Methodology of determining the metabolomic profile of tumor-associated macrophages and monocytes in oncological diseases

About authors

1 Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Moscow, Russia

2 Laboratory of translational cellular and molecular biomedicine, National Research Tomsk State University, Tomsk, Russia

3 Laboratory of Genetic Technologies, Siberian State Medical University, Tomsk, Russia

4 Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, Tomsk, Russia

5 Institute of Transfusion Medicine and Immunology, MI3, Mannheim Faculty of Medicine, University of Heidelberg, Germany

6 German Red Cross Blood Service Baden-Württemberg–Hessen, Mannheim, Germany

Correspondence should be addressed: Vladimir E. Frankevich
Akademika Oparina, 4, Moscow, 117997, Russia; moc.liamg@hciveknarfv

About paper

Funding: the study was financially supported by the Russian Federation represented by the Ministry of Science and Higher Education of the Russian Federation (agreement dated 29 September 2021 № 075-15-2021-1073 on the topic "Genetic and epigenetic editing of tumor cells and the microenvironment in order to block metastasis ").

Author contribution: Frankevich VE, Kzhyshkowska JG — study planning and coordination, manuscript writing; Bragina OD — cancer patient selection and clinical characteristic preparation; Novoselova AV — sample preparation, HPLC-MS/MS; Larionova IV, Patysheva MR — monocyte and macrophage sample preparation and characterization, model TAM system set-up; Rakina MA — obtaining conditioned media of breast cancer cell lines; Starodubtseva NL — HPLC-MS/MS data analysis, manuscript writing; Frankevich VE, Larionova IV, Patysheva MR — discussion of results; Frankevich VE, Kzhyshkowska JG, Starodubtseva NL — manuscript editing.

Compliance with ethical standards: the study was approved by ethical review board of the Federal State Budgetary Educational Institution of Higher Education of the Siberian State Medical University of the Ministry of Health of Russia (protocol № 7 of 14 January 2017), federal laws of the Russian Federation (№ 152, 323, and others), and the 1964 Declaration of Helsinki.

Received: 2022-08-22 Accepted: 2022-09-13 Published online: 2022-10-13

Breast cancer is the leading cause of cancer-related death among women worldwide. Tumor-associated macrophages (TAMs) constitute the primary component of innate immunity in breast cancer tissue. During the development of new approaches for breast cancer treatment aimed at editing the epigenome of TAM, precise methods for the analysis of macrophage metabolome are required to examine the effect on new approaches on macrophage metabolism. Our study aimed to develop an HPLC-MS/MS-based analytical approach to characterize the metabolome of human innate immune cells (TAMs and their precursors, monocytes). Analysis of lipid extracts was conducted on a Dionex UltiMate 3000 liquid chromatograph connected to a Maxis Impact qTOF mass analyzer with an ESI ion source. Quantitative analysis of 38 amino acids in the cells was conducted using the Jasem Amino Acids LC-MS/MS Analysis Kit and an HPLC-MS/MS chromatographic system consisting out of an Agilent 6460 triple quadrupole mass spectrometric detector (Agilent), and an Agilent 1260 II liquid chromatograph (Agilent ) with Amino acids-HPLC Column (Jasem). The modified Folch method with double extraction was found to be the optimal approached for the sample preparation, since it enables to simultaneously isolate the lipid extract and water-soluble substances, in particular, amino acids. The method of reversed-phase chromatography yielded more useful data on the cell lipid composition than the method of hydrophilic interaction liquid chromatography (HILIC). The minimum number of cells required to determine the metabolome of immune system cells (TAM and monocytes) was identified as 2 × 106. Thus, we have developed the approach to determine the lipid and amino acid composition of  modelled human TAMs and primary monocytes isolated out of breast cancer patients using minimal amount of clinical material.