For decades, the focus of oncology has been on genetic mutations responsible for the initial development and progression of tumors [1]. However, as our understanding of molecular biology has deepened, it has become clear that disrupted metabolism play a key role in the functioning of tumor cells [2]. A key hallmark of malignancy is altered metabolic properties of cells [3]. These changes include dysregulation of glucose and amino acid utilization, activation of “new” pathways for nutrient acquisition, utilization of glycolytic and citric acid cycle intermediates for nicotinamide adenine dinucleotide phosphate biosynthesis, increased nitrogen requirements, changes in the regulation of metabolic genes, and metabolic interactions with the microenvironment [4]. Key metabolic changes include increased glycolysis instead of oxidative phosphorylation for energy production, higher rates of lipid synthesis to support rapid cell membrane formation, and increased levels of amino acids such as glutamine (Gln) for rapid growth and proliferation [5]. Overall, the metabolic profile of cancer includes changes in energy and plastic metabolism, and redox balance characteristic of tumor cells [6].

Tumor cell metabolism is a complex and flexible process with significant heterogeneity depending on various factors [7, 8]. Currently, attempts are being made to identify key targets of tumor cell energy homeostasis for therapeutic intervention to increase the life expectancy of patients [9]. Undoubtedly, this approach is a promising strategy for drug therapy of malignant tumors [10]. However, there are a number of problems that require solutions, in particular the general attitude towards cancer as a genetic disease without taking into account the metabolic consequences of these molecular genetic disorders, the presence of parallel pathways and the plasticity of tumor processes, inter- and intratumoral heterogeneity of metabolism, the possibility of switching metabolism in the absence of the necessary energy sources and biomaterial, the similarity of metabolic processes in tumor and healthy tissue, as well as the lack of an effective model for studying metabolic therapy [11].

Influencing the metabolic pathways of cancer cells not only inhibits their development but also improves existing treatments, including chemotherapy and immunotherapy [12, 13]. Moreover, since tumor metabolism is accompanied by specific biochemical changes, it can also be used for diagnosis, prognostic assessment, and monitoring of therapy outcome [14].

Breast cancer (BC) is one of the most common female cancers [15, 16]. The most important feature of the systemic action of BC is that the tumor is capable of inducing profound morphofunctional changes throughout the body [17, 18], which will also be reflected in changes in the salivary composition [19, 20, 21, 22, 23]. Evaluation of systemic changes in breast cancer can be carried out using saliva, which has not been demonstrated to date [24]. Saliva can also be used as a convenient and effective model for evaluating metabolic therapy. Understanding the diagnostic and prognostic value of metabolites is crucial for improving cancer detection and improving patient treatment outcomes. The integration of metabolomics into clinical practice contributes to the development of personalized medicine, providing individualized treatment based on individual metabolic profiles [25].

A hypothesis was formulated that all components of cancer cell metabolism, including changes in energy metabolism, biosynthesis, and regulation of metabolic processes, would be reflected in changes in the metabolic composition of saliva across different BC phenotypes. Particular attention was proposed to be paid to the use of glutamine and fatty acids as additional or alternative energy substrates to glucose (amino acids, proteins), high levels of lipid biosynthesis (lipids), increased production of reactive oxygen and nitrogen species and, consequently, permanent activation of antioxidant defense mechanisms (enzymes, lipoperoxides, NO, DNA damage), as well as the immune response (cytokines). A comprehensive study of these salivary parameters in a single patient cohort has not yet been conducted.

The aim of the study was to identify clinically significant metabolic salivary features depending on the BC phenotype.

BC has been shown to cause both local and systemic metabolic changes, which can be assessed by changes in the composition of mixed saliva. These metabolic changes involve all components of metabolism: energy and plastic metabolism, as well as redox balance.

Energy metabolism. In BC, increased levels of glycolysis are observed compared to normal tissues (Warburg effect), and the balance between glycolysis and oxidative phosphorylation is disrupted [34]. Thus, we observed a change in the salivary amino acids profile. It is known that malignant neoplasms cause changes in the amino acid levels both in tumor tissues and in the blood. Elevated amino acid levels are due to pro-inflammatory and immunological factors and are associated with a more aggressive phenotype of BC [35]. Amino acids can enter saliva from the blood via extracellular and intercellular pathways, due to ultrafiltration, passive diffusion, and active transport, as well as from the excretory ducts of the salivary glands during the biosynthesis of protein secretion in acinar cells using sodium-dependent membrane transporters [36]. Luminal B HER2-positive and TNBC were characterized by the highest levels of salivary amino acids. An additional calculation of the Gln/Glu ratio revealed a different variation across different BC phenotypes. For example, for luminal A and B HER2-negative subtypes, this ratio was lower than in HC, while for luminal B HER2-positive and TNBC, it exceeded the control by more than twofold. Thus, Gln was more prevalent in saliva than Glu in these BC subtypes. High Gln levels in the blood provide cancer cells with carbon and nitrogen sources, stimulating biosynthesis, energy replenishment and cellular homeostasis during tumor growth [37]. It has been shown that the immune characteristics of breast cancer subgroups, identified on the basis of Gln metabolism, may be of importance for prognosis and personalized immunotherapy [38]. Previously, two main metabolic phenotypes of BC were identified: (1) dependent predominantly on glycolysis and the pentose phosphate pathway and associated with poor survival (HER2-positive and TNBC); (2) dependent on fatty acid oxidation and glutaminolysis (luminal A subtype of BC) [39].

Increased levels of salivary Gly were observed in luminal B HER2-positive and TNBC. Cao et al. [40] found that high levels of Gly and Ala are associated with HER2-positive BC. Ala is involved in the synthesis of Gly from pyruvate and Ser. High levels of Gly are also known to correlate with an unfavorable prognosis in BC [41], since the synthesis of Gly from glucose correlates with rapid cancer cell proliferation [42]. Thus, higher levels of salivary Gly in BC can be considered a marker of tumor aggressiveness.

We showed that Met levels increased only to a greater extent in the luminal A and luminal B HER2-negative BC subgroups, while Cys levels did not change significantly in these subgroups. For HER2-positive subtypes, a slight increase in Met levels was noted with a significant decrease in Cys levels. On the one hand, tumor cells are more dependent on exogenous Met than normal cells [43]. On the other hand, Met is a crucial component of antitumor immunity [44, 45]. Cys, obtained from food or synthesized from Met, is a limiting factor for the synthesis of glutathione (GSH), which in turn maintains redox homeostasis and protects cells from oxidative stress caused by reactive oxygen species (ROS) and lipid peroxides [46, 47]. Due to increased ROS production, many cancer cells have high GSH levels, which contributes to their resistance to anticancer therapy [48]. It can be speculated that unfavorable prognostic subtypes of BC are characterized by decreased antitumor immunity due to decreased Met levels, as well as impaired redox homeostasis due to decreased Cys levels.

Plastic metabolism. In cancer, Gln and fatty acids are utilized as additional or alternative energy substrates to glucose, and higher levels of lipid biosynthesis are also observed [49]. This is due to the need for energy and plastic support for enhanced anabolic processes and reactions synthesizing the structures of rapidly dividing cells [50]. Activation of the pentose phosphate cycle and the synthesis of pentoses, necessary for the construction of nucleic acids, is also observed.

One of the key aspects of lipid metabolism in carcinogenesis is the increased synthesis of fatty acids. We have shown a decrease in the absorbance in BC compared to healthy controls. It is known that lipids enter the oral cavity mainly with the secretions of the parotid and submandibular salivary glands [51]. Some lipids, such as cholesterol and free fatty acids, enter saliva from the blood serum [52, 53]. The source of a number of lipids in saliva are also the membranes of secretory vesicles and fragments of bacterial cell membranes. Lipids are one of the main components of cell membranes, so changes in the lipid composition of saliva may reflect changes in the composition of the cell membranes of the salivary glands [54]. The general decrease in the intensity of oscillations of methyl and methylene groups in the lipid structure may be the result of a decrease in their saturation. It is known that an increase in lipid saturation increases the rigidity of membranes, thereby reducing their permeability [55]. Lipid unsaturation increases the fluidity and flexibility of the cell membrane, facilitating its remodeling and the movement of membrane-bound proteins and lipids [56, 57].

A number of studies indicate a relationship between lipid metabolism, the tumor microenvironment, and BC aggressiveness [58]. Lipid composition significantly depends on the BC phenotype [59, 60]. The range of 3050–2800 cm^-1 is used to assess cell membrane permeability and protein oxidative modification processes. This range corresponds to vibrations of the methyl and methylene groups of saturated and unsaturated alkyl chains. The ratio 2923/2957 cm^-1, which shows the ratio of unbranched and branched lipid and fatty acid molecules (CH₂/CH₃), may be potentially informative. In non-luminal BC, this ratio decreases, which may be associated with a more branched structure of lipids, as well as their shorter chains. The non-luminal subtype also showed minimal changes in salivary amino acid content, which may be a result of a shift in energy metabolism toward fatty acids, unlike other BC subtypes. Branched structures are found in various types of lipids, including fatty acids, triacylglycerides, and phospholipids. However, branched structures are rare in natural lipids, primarily found in the membranes of certain bacteria. It is likely that the altered lipid structure in non-luminal BC is due to changes in the oral microbiome.

It should be noted that salivary lipids perform many functions, the disruption of which can lead to disintegration throughout the oral cavity [61]. They also perform a protective function in relation to the oral mucosa. Both a decrease in salivary secretion and a weakening of the protective function can subsequently lead to a number of disorders, including dry mouth, as well as an increased risk of bacterial, fungal and viral infections of the gums and oral mucosa [61]. Moreover, a disruption of lipid metabolism can lead to the accumulation of free fatty acids, which cause oxidative stress, increasing the ROS production [62]. All this can be reflected in a decrease in local immunity of the oral cavity and requires timely correction in patients, regardless of the molecular biological subtype of BC.

Redox balance includes an increase in ROS, which stimulate and support cancer cell proliferation, as well as an enhancement of the antioxidant mechanism, which allows cancer cells to survive in a prooxidant environment [63]. However, this prooxidant state promotes DNA damage and genomic instability, which enhances the malignant properties of cancer cells [64].

Redox biomarkers in saliva are known to correlate poorly with their plasma levels, but the 8-OHdG level in saliva reflects its concentration in the blood [65]. 8-OHdG can be formed from 8-oxo-dGTP in the nucleotide pool or as a result of direct oxidation of guanine in DNA [66]. We demonstrated an increase in 8-OHdG concentration in saliva, most pronounced in HER2-positive and TNBC, which is consistent with a higher level of oxidative DNA damage in these subgroups.

BC subtypes are characterized by different ROS production and susceptibility to antioxidant treatment [67]. We have shown that salivary catalase activity decreases more significantly in luminal BC subtypes, while AOA decreases to a greater extent in HER2-positive and TNBC. Estrogens and their metabolites are known to alter the redox balance through increased generation of superoxide radicals with their signaling and damaging effects [68, 69], which may cause a more noticeable depletion of antioxidant defense in patients with estrogen-positive BC. It has been shown that salivary peroxidase activity increased inversely proportional to the decrease in catalase activity. Salivary peroxidase is responsible for the breakdown of cytotoxic hydrogen peroxide, while it has a bactericidal effect on the oral microbiota [70]. Thus, salivary peroxidase activity may reflect the involvement of salivary glands in the prevention of oxidative stress. Increased salivary peroxidase activity in BC indicates enhanced enzymatic antioxidant defense, which protects the salivary glands and the entire oral cavity from oxidative damage.

An accumulation of more toxic Schiff bases was observed compared to primary and secondary lipid peroxidation products. The lowest levels of lipid peroxidation products were observed in HER2-positive BC subtypes. This may be related to higher levels of free amino acids with antioxidant properties (Asp, Gln, Gly, Pro, and Tyr), which were significantly higher in this group of patients.

At the systemic level, the presence of a chronic inflammatory process is accompanied by an increase in the blood levels of inflammatory mediators (cytokines, chemokines, eicosanoids, reactive oxygen and nitrogen species), acute-phase proteins, and growth factors, as well as the activation of blood coagulation factors [71]. To counteract hypoxia, tumor cells stimulate angiogenesis by increasing the levels of VEGF and other proangiogenic factors [72]. For the luminal A BC subtype, we observe both an increase in the concentration of VEGF, IL-1$\beta$, and INF-$\gamma$, as well as an increase in the level of salivary CRP, which characterizes more pronounced chronic inflammatory processes.

From a biological point of view, INF-$\gamma$ has antiviral, antitumor and immunomodulatory functions, which ensures its important role in coordinating the innate and adaptive immune response [73]. Saliva showed a uniform increase in INF-$\alpha$ concentrations regardless of the clinical, pathological, and molecular biological characteristics of BC. Conversely, despite the absence of statistically significant differences between BC and control subgroups in INF-$\gamma$ levels, significant differences were observed within the subgroups. Thus, salivary INF-$\gamma$ levels increased in tumors with high and intermediate differentiation (G I and G II) and low proliferative activity (Ki-67 20%), ER/PR-positive and HER2-negative status, which corresponds to the prognostically favorable luminal A subtype of BC. Apparently, this subtype exhibits an active anti-inflammatory response at the systemic level, which is reflected in changes in saliva composition.

The overwhelming majority of cancer patients develop secondary immunodeficiency of varying severity with a decrease in all components of anti-infective immunity. Thus, an increase in the concentration of both pro-inflammatory (IL-1$\beta$, IL-2) and anti-inflammatory (IL-4, IL-10) cytokines in the saliva of patients with BC was observed. However, higher concentrations of IL-2, IL-4 and IL-10 were observed in saliva for HER2-positive and TNBC. For the same subtypes, the decrease in the IL-8 concentration is more pronounced compared to the healthy controls. For INF-$\gamma$ and IL-18, an increase in concentration is observed with ER-positive expression and a decrease in ER-negative status. It is known that IL-18 affects the differentiation of predominantly type 1 T-helpers and activates NK cells, participating in the formation of a cellular immune response and antitumor immunity [74]. Initiation of IL-18 synthesis may lead to an increase in the INF-$\gamma$/IL-18 cycle: IL-18 induces the production of INF-$\gamma$ by lymphocytes, which stimulates monocytes/macrophages to increase their ICE activity, which promotes the synthesis of IL-18 [75, 76]. Apparently, a subgroup of non-luminal and TNBC is characterized by the presence of a less effective antitumor immune response due to a decrease in the level of IL-18 production by immunocompetent cells, as well as a less pronounced anti-inflammatory immune response due to a decrease in the level of INF-$\gamma$.

Recent studies have shown that the cytokine content in saliva does not correlate with their blood levels, indicating a certain autonomy of local immunity in the oral cavity, while reflecting general trends in the cytokine cascade in the patient’s body [77, 78]. Cytokines can enter saliva from the following sources: lymphocytes and accessory cells of the immune system embedded in the epithelium of the mucous membranes; serum transudate penetrating through the gingival pockets [79]; salivary glands, in which cytokines are formed and enter saliva with their secretions; epithelial cells of the oral mucosa, which produce cytokines upon contact with microorganisms [80]. In this regard, the identified patterns of changes in the level of salivary cytokines describe both local and systemic changes in BC phenotypes. It should be noted that with more prognostically favorable luminal A and B HER2-negative subtypes, a more pronounced inflammatory immune response is observed, whereas with less prognostically favorable subtypes (HER2-positive and TNBC), an increase in the level of anti-inflammatory cytokines (IL-4, IL-10) is observed. In these same subgroups, an increase in the concentration of IL-2 is observed. IL-2 is known to play a complex and sometimes opposing role in tumor progression and the immune response [81]. Although IL-2 can stimulate immune cells to attack tumors, it can also promote the proliferation of regulatory T cells, which suppress antitumor immunity [82]. Perhaps, an increase in the concentration of IL-2 indicates an increase in the antitumor immune response in a subgroup of patients with HER2-positive and TNBC.

Limitations of the study include the lack of ability to analyze the composition of the oral microbiome, as well as the lack of parallel determination of the composition of saliva and blood to confirm or refute the existence of a correlation between these biological environments. The limitations of the study also include the lack of a quantitative assessment of the oral cavity condition, since at this stage the study design only aimed to exclude an active inflammatory process in the oral cavity. Other limitations of the study include its single-center nature, the lack of longitudinal follow-up, and the lack of assessment of changes in salivary parameters during treatment. At this stage of the study, the primary objective was to select potentially informative salivary markers for assessing metabolic changes in different BC phenotypes. In the continuation of the study, we plan to conduct parallel determinations of concentrations in blood and saliva for these parameters, as well as conduct multicenter validation, evaluate correlations with the oral microbiome, and conduct dynamic monitoring before and after treatment. Although a subgroup reflecting the structure of the overall sample was formed for each indicator, limitations of the study also include the fact that saliva composition analysis was not performed on all samples for each indicator. An additional limitation may be the small sample size for individual salivary indicators.

Thus, the composition of saliva reflects systemic metabolic changes in BC, allowing for the development of a metabolic profile of BC, including consideration of the tumor’s molecular subtype. The salivary amino acids profile has been shown to correlate with receptor status, phenotype, and aggressiveness of BC. This confirms the existence of BC metabolic heterogeneity, which can be assessed by analyzing mixed saliva parameters. Furthermore, changes in salivary composition can be used to track changes in key components of BC energy homeostasis, which can be targeted by metabolic therapy.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

LB designed the research study and performed the research. LB performed the experiment and analyzed the data. The author contributed to drafting the manuscript. The author read and approved the final manuscript. The author has participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The study was conducted in accordance with the Declaration of Helsinki. The study focusing on the biochemical characteristics of saliva in oncological patients was approved by the Ethics Committee of Omsk Regional Clinical Oncological Dispensary (21 July 2016, protocol code 15). Because the Ethics Committee of Omsk Regional Clinical Oncological Dispensary later closed, the expanded study was approved by the Ethics Committee of Omsk State Pedagogical University (20 March 2024, protocol code 46-04/2). The study was expanded to broaden the scope of metabolite detection in patients. Written informed consent was obtained from all patients or their families/legal guardians.

Thanks to all the peer reviewers for their opinions and suggestions.

This research was funded by Russian Science Foundation, grant number 23-15-00188, https://rscf.ru/project/23-15-00188/.

The author declares no conflict of interest.