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.

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Monocytes are large circulating white blood cells that are the main precursors of tissue macrophages as well as tumor-associated macrophages in the adult body. Different types of monocytes have multidirectional effects on the growth and metastatic spread of cancer cells, both activating and inhibiting these processes. Tumor progression is associated with the triggering of a whole cascade of inflammatory and immune reactions. These pathological processes are associated with changes in the amino acid content of monocytes, which can lead to disruption of their function, in particular their migration, division and maturation. The aim of the work was to profile the amino acids of monocytes, followed by a study of the amino acid composition of monocytes from patients with breast cancer using liquid chromatography with mass spectrometric detection. Significant differences in metabolite levels in monocytes of breast cancer patients and monocytes of healthy donors were found for glycine (p-value = 0.0127), asparagine (p-value = 0.0197), proline (p-value = 0.0159), methionine (p-value = 0.0357), tryptophan (p-value = 0.0028), tyrosine (p-value = 0.0127). In the study, we identified biological networks that could potentially be involved in altering the phenotype of monocytes affected by breast cancer (BC), using bioinformatic analysis of metabolic pathways involving the discovered amino acids. Mathematical models based on amino acid combinations with 100% sensitivity and specificity have been developed. Features of immune system cell metabolism in BC have been identified and potential diagnostic biomarkers have been proposed.

Novorozhdennye ot materej s COVID-19 podverzheny risku zarazheniya, mogut imet' vysokij risk oslozhnenij v rannem neonatal'nom periode i otdalennye posledstviya dlya zdorov'ya. Cel' issledovaniya — opredelit' aminokislotnyj profil' pupovinnoj plazmy i amnioticheskoj zhidkosti pacientov s COVID-19 dlya analiza svyazi vliyaniya perenesennogo antenatal'no COVID-19 na izmeneniya v metabolome «vnutriutrobnogo pacienta». Dlya ocenki urovnya 31 aminokisloty v obrazcah amnioticheskoj zhidkosti i pupovinnoj plazmy beremennyh s COVID-19, poluchennyh pri rodorazreshenii, primenyali vysokoeffektivnuyu zhidkostnuyu hromatografiyu s mass-spektrometricheskoj detekciej. Osnovnuyu gruppu sostavili 29 pacientok s podtverzhdennym diagnozom COVID-19; kontrol'nuyu — 17 somaticheski zdorovyh zhenshchin s beremennost'yu bez oslozhnenij. Koncentracii vos'mi aminokislot v amnioticheskoj zhidkosti statisticheski znachimo (p < 0,05) razlichalis' mezhdu issleduemymi gruppami. Razrabotany modeli logisticheskoj regressii (chuvstvitel'nost' 0,84; specifichnost' — 1), pozvolyayushchie opredelyat', chto analiziruemaya amnioticheskaya zhidkost' vzyata ot pacientok, perenesshih COVID-19. V plazme pupovinnoj krovi znachimye razlichiya obnaruzheny dlya chetyrekh aminokislot. Postroennye modeli pozvolyayut vyyavlyat' prinadlezhnost' issleduemoj pupovinnoj plazmy pacientam gruppy kontrolya ili COVID-19 (chuvstvitel'nost' i specifichnost' — 1). Odnovremenno v dvuh tochkah (amnioticheskaya zhidkost' i pupovinnaya plazma), kompleksno otobrazhayushchih metabolom ploda, byli vyyavleny i statisticheski znachimo otlichalis' pri COVID-19 tri aminokisloty. Vozdejstvie virusa na organizm privodit k vyrazhennym izmeneniyam v metabolome amnioticheskoj zhidkosti i pupovinnoj plazmy ploda, chto mozhet privesti k narusheniyu programmirovaniya proizvodstva belkovyh molekul, no ne proyavlyaetsya pri rozhdenii.

Neonates born to mothers with COVID-19 are at risk for infection, they may have high risk of complications during the neonatal period, and long-term health consequences. The study was aimed to define the amino acid profile of blood plasma and amniotic fluid in patients with COVID-19 in order to assess the relationship between the COVID-19 infection during the antenatal period, and metabolomic alterations in the “intrauterine” patient. The levels of 31 amino acids in the samples of amniotic fluid and cord blood plasma of pregnant women with COVID-19, obtained during delivery, were assessed by high-performance liquid chromatography-mass spectrometry. The index group included 29 patients with confirmed diagnosis of COVID-19, and the control group included 17 healthy women with uncomplicated pregnancies. There were significant (p < 0.05) differences in the concentrations of eight amino acids between the studied groups. Logistic regression models were developed (sensitivity 0.84; specificity 1) making it possible to define, whether the assessed amniotic fluid was obtained from COVID-19 patients. Significant differences in the concentrations of four amino acids were observed in the umbilical cord blood. The models developed made it possible to define whether the studied cord blood plasma belonged to controls or to COVID-19 patients (sensitivity and specificity 1). Three amino acids were detected, and their levels were significantly different in COVID-19 patients simultaneously in two points (amniotic fluid and cord blood plasma), depicting the fetal metabolome in a holistic manner. The impact of the virus on those infected results in pronounced metabolomic alterations in the amniotic fluid and the fetal cord blood plasma, which may lead to impaired programming of protein production, but never show up at birth.