In this study, we performed targeted and untargeted metabolic profiling of tissues obtained from patients diagnosed with BC. Our objective was to generate distinctive metabolic signatures that could aid in early and accurate detection of BC using NMR and 109AgNPs LDI-MS techniques. We performed targeted 1H NMR analysis of normal and neoplastic tissues to identify a panel of 43 metabolites. Thirty-four of these compounds were present in higher concentrations and nine at lower concentrations in the cancer tissue compared to adjacent normal one (see Tables S2,3 in Supplementary data). The elevated levels of these 34 metabolites may indicate an increased synthesis of tumor-related metabolites that are secreted by cancer cells or changes in the composition of non-cancerous tissues caused by tumor infiltration through the epithelial barrier. In addition, the presence of tumors may trigger inflammatory responses that contribute to the elevation of certain metabolites. The metabolomics data obtained through 1H NMR analysis showed that six compounds exhibited higher concentrations in cancerous tissue than in normal tissue, and these compounds effectively differentiated between the two groups with significant discriminating power (Table 1). These included lactate, glutamine, glutamate, hypoxanthine, serine, and threonine.
Among the metabolites that effectively discriminated between cancerous and normal tissue samples, lactate emerged as a particularly significant biomolecule with a high VIP value. As the anion of a hydroxy carboxylic acid, lactate plays a key role in human metabolism and serves as a crucial energy reservoir (Rosenstein et al. 2018). By enabling the maintenance of ATP production and mitigating acidosis caused by ATP hydrolysis, lactate plays a vital function in cellular metabolism (Rosenstein et al. 2018). Furthermore, lactate has been identified as a significant contributor to acidosis in the tumor microenvironment (TME), which is associated with an acid-resistant phenotype that enables cancer cells to promote their own survival (Afonso et al. 2020). To sustain the uncontrolled growth and proliferation of cancer cells, including urothelial carcinoma cells, glycolysis is the primary source of energy. Consequently, a high glycolytic flux is dependent on the overexpression of genes related to glycolysis, which leads to the overproduction of pyruvate, alanine, and lactate (Massari et al. 2016). Research suggests that elevated lactate levels and acidification resulting from cancer cells and glycolytic metabolism can promote carcinogenesis by causing matrix degradation and cancer cell invasiveness (Gatenby et al. 2006). Furthermore, lactate has been implicated in metastasis and resistance to chemo-radiotherapy (Fischer et al. 2007). Due to its involvement in various metabolic pathways, lactate holds promise as a potential biomarker for cancer diagnosis and prognosis (Liu et al. 2016; Massari et al. 2016). Our study found that levels of lactate were significantly higher in cancer tissues compared to normal tissues in patients with BC. This aligns with previous findings from the analysis of urine and serum samples from BC patients using both NMR and LC-MS (Bansal et al. 2013; Wittmann et al. 2014). Additionally, Tripathi et al reported higher levels of lactate in tumor samples compared to benign disease in their studies of BC tissues also using NMR (Tripathi et al. 2013). These consistent results across different studies suggest that lactate could be a potential biomarker for BC diagnosis and monitoring.
The second most differentiating cancer group and the normal metabolite with the highest VIP value was glutamine. This compound is among the most abundant free amino acids in the body and plays a crucial role in the transport of nitrogen and maintaining acid-base balance (Hall et al. 1996). It is a primary energy source for rapidly dividing cells and is involved in excreting nitrogen compounds (Labow and Souba 2000). Furthermore, glutamine is metabolized to fuel the tricarboxylic acid (TCA) cycle, a vital process for obtaining energy (Deberardinis et al. 2007). Cancer cells heavily rely on glutamine as an energy substrate and use it to synthesize nucleotides and other amino acids (Wu et al. 2020). Studies on bladder cancer suggest that glutamine may promote tumor metabolism and increase the aggressiveness of cancer cells. However, the mechanisms and effects of glutamine metabolism in cancer are still being actively researched (Sun et al. 2019). More recently, further studies have provided knowledge of the potential use of glutamine as a biomarker for bladder cancer (Alba Loras et al. 2019a). Our findings, which demonstrate elevated levels of glutamine in tumor tissue, are in line with numerous prior studies that have revealed a significant increase in glutamine levels in serum and urine samples from bladder cancer patients in comparison to controls (Bansal et al. 2013; Wittmann et al. 2014). Furthermore, a previous NMR-based study of bladder cancer tissue also reported higher glutamine levels in tumor tissue relative to benign disease (Tripathi et al. 2013). Furthermore, in time series metabolomics analyses of urine and serum samples obtained from bladder cancer patients pre- and post-resection, glutamine demonstrated significant potential in differentiating neoplastic samples from healthy ones (Gupta et al. 2020; Jacyna et al. 2022).
Another potentially important marker of BC is the glutamate, which is a fundamental metabolite in the human body derived from alpha-amino acid anions and is the conjugate base of glutamic acid. It contains anionic carboxyl groups and a cationic amino group and plays a crucial role in both normal and abnormal brain functioning, as well as in peripheral organs (Danbolt 2001). Cancer cells alter metabolic pathways, shifting glucose conversion towards pathways required for cell proliferation and leading to increased synthesis of proteins, including glutamine (Guin et al. 2014). This feature is consistent with the increased level of glutamate observed in the samples of diseased individuals, indicating that glutamate could be a useful biomarker for BC with high diagnostic value and the ability to report on disease recurrence (Y. Cheng et al. 2015b). This conclusion was also confirmed by our analysis, which found that the tissue level of glutamate is higher in cancer tissue than normal tissue. Similarly, Wittmann et al. found that urinary levels of glutamate were significantly elevated in patients with bladder cancer compared to healthy controls, indicating their potential as diagnostic biomarkers for the disease (Wittmann et al. 2014).
Hypoxanthine is a natural purine base that is produced during purine degradation and can be converted to xanthine and uric acid while generating reactive oxygen species through the action of the xanthine oxidase enzyme (Lawal and Adeloju 2012). Due to its diminutive and polar structure, hypoxanthine can easily accumulate in biological fluids and tissues, making it a potential indicator for medical diagnosis (Garg et al. 2022). Specifically, hypoxanthine is a significant product that is generated during the breakdown of nucleotides and can serve as a precursor of uric acid and is an intermediate in the breakdown of purines (Pasikanti et al. 2010). As such, its quantification is highly valuable for relevant clinical diagnoses (Dervisevic et al. 2016). Furthermore, increased levels of hypoxanthine are associated with decreased levels of uric acid, adenosine, and inosinic acid. In cancer cells, this pathway is often disrupted, leading to the accumulation of hypoxanthine. Therefore, the measurement of hypoxanthine levels can not only serve as a potential biomarker for medical diagnosis, but may also provide important information about the underlying metabolic changes in cancer cells (Rodrigues et al. 2016). In our studies, hypoxanthine levels were higher in cancer tissue compared to normal. This compound has also been previously detected in higher amounts in the urine and serum of BC patients and suggested to be a potential bladder cancer biomarker (Alberice et al. 2013; Gao et al. 2012; Hu et al. 2021; A. Loras et al. 2018; Tan et al. 2017; Wittmann et al. 2014).
Serine and threonine are amino acids that were also found to be in high concentrations in BC tissue compared to normal in our study. Serine is an endogenous amino acid that plays a significant role in various biosynthetic pathways in the human body, such as pyrimidine, purine, creatine, and porphyrin biosynthesis. Cancer cells utilize serine as the primary source of one-carbon units, which are necessary for the production of cellular components and proliferation (Newman and Maddocks 2017). Additionally, serine protease is involved in tumor invasion and metastasis in oncogenesis (Sanguedolce et al. 2015). On the other hand, threonine is an essential amino acid that is crucial for the formation of various building blocks of proteins, including tooth enamel, collagen, and elastin. It also plays an essential role in the nervous system and several metabolic pathways. Both serine and threonine are critical elements of a serine/threonine-protein kinase, which has been identified as a potential biomarker for bladder cancer (Hentschel et al. 2021). The increased levels of both serine and threonine in our study were also observed by other researchers in blood serum (Amara et al. 2019; Vantaku et al. 2019) and also in urine (Kim et al. 2010).
Utilizing modified silver-109 targets in LDI-MS experiments enabled to measure the amount of polar and non-polar metabolites in tissue extracts. By employing this approach, analysis of tissue metabolites using MS allowed for the identification of 31 compounds that exhibited lower abundance in cancer tissue in comparison to normal while one compound was found to be present in higher concentrations. Most of these compounds were putatively identified as peptides and lipids. Among the lipids found to be elevated in the normal tissue of BC patients, five belonged to the fatty acyl class, while the remaining three were classified as diradylglycerols, and one as a glycerophosphocholine.
The level of lipids in cancerous tissues can be influenced by various factors, including altered metabolism, changes in lipid transport and uptake, and increased utilization of lipids for energy production (C. Cheng et al. 2018). Cancer cells tend to exhibit increased aerobic glycolysis, also known as the Warburg effect, which can result in a reduction of lipid biosynthesis and accumulation in the cells (Broadfield et al. 2021). Additionally, cancer cells may rely on increased uptake of lipids from the extracellular environment to support their growth and proliferation. Moreover, cancer cells can utilize lipids as an energy source, which may contribute to a decrease in lipid levels in the tissue (Menendez and Lupu 2007).
In an effort to identify cellular markers that could distinguish between the various grades and stages of BC, several metabolomics studies of urine and blood of BC patients have been reported (Di Meo et al. 2022; Petrella et al. 2021). To our knowledge, however, only three studies have investigated the connections between changes in metabolite levels in tissues from BC patients and the distinct grades and/or stages of tumor development (Piyarathna et al. 2018; Sahu et al. 2017; Tripathi et al. 2013).
In our study, significantly higher concentrations of lactate and ethanolamine were measured in the HG cancer tissue of BC patients compared to the levels found in the normal tissue group (Fig. 3, Table 1). We found that lactate is one of the most differentiating metabolites between normal and neoplastic tissue, regardless of the stage of cancer. Ethanolamine is a component of certain phospholipids that make up the structure of cell membranes and plays an important role in the structure and function of cell membranes. These lipids are also involved in cell signaling and other cellular processes (Vance and Tasseva 2013). In some types of cancer, there is evidence that the levels of ethanolamine differ significantly between samples collected from cancer patients compared to controls (Swanson et al. 2008). The higher level of ethanolamine may be related to the increased cell proliferation and growth that is characteristic of cancer. Cancer cells may require more ethanolamine to support the synthesis of new cell membranes and other cellular structures as they divide and multiply (M. Cheng et al. 2016).
Our research has identified a panel of 11 metabolites that, when considered together, may be good discriminators of low-grade cancer tissue versus adjacent normal tissue in bladder cancer patients. These metabolites include lactate, alanine, choline, glutamine, hypoxanthine, leucine, methionine, phenylalanine, serine, threonine, and tyrosine, eight of which are alpha-amino acids.
One of the most differentiating compounds between LG cancer and normal tissue from BC patients is choline. It is a crucial water-soluble quaternary amine that is often classified as a B vitamin due to its similar chemical structure. Choline has several important functions within the human body, particularly in neurochemical processes (Tayebati et al. 2017). Choline plays a critical role in the production of phospholipids and the metabolism of triglycerides, making it essential for the proper structure and function of cell membranes. Our study found that cancer tissue from patients with bladder cancer had higher levels of choline compared to normal tissue, which could be due to increased absorption of choline by cancer cells. Our findings are consistent with previous research demonstrating that cancer cells tend to increase fatty acid synthesis, which can then act as a substrate for phosphatidylcholine synthesis, leading to its elevation in tumor cells (Koundouros and Poulogiannis 2019; Saito et al. 2022). Interestingly, we observed the same trend in urine samples, with increased levels of choline observed in patients with BC (Li et al. 2021; Alba Loras et al. 2019b). Moreover, one of our previous studies revealed that the increase in tissue choline levels among cancer patients is consistent with the decrease in choline levels found in the serum of patients with BC compared to control individuals (Ossoliński et al. 2022).
Our current study has indicated that tissue-based metabolite profiling can accurately discriminate different stages of cancer tissue (pTa and pT1) from normal tissue from BC patients (Table 1, Fig. 4). In the tissue extracts of patients with pTa and pT1 stages of BC, we identified 13 significant metabolites that were good discriminators of the different cancer stage groups compared to the normal tissue group, most of which are alpha-amino acids and have also been reported in the literature, as described above, in relation to the occurrence of cancer.