Oral cancer, one of the most common form of head and neck cancers, has an annual incidence rate of ̴380,000 and it holds the sixteenth position in malignancies worldwide (Chamoli et al., 2021). Oral squamous cell carcinoma (OSCC) accounts for more than 90% of the oral malignancies (Badwelan et al., 2023). OSCC development is a multistep process that seems to be a result of patients’ genetic predisposition, environmental factors, age and epigenetic modifications (Aghiorghiesei et al., 2022). The major risk factors for OSCC development are tobacco use, betel quid usage, and alcohol misuse (Warnakulasuriya, 2009). These factors may act individually or interdependently with multiplicative effect on the cancer risk (Lissowska et al., 2003; Subapriya et al., 2007). Usually, OSCC is preceded by oral potentially malignant disorders (OPMD), like oral leukoplakia, having the same etiological risk factors as OSCC (Bewley and Farwell, 2017).

It is becoming clear that the development and progression of cancer, including OSCC, involve not only genetic/epigenetic abnormalities in the cancer cells, but also require a mutual interplay between the tumor cells and adjacent stromal cells such as the cancer associated fibroblasts (CAFs), immune cells, and endothelial cells. This complex network of various cell types in the vicinity of the tumor is referred to as the tumor microenvironment (TME) (Wang et al., 2021). The cells in the TME may undergo reprogramming to provide a tumor-supportive milieu and produce signaling molecules that can for example, promote cancer cell proliferation and metastasis (Shan et al., 2021). CAFs are involved in several biological processes such as cancer cell proliferation and migration, angiogenesis, and extracellular matrix (ECM) remodeling, thereby contributing to tumor progression and metastasis. In some types of cancer, CAFs are considered a central component of the TME (Arima et al., 2023) and in OSCC their presence has been linked to increased tumor invasion and worse prognosis (Fujii et al., 2012; Liu et al., 2016). Due to the complex and varied TME composition, the crosstalk between its elements is also complex and multi-leveled. Within the TME, cells can communicate directly via cell-to-cell contact and adhesion molecules, electrical coupling through gap junctions, or indirectly through releasing cytokines, growth factors, and extracellular vesicles (EVs) (Dominiak et al., 2020).

EVs are a heterogeneous population of lipid bilayered vesicles, ranging in diameter from 30 to 10,000 nm. As naturally occurring and released by all cell types, EVs have emerged as an important player in intercellular communication, mainly through their ability to transfer their biological cargo of proteins, lipids, and nucleic acids to recipient cells (Valadi et al., 2007; Subra et al., 2010). The physiological function of EVs include regulation of various processes such as angiogenesis, cell survival, proliferation, and apoptosis (Li et al., 2022). However, EVs may also be involved in pathophysiological processes, and could be part of the background for disease onset and progression (Van Niel et al., 2018).

The molecular pathogenesis of OSCC is complex as it involves altered expression of specific genes and proteins, but also comprises changes in metabolic processes (Wei et al., 2011; Xie et al., 2012). In particular, the metabolome, reflecting the interactions between genome, microbiome, and environmental factors, is dynamic and diverse (Beger et al., 2016). In general, one of the most characteristic metabolic features of the cancer cells is increased glucose uptake and its altered metabolism. After metabolizing glucose to pyruvate (glycolysis), cancer cells switch from oxidative phosphorylation (OXPHOS) of pyruvate in the citric acid cycle (TCA) to fermentation of pyruvate into lactic acid. The latter is an anaerobic mechanism preferred by cancer cells even when adequate amounts of oxygen is present. Since it is less effective in energy production than OXPHOS, it must have other benefits (Gray et al., 2014). Recent studies have revealed that normal oral fibroblasts (NOFs) co-cultured with OSCC cells, change their metabolic phenotype into a pro-tumorigenic one. This change was characterized by increased rate of aerobic glycolysis, increased levels of reactive oxygen species and lactate, and overexpression of the lactate transporter MCT-4 in the NOFs (Zhang et al., 2020), termed the reverse Warburg effect. MCT-4 exports lactate out of the fibroblasts, making it available as fuel for cancer cells and at the same time acidifying the tumor environment. However, the mechanism of this fibroblast phenotype switch is not fully understood, nor is the nature of interaction between cancer cells, cells that constitute the TME, and normal cells in the tumor vicinity. We hypothesize that EVs derived from OSCC cells can affect normal oral cells and modify them towards a pro-tumorigenic phenotype. Therefore, the aim of the project was to characterize the effect of OSCC EVs on the metabolic profile of normal oral fibroblasts (NOFs).

The present study explored the metabolic profile in normal oral fibroblasts (NOFs) exposed to EVs of OSCC origin. We identified dysregulation of several key metabolites (Figure 7) associated with pathways (Figure 6) that may be important for metabolic reprogramming of NOFs into a pro-tumorigenic phenotype.

Among the metabolites that were significantly dysregulated, an increase in pyruvate level was seen in NOFs treated with EVs for 24 h, and as pyruvate is the end-product of glycolysis, it may indicate dysregulation of this major metabolic pathway. Pyruvate can be oxidized and further processed in the TCA cycle, or alternatively it can undergo fermentation to lactic acid (Gray et al., 2014). Our results suggests that exposure of NOFs to OSCC EVs may have caused a switch in NOFs from pyruvate oxidation to fermentation. The pathway analyses indicate that besides glycolysis, the most enriched pathways in NOFs after exposure to OSCC EVs include pyruvate metabolism and Warburg effect, which is manifested by pyruvate fermentation and lactate accumulation during glucose metabolism even in the presence of abundant oxygen (Warburg, 1956). In the cancer cells the majority of glucose is metabolized through fermentation (66%), but it is still unclear why this energetically highly inefficient process is preferred instead of OXPHOS (Koppenol et al., 2011). Although fermentation is less effective, it is ten times faster than full glucose oxidation, so it may support rapid ATP production to meet an increasing demand in intensively proliferating cancer cells (Devic, 2016; Liberti and Locasale, 2016). Furthermore, reducing OXPHOS may protect the cell from ROS produced by mitochondria during electron chain activity (Napolitano et al., 2021). Another reason for the preference for fermentation over OXPHOS may be associated with extracellular acidification due to the increased lactate that confers immune evasion ability onto cancer cells and creates a favorable TME (Boedtkjer and Pedersen, 2020; Chen et al., 2021). In accordance with this, we found that the lactate level was increased in the NOFs exposed to the EVs, but the difference was not statistically significant. The level of lactate in each individual sample revealed a large variation, which explain the lack of significance. This may be due to the cells having already secreted a large amount of lactate as part of a “reverse Warburg effect” (Jiang et al., 2019). In the reverse Warburg effect, stromal cells undergo aerobic glycolysis, but instead of using the produced intermediates such as lactate and pyruvate themselves, the cells secrete those energy metabolites to fuel cancer cells that have high energy demands due to their rapid proliferation (Pavlides et al., 2009). This finding is in line with the report of Zhang et al. (2020) which revealed that in co-culture with OSCC cells, NOFs undergo aerobic glycolysis, secrete lactate, and overexpress the lactate transporter MCT-4. This metabolically altered NOF phenotype possibly corresponds with impaired mitochondrial activity (Zhang et al., 2020), and may be an effect of EV cargo on recipient cells.

The present study revealed that in addition to pyruvate and lactate, several other metabolites involved in energy production, such as ATP, coenzyme A, dihydroxyacetone phosphate, UTP, NAD, FAD, cADPR, and docosahexaenoic acid, were dysregulated in NOFs following OSCC EVs exposure. Results of the metabolites detection with LC-MS were verified with Western blotting for PKM2 and ALDOA. Moreover, these findings are supported by the functional analysis demonstrating enrichment of glycolysis (related with pyruvate, ATP, dihydroxyacetone phosphate, and NAD), TCA cycle (related with pyruvate, ATP, coenzyme A, NAD, and FAD) and mitochondria electron transport chain (related with ATP, dihydroxyacetone phosphate, NAD, and FAD). Of interest, in addition to the established role of the TCA cycle in biosynthesis and bioenergetics, the TCA intermediates like α-ketoglutarate, fumarate, lactate, and succinate can affect various aspects of cancer progression or even act similarly to cytokines (Zasłona and O’Neill, 2020; Eniafe and Jiang, 2021). These metabolites were detected in the present study but were not significantly changed. However, even if not significant, disturbed levels of these metabolites could contribute to the overall picture of differences between control and OSCC EV-exposed fibroblasts. Furthermore, dysregulation of the TCA cycle and mitochondrial electron transport chain is considered another feature of the OSCC metabolic hallmark (Vitório et al., 2020; Bai et al., 2022). However, this finding may also be a consequence of the glycolysis dysregulation and Warburg effect (Jiang et al., 2019) rather than an independent event. Still, it is interesting that a single exposure to the OSCC EVs was sufficient to trigger substantial changes in NOFs.

Several fatty acids (FAs), such as nervonic acid, linoleate, oleate, palmitoleic acid, and docosahexaenoic acid, were significantly downregulated in NOFs exposed to OSCC EVs. Cancer cells redirect FAs from energy metabolism towards de novo lipogenesis to promote proliferation and lipid signaling (Louie et al., 2013). Nervonic acid plays a role in the immune response as it regulates transformation and proliferation of splenic lymphocytes, and yield of pro-inflammatory chemokines and cytokines (Borish and Steinke, 2003; Yamazaki et al., 2014; Yuan et al., 2023), which could affect the NOFs response to EV cargo and direct them towards a pro-tumorigenic phenotype. While oleate and linoleate stimulate proliferation in breast cancer (Yonezawa et al., 2004), oleate has anticancer effect in various other types of cancers (Carrillo et al., 2012). For example, in tongue squamous cell carcinoma, oleate induces apoptosis and autophagy (Jiang et al., 2017). Regardless of the role of FAs as pro- or anti-cancer agents (Denko, 2008; Jiang et al., 2017; Souza et al., 2017), it is possible that at the initial step of reprogramming toward a pro-tumorigenic phenotype of NOFs, detected downregulation of nervonic acid, linoleate, oleate, palmitoleic acid, and docosahexaenoic acid may rather reflect dysregulation of immune processes in fibroblasts as part of a defensive response to the OSCC EVs.

We also observed that the reaction of fibroblasts to OSCC EV exposure was time dependent. There was an early response already showing after 15 min, but the response was more pronounced after 24 h. However, at 48 h, the metabolic profile seemed to normalize in the EV-exposed fibroblasts. This may indicate that a single exposure to the molecular cargo carried by the EVs may not be sufficient to achieve a long-lasting change in metabolic status of NOFs. It is difficult to estimate the pattern of EV release during the course of cancer development and how frequent the stimulation of nearby cells is, but it can be expected that this is not an on-off phenomenon (Mathieu et al., 2021; Auber and Svenningsen, 2022). Therefore, it is reasonable to assume that normal cells subjected to long-term exposure to the cancer-derived EVs will undergo phenotype change, and that this probably will be a long-term effect, ultimately contributing to transformation of NOFs e.g., into CAFs.

EVs are carriers of the diversified molecules that can affect biological processes such as proliferation, apoptosis, DNA repair, angiogenesis, and immune response (Becker et al., 2016; Ciardiello et al., 2016; Søland et al., 2024). To date studies on OSCC EV profiling have mainly focused on the nucleic acid and protein cargo (Leung et al., 2021). In monocytes, the OSCC EV signature miR-21 and miR-27 led to activation of the inflammatory pathway, that in turn contributed to the establishment of a pro-inflammatory and protumorigenic milieu (Momen-Heravi and Bala, 2018). Such oncogenic miRNAs are considered as promising diagnostic markers for OSCC and other cancers (Li et al., 2016; Leung et al., 2021). On the other hand, more detailed multi-omic analysis of the OSCC EVs, covering also metabolomic and lipidomic data, revealed 11 hub proteins - candidates as prognostic markers (Busso-Lopes et al., 2021). Our current research revealed that OSCC EVs can affect NOF metabolome. Thus, the continuation of this study could be a similar analysis of the transcriptome and proteome to identify pathways that are affected by the OSCC. Such an approach could be beneficial to extend the knowledge on EV effects on normal cells and indicate potential targets for OSCC therapy. As a metabolomics profiling approach based on the LC-method and annotation by mass accuracy and LC retention times has its limitations, it could in future work be considered to introduce a labelled internal standard. It might be helpful to confirm significant results by validation of variations in sample preparation, extraction, and instrument performance and matrix effect. However, an internal standard needs to be selected carefully not to cause any ion suppression for analyzed metabolites. Therefore, more studies are needed to address all challenges associated with potential EV use in diagnosis, prognosis, and therapy of OSCC.

In summary, the present study identified a range of changes in NOF metabolites after exposure to OSCC-derived EVs. Various metabolites involved in glycolysis and amino acid and lipid metabolism, crucial for proper cell functioning, were affected. Nonetheless, it should be highlighted that the impact of cancer-derived EVs may go far beyond this and affect cell functioning at the level of transcriptome and proteome to induce changes that in the light of the obtained results can be considered as acquiring a pro-tumorigenic phenotype. Further studies are needed to identify molecular mechanisms involved in this phenotype switch. In the long-term perspective, a broad characterization of the effects of cancer-derived EVs on healthy cells should enable identification of potential biomarkers or targets for future anti-OSCC therapies.