully untargeted 4D metabolomics approach. Additionally, a targeted flow cytometry-based cytokine panel was used to screen for inflammatory markers. Uni-and multivariate statistics were applied to identify significantly altered metabolites and lipids between cancer types and tumor stages. Non-endogenous, and thereof potentially microbiota-derived metabolites were identified using the Human Microbial Metabolome Database (MiMeDB). Results: Distinct metabolic differences were observed between ovarian cancer (OC) stages II-III and gastrointestinal cancer (GI) groups, while stage IV OC showed metabolic profiles similar to GI cancers. In GI patients, microbiota derived metabolites showed higher levels of 3-methylindole, 3-methylxanthine, caffeine, D-glucurono-6,3-lactone, D-tagatose, glucosamine, LPCs, and trimethylamine N-oxide, with lower levels of benzamide, phosphocholine, sphinganine, and thymol compared to OC. Stage IV patients exhibited elevated concentrations of 1-methylhistidine, 3-hydroxyanthranilic acid, 4-pyridoxic acid, biliverdin, butyryl-L-carnitine, indole, LPI 18:1, mevalonic acid, and phenol, with reduced levels of naringenin, o-cresol, and octadecanedioic acid compared to OC stage II-III. Correlation analysis with cytokines showed a positive correlation of glucosamine, D-tagatose, trimethylamine N-oxide, caffeine, LPC 22:4, and LPC 20:1 with IL-10, while benzamide and thymol negatively correlated. Furthermore benzyl alcohol, naringenin, and phenol correlated positively with MCP-1, while 1-methylhistidine, 4-pyridoxic acid, and mevalonic acid showed negative correlations. These altered metabolites can be mostly linked to lipid metabolism and inflammatory pathways. Conclusion: Dysregulated lipid metabolism plays a crucial role in ovarian cancer progression. Furthermore, 3-hydroxyanthranilic acid, indole, and naringenin indicate heightened inflammation and metabolic stress, serving as potential markers of disease progression. Deep metabolic phenotyping of ascites by timsTOF based 4D metabolomics elucidated the presence of microbiota-derived metabolites in ascites and distinct metabolic signatures in OC and GI. Involvement in typical tumor-related pathways pinpoints the relevance of these metabolites in the interaction between gut microbiota, the tumor microenvironment, and cancer biology and underscores the significant influence of microbiota in shaping malignant ascites.

The authors have declared no competing interest.

This study was funded by a research grant by Bruker Switzerland.

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This study was conducted in accordance with the principles outlined in the Declaration of Helsinki and approved by the Ethics Committee, Faculty of Medicine, University of Tuebingen, Germany (Ref. Nr. 696/2016BO2 and 117/2020BO1). Written informed consent was obtained from all participating patients. The collection of samples did not interfere with or alter patient treatment plans. All data were anonymized in compliance with the European General Data Protection Regulation (GDPR) and applicable German data protection laws.

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The data supporting this study are available from the corresponding author upon reasonable request.