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Targeted Microchip Capillary Electrophoresis-Orbitrap Mass Spectrometry Metabolomics to Monitor Ovarian Cancer Progression (calibration standards)

ABSTRACT: The lack of effective screening strategies for high-grade serous carcinoma (HGSC), a subtype of ovarian cancer (OC) responsible for 80% of OC related deaths, emphasizes the need for new diagnostic markers and a better understanding of disease pathogenesis. Capillary electrophoresis (CE) coupled with high-resolution mass spectrometry (HRMS) offers high selectivity and sensitivity, thereby increasing metabolite coverage and consequently enhancing biomarker discovery. Recent advances in CE-MS include small, chip-based CE systems coupled with nanoelectrospray ionization (nanoESI) to provide rapid, high-resolution analysis of biological specimens. Here, we describe the development of a targeted microchip (µ) CE-HRMS method to analyze 40 target metabolites in serum samples from a triple-mutant (TKO) mouse model of HGSC, with an acquisition time of only 3 min. Extracted ion electropherograms showed sharp, highly resolved peak shapes, even for structural isomers such as leucine and isoleucine. All analytes maintained good linearity with an average R2 of 0.994, while detection limits were in the nM range. Thirty metabolites were detected in mice serum, with recoveries ranging from 78 to 120 %, indicating minimal ionization suppression and good accuracy. We applied the µCE-HRMS method to sequentially-collected serum samples from TKO and TKO-control mice. Time-resolved analysis revealed characteristic temporal trends for amino acids, nucleosides, and amino acids derivatives associated with HGSC progression. Comparison of the µCE-HRMS dataset with non-targeted ultra-high performance liquid chromatography (UHPLC) – MS results revealed identical temporal trends for the 5 metabolites detected on both platforms, indicating the µCE-HRMS method performed satisfactorily in terms of capturing metabolic reprogramming due to HGSC progression, while reducing the total analysis time 3-fold.

ORGANISM(S): Synthetic

DISEASE(S): Cancer

SUBMITTER: Samyukta Sah

PROVIDER: ST002136 | MetabolomicsWorkbench | Mon Apr 11 00:00:00 BST 2022

REPOSITORIES: MetabolomicsWorkbench

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Project description:Ovarian cancer (OC) ranks among the deadliest cancers in women. Lack of symptoms, rapid spread of metastases and common chemoresistance contribute to unfortunate fate of majority of OC patients, especially those having high-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of OC. Many HGSC patients have excess fluid in the peritoneum at the stage of diagnosis called ascites. Ascites is basically a tumor microenvironment (TME) in the liquid form containing various cells, proteins and also extracellular vesicles (EVs). EVs are small membrane-bound particles that convey proteins, lipids and nucleic acids between cells and their cargo reflects the cell of origin. EVs play important role in cancerogenesis and hold great promise as disease biomarkers as well as potential therapeutic targets. Small size and polydispersity of EVs bring various challenges to their isolation and characterization, including method-dependent enrichment of different EV subtypes as well as contaminants. We isolated EVs from ascites of 11 patients by two different methods (ultracentrifugation coupled to sucrose cushion and size-exclusion chromatography, qEV column) and analyzed them by mass spectrometry. We identified core ascitic EV proteins present in all patients that contain typical EV markers and are devoid of method-dependent contaminants. To cover interpatient heterogeneity, we expanded this “core proteome” with proteins found in majority of patients. Next, we compared them with proteins of EVs from related control fluids and uncovered proteins present only on EVs from HGSC patients. We believe this list of proteins contain both important players of HGSC progression as well as potential biomarkers. Using single cell RNA sequencing data, we mapped the origin of EVs to different types of cells present in malignant ascites. Our results suggest that EVs in ascites do not come predominantly from tumor cells, but rather from variety of non-malignant cell types including cancer-associated fibroblasts and tumor-associated macrophages, which presence in ascites was confirmed by flow cytometry. This emphasizes the recently appreciated role of TME in the progression of HGSC. To conclude, this is the first study attempting to link EV composition to the cell types producing it. As such it opens numerous avenues both for better understanding of EV role in tumor promotion/prevention and for the improved HGSC diagnostics.

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Targeted Microchip Capillary Electrophoresis-Orbitrap Mass Spectrometry Metabolomics to Monitor Ovarian Cancer Progression (calibration standards)

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