Head and/or Neck Cancer Deviates the Tryptophan, Arginine and Glutamine Pathways: Applications in Diagnosis and Prognosis

Abstract

Background: This study aimed to identify metabolic alterations in the tryptophan, arginine, and glutamine pathways in patients with head and neck carcinoma (HNC) that could provide diagnostic and prognostic biomarkers.

Materials and methods: Serum levels of tryptophan, kynurenine, arginine, putrescine, glutamate, and glutamine were measured using micellar electrokinetic chromatography with laser-induced fluorescence detection (MEKC-LIFD). The study cohort included five treatment-naïve HNC patients, four treated HNC patients, three patients with benign tumors, and seven healthy controls.

Results: Serum concentrations of tryptophan and kynurenine were significantly elevated in both untreated and treated HNC patients compared to controls (p = 0.01 and p = 0.002, respectively). While both metabolites remained elevated post-treatment compared to controls (p = 0.002 and p = 0.0003, respectively), tryptophan decreased significantly after treatment. Serum arginine was significantly higher in patient groups versus controls (p = 0.04 and p = 0.002) but did not change with treatment. In contrast, serum putrescine increased after treatment compared to both controls (p = 0.035) and untreated patients (p = 0.05). The arginine/putrescine ratio was significantly lower in untreated versus treated patients. Furthermore, untreated HNC patients exhibited a significant decrease in glutamine and a significant increase in glutamate, a finding not present in treated patients. Serum glutamate was also elevated in benign tumor patients (p = 0.04).

Conclusion: These findings reveal significant dysregulation of tryptophan metabolism in HNC and identify serum tryptophan and putrescine as potential markers for monitoring treatment efficacy. Altered arginine metabolism and a pronounced shift in the glutamine-glutamate axis, indicative of increased glutaminolysis, further characterize the metabolic profile of active HNC tumors.

Graphical abstract

Keywords

head and neck cancer / metabolic biomarkers / tryptophan / kynurenine / arginine / glutamine / capillary electrophoresis

Introduction

Head and neck cancers are a heterogeneous type of tumors found in the oral cavity, oropharynx, hypopharynx, larynx, and several other subregions[1]. Approximately 750,000 new cases and 360,000 deaths occur worldwide each year[2]. Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck cancer[3]. HNSCC accounts for approximately 3% of new cancer cases and 3% of deaths worldwide[4]. Approximately 30% to 40% of patients with HNSCC have early-stage (stage I/II) disease at diagnosis[4]. More than 60% of patients with head and neck squamous cell carcinoma are initially diagnosed as locally advanced[4]. In summary, HNSCC has a high risk of local recurrence and a poor prognosis and is generally treated with combinations of surgery, radiation therapy (RT), and systemic therapy[5].

Advances in technology and computing now enable high-throughput analysis of multiple domains of biological information, including the genome, transcriptome, proteome, and metabolome[6].

Metabolomics is a rapidly growing field with potential applications across diverse disciplines. Metabolomics has received special attention in biomarker discovery and diagnostics[7]. Among the small molecules, arginine seems to play a crucial role in head and neck carcinoma (HNC)[8]. In addition, arginine succinate synthetase, the main enzyme for arginine synthesis, is highly expressed in HNC[9]. Consequently, it has been proposed that arginine deprivation should be used as a new treatment strategy for HNC[10]. Furthermore, one of the main metabolites of arginine is putrescine and this polyamine has been found elevated in HNC tumor cells[11]. Based upon these findings, we explored two small molecules, i.e., arginine and putrescine in HNC patients.

Another small molecule of interest is tryptophan, an essential amino acid which main metabolic product (quantitatively speaking) is kynurenine[12]. The synthesis of kynurenine is accomplished by the enzyme indoleamine 2,3-dioxygenase which plays a role in HNC[13]. The kynurenine pathway and associated catabolites promote tumor progression and modulate the host’s antitumor immune response[14]. For these reasons, considerable attention has been paid to the role of the enzyme indoleamine 2,3-dioxygenase 1 and its catabolite, kynurenine. These advances in our understanding of the tumor-promoting role of the kynurenine pathway have led to the development of new therapeutic strategies to achieve anticancer effects and reverse immune escape[15]. In view of these facts, we included tryptophan and kynurenine analysis in the present study of small molecules relevant to HNC.

Finally, glutamine, another small molecule, has become a target for metabolomic studies in HNC because the tumor cells switch toward glutamine degradation as their main way to fulfill its energetic needs[16]. In tumor cells, glutamine is converted first to glutamate and then to α-ketoglutarate and incorporated to the tricarboxylic acid cycle to generate energy[17]. These findings led us to include the analysis of glutamate and glutamine in the present study.

The metabolic landscape of cancer is characterized by profound reprogramming to support rapid proliferation, survival, and immune evasion. This study simultaneously investigates three interconnected amino acid pathways—tryptophan, arginine, and glutamine—that are frequently co-opted in tumors. The tryptophan-kynurenine axis is a key immunosuppressive pathway, arginine metabolism fuels polyamine synthesis for cell growth, and the glutamine-glutamate axis (glutaminolysis) provides energy and biosynthetic precursors. Investigating these pathways together provides a more holistic view of the metabolic adaptations in HNC and their collective impact on tumor biology and patient immunity.

Materials and methods

Patient cohort and clinical characteristics

This study included a total of 19 subjects. The patient groups consisted of five treatment-naïve patients with histologically confirmed HNC, four HNC patients who had completed a primary course of therapy, and three patients with benign head and neck tumors. A control group of seven healthy subjects with no history of cancer or chronic inflammatory diseases was also recruited.

Newly diagnosed, treatment-naïve patients with HNC were eligible for the untreated group. Exclusion criteria for all participants included a prior history of other malignancies, severe renal or hepatic dysfunction, and current use of immunomodulatory drugs.

Patients in the “treated” group had completed a primary course of therapy. The treatments included surgery alone (n = 2), radiotherapy with concurrent chemotherapy (n = 1), or surgery followed by adjuvant radiotherapy with concurrent chemotherapy (n = 1). Blood samples for this group were collected during follow-up visits at a median of 3 months (range: 1–6 months) after the completion of their primary treatment.

The distribution of tumor types is summarized in Table 1. A detailed summary of the available clinical characteristics for the HNC patients is provided in Table 2. We note that performance status was not systematically recorded for all patients, which we acknowledge as a limitation in the Discussion.

Chemicals and reagents

L-arginine, putrescine, tryptophan, kynurenine, glutamate, glutamine, fluorescein isothiocyanate (FITC) isomer 1, sodium bicarbonate, sodium carbonate, and sodium hydroxide were purchased from Sigma Chemical Co. Acetone and acetonitrile were purchased from J.T. Baker. Milli-Q water was used throughout.

Apparatus, electrophoretic conditions, and data acquisition

Measurements were performed on an R2D2-1 CZE-LIFD apparatus (Meridialysis Co., Mérida, Venezuela)[18] equipped with a 40 mW solid-state laser (Cobolt, Sweden) and a 50 cm fused silica capillary with an ID of 40 µm and OD of 150 µm. The 488 nm wavelength was used. Samples were hydrodynamically injected into the anodic end of the capillary by applying a negative pressure of −12 psi at the cathodic end of the capillary for 1 s. After injection, the anodic end of the capillary was immersed in a reservoir filled with borate–SDS buffer equipped with a platinum–iridium electrode while the cathodic end remained in the borate–SDS buffer filled reservoir at the cathode. A positive 27 kV was applied at the anode for electrophoretic separation. This procedure created an electroosmotic flow of 0.75 m²·V⁻¹·s⁻¹ in the anode-cathode direction with the suspended SDS micelles as a pseudo stationary phase for solvophobic separation.

Data were collected for 30 min at 41 points per second and digitally filtered using the moving average method. Peak heights were measured, as this proved to be a satisfactory and reliable measurement proportional to the analyte concentration. Results were analyzed by curve fitting using SPSS 9.0 for Windows. The significance level was set at p = 0.05.

After each analysis, the capillary was washed with 1 mol/L NaOH (4 min), followed by 18 mΩ water (4 min), and 40/20 mmol/L borate–SDS buffer (5 min). All solutions were filtered through 0.22 µm pore-size membranes before injection into the capillary.

Identification of arginine, putrescine, tryptophan, kynurenine, glutamate, and glutamine

One milligram of each analyte was dissolved in 1 mL of 20 mmol/L carbonate buffer (prepared by mixing 5 mL of 200 mmol/L sodium carbonate solution with 5 mL of 200 mmol/L sodium bicarbonate solution and water to 100 mL). A derivatization solution was prepared by dissolving 1 mg of FITC in 1 mL of acetone and mixing it with 1 mL of 20 mmol/L carbonate buffer. Five microliters of the FITC derivatization solution were added to each analyte dilution. A blank solution was prepared by adding 5 µL of the FITC derivatization solution to 1 mL of 20 mmol/L carbonate buffer. A mixture of the sample and the derivatized analyte was injected and the corresponding chemical was identified by enlarging a peak in the electropherogram.

Blood sample treatment

Blood samples were collected from all subjects after an overnight fast (≥ 8 h) to minimize the confounding effects of recent dietary intake on serum metabolite levels. The blood sample was collected in a tube and centrifuged for 5 min at 375 g to separate the plasma from the cells. A 200 µL volume of the supernatant was mixed with 200 µL of cold acetonitrile to precipitate proteins. The sample was centrifuged again at 375 g for 5 min, and 20 µL of the supernatant was collected and reacted with 20 µL of the derivatization solution. For the detection of amino acids and their metabolites, an aliquot (20 µL) of each sample was derivatized with an equal volume of the FITC solution in acetone. After 16 h in the dark, the samples and standards were ready for CZE analysis.

Data analysis

The electropherograms were analyzed using software capable of filtering with a digital moving average filter and horizontal and vertical shifting capabilities for automatic electropherogram alignment. The same software also averages an unlimited number of electropherograms[19]. Statistical comparisons between multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for pairwise comparisons. For comparisons between two groups, unpaired or paired two-tailed Student’s t-tests were used as appropriate. A p-value < 0.05 was considered statistically significant. Given the exploratory nature of this study and the small sample size, which makes classic corrections for multiple comparisons (e.g., Bonferroni) overly conservative, we report uncorrected p-values but interpret the results with caution, emphasizing the need for validation in larger cohorts.

Results

In the tryptophan pathway (see Figure 1), serum tryptophan levels in patients with malignant tumors were significantly higher than in the serum of healthy controls (133 ± 20.7 μmol/L vs. 57 ± 2.7 μmol/L, respectively), and the probability value in favor of the null hypothesis (p) was p = 0.0095. In treated patients, serum tryptophan levels remained significantly higher than in healthy controls (post-treatment patients: 78.4 ± 2.7 μmol/L vs. healthy controls: 57.0 ± 2.7 μmol/L, p = 0.0015). However, tryptophan levels decreased with treatment (pre-treatment: 133.0 ± 20.7 μmol/L vs. post-treatment: 78.4 ± 2.7 μmol/L, and the difference was significant, p = 0.0292). The difference in serum tryptophan levels between patients with benign tumors (100.0 ± 16.0 μmol/L) and healthy controls (57.0 ± 2.7 μmol/L) was not significant (p = 0.1) and the difference in serum tryptophan levels between patients with malignant tumor (133.0 ± 20.7 μmol/L) and patients with benign tumor (100.0 ± 16.0 μmol/L) was also not significant (p = 0.2).

Quantitatively, the main metabolite of tryptophan is kynurenine. The differences in serum kynurenine levels in the four groups studied followed the same trend as those for tryptophan. Serum kynurenine levels were significantly higher in patients with malignant tumor (3.6 ± 0.3 μmol/L) vs. healthy controls (1.5 ± 0.2 μmol/L) both before treatment (p = 0.0004) and after treatment (3.1 ± 0.2 μmol/L, p = 0.0051), but were not modified with treatment (patients with malignant tumor before treatment, 3.6 ± 0.3 μmol/L vs. patients with malignant tumor after treatment, 3.1 ± 0.2 μmol/L, NS). There was also no significant difference between patients with benign tumors and healthy controls (2.6 ± 0.3 μmol/L vs. 1.5 ± 0.2 μmol/L, respectively, NS), nor between patients with malignant tumor vs. patients with benign tumor (NS).

The metabolites studied in the arginine pathway were arginine and putrescine (see Figure 2). Serum arginine levels were significantly increased in patients with malignant tumor both before treatment (patients with malignant tumor before treatment: 170.2 ± 11.8 μmol/L vs. healthy controls: 83.1 ± 17.9 μmol/L, p = 0.002) and after treatment (patients with malignant tumor treated: 318.8 ± 17.9 μmol/L vs. healthy controls: 83.1 ± 17.9 μmol/L, p = 0.05.

Serum arginine levels were not different when comparing patients with benign tumors with healthy controls (601.9 ± 289 μmol/L vs. healthy controls: 83.1 ± 17.9 μmol/L respectively, NS) or with patients with malignant tumors (601.9 ± 289.0 μmol/L vs. 170.2 ± 11.8 μmol/L respectively, NS). There was also no significant difference between patients with malignant tumors before vs. after treatment (170.2 ± 11.8 μmol/L vs. 318.8 ± 71.0 μmol/L respectively, NS).

Serum putrescine levels were only significantly different in treated patients with malignant tumors vs. healthy controls (0.99 ± 0.20 μmol/L vs. 0.23 ± 0.03 μmol/L, respectively, p = 0.033) and in treated patients before vs. after treatment (0.34 ± 0.08 μmol/L vs. 0.99 ± 0.2 μmol/L, respectively, p = 0.045). Other comparisons were not statistically significant.

Given that arginine is a precursor to putrescine via the action of arginase and ornithine decarboxylase (ODC), we hypothesized that the ratio of serum arginine to putrescine could provide a more integrated view of the metabolic flux through this pathway and its potential dysregulation in HNC. Interestingly, significant differences were found when comparing the ratios of patients with malignant tumors vs. healthy controls (637.6 ± 101 vs. 338.4 ± 57, respectively, p = 0.014) and patients with malignant tumors before and after treatment (637.6 ± 101.0 vs. 324.0 ± 62.0, respectively, p = 0.0264).

In the glutamate pathway we measured glutamate and found a statistically significant difference when comparing the serum levels of patients with malignant tumors vs. healthy controls (see Figure 3) (85.0 ± 15.8 micromolar vs. 49.7 ± 10.0 μmol/L respectively, p = 0.0285) and benign tumors vs. healthy controls (133.5 ± 16.0 μmol/L vs. 49.7 ± 10.0 μmol/L respectively, p = 0.04).

In contrast to the variation in serum glutamate, the concentration of serum glutamine was significantly lower in the patients with malignant tumors vs. healthy controls (7.1 ± 2.6 micromolar vs. 25.1 ± 5.3 μmol/L respectively, p = 0.0265). Patients with benign tumors had higher serum glutamine concentrations than patients with malignant tumors (27.5 ± 8.7 micromolar vs. 7.1 ± 2.6 micromolar respectively, p = 0.016). Notably, there was no significant difference in serum glutamine levels between patients with benign tumors and healthy controls (27.5 ± 8.7 μmol/L vs. 25.1 ± 5.3 μmol/L, p = 0.78), underscoring that glutamine depletion is a specific feature of malignant HNC tumors in our cohort.

Discussion

In the patients with malignant tumors included in this study, both tryptophan and kynurenine levels are significantly increased, and this phenomenon has been reported in patients with various types of cancer[20–23]. We found significantly increased serum tryptophan levels in HNC patients, both in untreated and treated malignant tumors, but not in patients with benign head or neck tumors. Being an essential amino acid, tryptophan is primarily acquired through the diet, and a small amount is synthesized by intestinal microorganisms[24]. Furthermore, the process of protein destruction in autophagosomes and lysosomes produces tryptophan in a sort of recycling process[25]. Consequently, an increase in serum tryptophan may be caused by an increase in protein intake, a decrease in tryptophan metabolism, overproduction by the intestinal bacterial flora, or an increase in autophagy[26]. In the case of patients with HNC, an increase in dietary intake can be ruled out because most of these patients are anorexic, and increased serum tryptophan has been reported in patients with other types of cancer and severe anorexia[27]. The contribution of the intestinal flora is too small to explain the increase in serum tryptophan in question. As we will see later, tryptophan metabolism is greatly increased in cancer patients, which should lead to decreased serum tryptophan levels in HNC patients. Therefore, the observed increase in serum tryptophan in HNC patients is paradoxical and suggests a complex dysregulation. While increased dietary intake can be ruled out due to cancer-associated anorexia, and the contribution of intestinal flora is likely minor, other mechanisms such as reduced hepatic clearance or, notably, an increase in autophagy with massive protein destruction, could be contributing factors[26,28]. Interestingly, increased autophagy in cancer cells has been reported[28] and is considered a key factor in tumor survival and metastasis. While our data does not directly measure autophagic flux, the elevated tryptophan levels are consistent with this hypothesis, which should be investigated in future studies.

However, increased serum tryptophan has several favorable consequences for cancer cell proliferation and adverse consequences for the patient. There is abundant evidence that tryptophan and its metabolites contribute to blocking the mechanism by which our immune system recognizes and eliminates cancer cells. Indeed, tryptophan is the substrate of the enzyme indoleamine dioxygenase (IDO), which transforms it into kynurenine. We found a significant increase in serum kynurenine in patients with HNC. In these patients, IDO activity is increased[29], which explains the increased kynurenine we found in our HNC patients. The consequences of these phenomena are unfavorable for patients. Indeed, kynurenine (the most abundant metabolite of tryptophan) is immunosuppressive. It is an agonist of the alkylated hydrocarbon receptor (AHR)[30], a transcription factor that directly interacts with the genome to promote gene expression of the proteins PD-L1[31], CTLA-4[32], Lag3[33], and CD39[34], all of which are involved in suppressing the immune response against cancer cells. We found that serum kynurenine concentration remained elevated in HNC patients after treatment. This persistent elevation, likely reflecting continued IDO activity, could contribute to a sustained immunosuppressive microenvironment, potentially impacting long-term survival and response to immunotherapy[39]. This underscores the need for further research into the specific kynurenine metabolites and enzymes active in HNC to develop selective therapeutic blockers. This significantly high elevated concentration of kynurenine might contribute to the poor overall 5 years survival of HNSCC patients.

From this, it follows that blocking kynurenine production can cure HNC. Unfortunately, the effects of kynurenine on HNC are not so straightforward[35]. Experimentally, IDO has been blocked and kynurenine decreased in mice with HNC with the intention of producing inflammation that would decrease tolerance to cancer cells, and the results do not show elimination of symptoms or the tumor[35]. The problem is that kynurenine’s actions are driven by its metabolites, and these result from two different metabolic pathways. In one pathway, kynurenine can be transformed into 3-hydroxykynurenine and in another it can be transformed into kynurenic acid[36]. 3-Hydroxykynurenine causes immunotolerance[37] while kynurenic acid causes inflammation[38]. It is then possible that when IDO is inhibited to decrease the tolerance produced by 3-hydroxykynurenine, the inflammation produced by kynurenic acid is also being inhibited. The present study suggests that kynurenine metabolites and the main enzymes in HNC should be studied in order to develop selective blockers of kynurenine metabolic pathways that are adjuvants to surgical treatment and immunotherapy of HNC[39].

The arginine pathway also showed significant changes in HNC patients. In general, patients had elevated serum arginine concentrations, both in untreated and treated patients, and one of its most important metabolites, putrescine as well. We found few reports on serum arginine concentrations in patients with HNC. Liu et al. reported that elevated serum arginine levels significantly increase the risk of HNC, but when these increases were corrected for with other risk factors, the association was no longer statistically significant[40]. This means that they found patients with elevated serum arginine and patients with low serum arginine concentration. We also found high variability of serum arginine concentration in HNC patients. In the HNC patients examined in the present work, there was a large dispersion of the data both in patients with untreated malignancies (130 μmol/L to 560 μmol/L) and in treated patients (94 μmol/L to 517 μmol/L) while in healthy controls the dispersion was smaller and the levels lower (16 μmol/L to 158 μmol/L with a mean of 83 μmol/L). Furthermore, the high variability in serum arginine levels within the benign tumor group, likely attributable to the small sample size, precluded the finding of a statistically significant difference compared to controls despite a large difference in means. Therefore, we consider this to be the first report of a significant increase in serum arginine levels in HNC patients. Such an increase may be explained by an exaggerated intake of arginine, a decrease in arginine metabolism, an increase in autophagy or by an increase in de novo synthesis of arginine[41]. Since patients with HNC are anorectic and dysphagic, and their arginine metabolism is elevated (as demonstrated by an increase in one of its metabolic products, putrescine), elevated of de novo arginine synthesis and increased autophagy are the most plausible explanations. Arginine synthesis in our body is due to the conversion of the amino acid citrulline into an arginine precursor called argininosuccinato by an enzyme called argininosuccinato synthetase (ASS). This metabolic product is then converted into arginine by another enzyme called argininosuccinato lyase. It has been reported that HNC tumors might have very low levels of argininosuccinato synthetase or very high levels of this enzyme. The former are called auxotrophic because their arginine supply depends on diet, while the latter are called non-auxotrophic for obvious reasons[8]. The heterogeneity of ASS1 expression in HNC tumors[8,9] is a critical factor that could explain the high variability in serum arginine levels we observed. This heterogeneity has profound therapeutic implications, as ASS1-deficient (auxotrophic) tumors may be vulnerable to arginine deprivation therapies, whereas ASS1-proficient (non-auxotrophic) tumors would be resistant[10]. It has been shown that the survival of patients with HNC is inversely proportional to the levels of argininosuccinato synthetase in their tumor cells[10]. This observation has both therapeutic and prognostic consequences. Therapeutic because if arginine promotes tumor growth and the tumor cells cannot produce it (they are auxotrophic) by limiting the supply of arginine or blocking its uptake by the tumor cells, the cells would be forced to enter programmed cell death. On the contrary, in non-auxotrophic tumor cells, reducing the supply of arginine should not have antitumor effects because they can produce the arginine they need. And HNC patients with high serum arginine levels will have a worse prognosis[10,42,43]. Arginine is metabolized by several metabolic pathways. One of the most prominent is the polyamine pathway. The enzyme arginase generates urea and ornithine from arginine. Ornithine is converted to putrescine by the enzyme ODC. Putrescine is then metabolized to produce spermine and spermidine and several highly active molecules derived from these 3 polyamines that are involved in cancer[44]. Although serum putrescine levels have not been reported in HNC patients, changes in the metabolites of this polyamine have been reported[44]. Specifically, urinary concentrations of the metabolites N-acetyl spermine, N-acetyl spermidine, and N, N-Acetildiespermidina were increased in HNC patients. The increased levels of polyamine metabolites in these patients should be accompanied by increased ODC activity. This phenomenon (increased ODC) has been reported in tumor biopsy cells from patients with HNC[45]. These authors found significantly higher levels of this enzyme in biopsies in which the malignant cells were poorly differentiated vs. those containing highly differentiated cells. This indicates that the degree of malignancy of HNC tumors is greater as their cells contain greater amounts of ODC, and therefore they should generate greater amounts of putrescine. In the present study, we did not find an increase in serum putrescine in patients with HNC. This indicates that more putrescine is being produced than is produced in the metabolism of normal cells, but that it is being rapidly metabolized, and for this reason, its metabolic products increase in patients with HNC. However, serum putrescine was significantly increased in patients who had been treated for HNC. This indicates that the arginine/putrescine ratio should decrease after treatment. Indeed, we found that this ratio was significantly increased in untreated HNC patients and significantly reduced in patients treated for HNC.The present work adds the Arginine/Putrescine ratio as a new marker of the effectiveness of HNC treatment.

Serum glutamate concentration was significantly higher (p = 0.0256) when comparing serum levels of HNC patients with healthy controls. The same finding has been previously reported[46]. There is abundant evidence that T lymphocytes express glutamate receptors on their surface[47] and that during T lymphocyte activation that occurs during the inflammatory response, the expression of glutamatergic receptors on CD4⁺ and CD8⁺ T cells increases[48]. Furthermore, in the cited work they show that the blockade of metabotropic and AMPA receptors decreases the ability of T lymphocytes to kill tumor cells in vivo.

Besides this immunological effects, glutamate is the metabolic precursor of glutamine due to the action of the enzyme glutamine synthetase that transforms glutamate into glutamine[49], which is considered a powerful supplier of energy and nitrogen for tumor cells in HNC[50]. An increase in glutaminase has been reported in HNSCC patients and such over expression of the glutaminase gene causes what has been named “glutamine addiction” in cancer cells[46]. The main consequence is that reducing glutamine metabolism can be a powerful tool to combat HNSCC because it increases glutamate which is pro-inflammatory and decreases one of the main energy suppliers for cancer cells which is glutamine.

This study has several limitations that must be acknowledged. The most significant is the small sample size, particularly in the patient subgroups (6 untreated HNC, 4 treated HNC, and 3 benign tumors), which limits statistical power, increases the risk of Type I and Type II errors, and restricts generalizability. Additionally, we did not perform detailed assessment of long-term dietary habits, nor did we directly measure autophagic activity, protein intake, gut flora composition, or hepatic/renal clearance rates, which prevents definitive mechanistic conclusions about the observed metabolic alterations. The heterogeneity in tumor types and treatments, while reflecting real-world clinical diversity, may introduce confounding variables. Furthermore, performance status was not systematically recorded for all patients. While we applied appropriate statistical tests for our cohort size, the exploratory nature of this study and small sample size made classic multiple comparisons corrections overly conservative. Therefore, all significant findings should be interpreted as preliminary and require validation in larger, well-characterized cohorts.

Conclusion

Tryptophan metabolism plays a pivotal role in HNC pathogenesis, serving as both a biomarker and therapeutic target. Our findings suggest that: elevated serum tryptophan reflects enhanced autophagy in HNSCC, providing substrates for tumor proliferation while paradoxically fueling immunosuppressive kynurenine production. Persistent kynurenine elevation post-treatment confirms IDO-driven immune evasion via chronic AHR activation and checkpoint induction (PD-L1/CTLA-4), necessitating selective pathway blockers. The arginine/putrescine ratio emerges as a dynamic biomarker of therapeutic response, with post-treatment decreases indicating reduced polyamine demand in tumor stress states.

Glutamine-to-glutamate shift signifies bioenergetic reprogramming that impairs T-cell cytotoxicity while supporting tumor growth—a vulnerability exploitable through glutaminase inhibition. Finally, our pilot study identifies significant dysregulations in the tryptophan, arginine, and glutamine pathways in HNC patients. We propose serum tryptophan and the arginine/putrescine ratio as potential dynamic biomarkers for monitoring disease activity and treatment response. The pronounced shift in the glutamine-glutamate axis signifies bioenergetic reprogramming in active tumors. Finally, the MEKC-LIFD technique used here is a relatively inexpensive and effective method for quantifying these metabolites. This work provides a foundational rationale for future larger-scale studies to validate these findings and explore the integration of such metabolic profiles with other data types for improved diagnosis and prognosis of HNC, particularly in resource-limited settings.

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