Background
Children with cancer receiving intensive chemotherapy frequently report cooccurring psychoneurological symptoms (PNS), including pain, fatigue, anxiety, depression, and cognitive dysfunction [
1]. Collectively, these symptoms are defined as the PNS cluster, which can develop up to 6 months after treatment and even continue into survivorship [
2]. Unfortunately, poor management and treatment of PNS can significantly reduce a child’s quality of life (QOL) and future psychosocial functioning [
3,
4].
A symptom experience framework presented by Hockenberry and Hooke identified multiple antecedents that influence children’s experience of PNS across cancer treatment, including personal (e.g., sex and developmental stage), environmental (e.g., child’s hospitalization), and disease-related (e.g., type of cancer, length of treatment, treatment frequency, and chemotherapy drugs) factors [
5]. Subsequent literature proposed that the PNS cluster may share common biological mechanisms [
6], such as proinflammatory cytokines (e.g., IL-6 and TNF-α), Hypothalamic–Pituitary–Adrenal (HPA) axis, and monoamine neurotransmission system [
7‐
9]. Nevertheless, the biological mechanisms of the PNS cluster are still largely unknown in cancer populations, particularly in pediatric oncology [
10]. Recently, investigations of the microbiome–gut–brain (MGB) axis [
11,
12] suggest that the gut microbiome (i.e., a collection of microorganisms and their genomes in the gastrointestinal tract) can signal the brain via functional metabolites and activation of other pathways (e.g., neurotransmitters), ultimately resulting in PNS for patients with cancer receiving chemotherapy [
13,
14].
Chemotherapy has the potential to negatively interfere the MGB axis through a diverse set of pathways, including dysregulating the diversity and composition of bacteria in lumen, altering the gut microbiome-derived metabolites, and activating neuroimmune signaling [
11,
12,
15]. As a commonly used treatment modality in children with cancer, chemotherapy can potentially lead to PNS via the MGB axis. Although limited, promising work has demonstrated enriched abundance of
Bacteroides among adult patients with low PNS and enriched abundance of
Blautia for those with high PNS [
16]. Additionally, adult patients with head and neck cancer with high PNS had higher abundance of gut microbial Bacteroidota,
Ruminiclostridium, and
Tyzzerella compared to those with low PNS, while patients with low PNS had higher abundance of
Lactococcus and
Phascolarctobacterium compared to those with high PNS [
13]. However, the role of the gut microbiome in PNS for children with cancer (CWC) has yet to be elucidated [
17].
Microbiome-derived metabolites represent the functional role of the gut microbiome, as they are the drivers of gut–brain communication and carry out signals of a disturbed gut microbiome [
18]. Communications between the gut and the brain occur following a network of pathways involving key microbial metabolites, such as short-chain fatty acids (SCFAs) [
19] and tryptophan for kynurenine pathway metabolism [
18]. SCFAs are part of a group of key microbial metabolome pathways associated with psychological functioning [
20]. Alterations in the SCFA metabolism can result in disturbances to the central nervous system [
21], although the effects of SCFAs on PNS have primarily been studied in animal models [
20]. Additionally, tryptophan, an essential amino acid, is another key metabolite in the MGB axis, with dual emphasis on the regulation of serotonin and melatonin synthesis, and the control of kynurenine pathway [
18,
22]. Tryptophan must be obtained from dietary or microbial sources [
18] and can be synthesized from chorismate by bacterial phyla Pseudomonadota, Actinomycetota, and Bacillota [
23].
In humans, untargeted metabolomics analysis showed that increased pain was associated with decreased tryptophan, and increased fatigue was associated with decreased arachidonic acid [
24] in women with breast cancer receiving chemotherapy. Targeted metabolomics analysis further indicated moderate-to-strong correlations between changes in pain and tryptophan, as well as between changes in depressive symptoms and serotonin levels [
24,
25]. Decreased tryptophan, increased kynurenine, and subsequent altered tryptophan/kynurenine ratio were associated with a higher level of PNS among cancer survivors [
25]. Among children with cancer receiving chemotherapy, fatty acids pathways were associated with pain, and both tryptophan and carnitine shuttle pathways were associated with the PNS cluster [
14].
A growing body of preclinical studies support the impact of the gut microbiome and microbial metabolites on the gut–brain communications via neuronal, immunological, and endocrinological pathways [
26]. However, research on this mechanistic pathway in the context of chemotherapy-related PNS is still very limited. Furthermore, current work primarily adopts single-omics approaches (e.g., microbiome analysis or metabolomics analysis independently) in human health and disease. On the other hand, multi-omics approaches provide an opportunity to examine multiple layers of molecules (e.g., microbiome and metabolites) [
27] to interpret health outcomes. Thus, there is paucity of research regarding how the interrelationship between the gut microbiome and their metabolites can influence PNS among patients with cancer receiving chemotherapy. Considering the severe PNS burden among children with chemotherapy and the unknown biological mechanisms of PNS, uncovering the multi-omics biological pathways within the MGB axis will pave a way for precision medicine (e.g., diet and probiotic interventions) to manage and treatment-related psychoneurological toxicities among CWC.
The purpose of this study was to investigate the associations between the gut microbiome–metabolome pathways and PNS among CWC receiving chemotherapy (pre-cycle two chemotherapy [T
0] and post-chemotherapy within 4 weeks [T
1]) compared to a group of healthy children (HC). An integrative multi-omics approach (i.e., metabolomics coupled to amplicon microbiome data) was adopted to examine the interrelationship of PNS-associated microbial taxa and their functional metabolites in CWC across chemotherapy. This study adopted a multi-omics network integration program xMWAS [
28] to analyze associations of microbiome–metabolome pathways with PNS.
Discussion
This study examined the microbiome–metabolome pathways associated with PNS among CWC receiving chemotherapy compared to HC in a network-based multi-omics analysis. We found that CWC post-chemotherapy showed the lowest number of correlated gut microbes, but more metabolites compared with those pre-cycle two chemotherapy and HC. Different patterns of microbiome–metabolite–PNS networks post-chemotherapy are associated with changes of PNS trajectories and the disturbed gut microbiome across cancer chemotherapy. Interestingly, PNS were clustered into two communities within the microbiome–metabolome networks in both study groups, revealing that specific gut microbial genera (e.g., Megasphaera, Ruminococcus, and Prevotella) were associated with the carnitine shuttle, fatty acid metabolism/activation, and tryptophan metabolic pathways. As the first of its kind, this study identified microbiome–metabolome pathways associated with PNS for CWC using a multi-omics approach. Although this study was limited by a small sample size, our findings provide promising microbiome–metabolome targets to validate in future studies with larger cohorts.
Compared with HC, CWC receiving chemotherapy reported more symptom burden, with particularly increased fatigue and cognitive dysfunction scores. Our findings partially reflected previous work regarding the overall trend of PNS across chemotherapy [
51,
52], including potential improvement of some symptoms, such as pain [
53,
54] and anxiety [
54] post-chemotherapy. This may be attributed to recovery from treatment-related procedures, acute chemotherapy toxicities, and discharge from the hospital after completion of chemotherapy. However, the worsened fatigue and cognitive dysfunction post-chemotherapy echo previous literature on the continuity of chemotherapy adverse events, particularly severe fatigue [
55] and cognitive impairment [
56,
57] in this population. Due to the significant influence of PNS on children’s future functional status and QOL, understanding the biological mechanisms of PNS trajectories during chemotherapy treatment is critical.
Recent innovations in the MGB axis propose that the gut microbiome can influence PNS via regulating specific metabolic pathways (e.g., SCFAs and tryptophan) [
12,
18,
58]. Chemotherapy has been reported to disturb the gut microbiome in CWC, reducing abundance of anaerobic bacteria (i.e.,
Bacteroides,
Clostridium cluster XIVa,
Faecalibacterium, and
Bifidobacterium), whereas
Enterococcus, often pathogenic, drastically increased [
31,
59,
60]. The disturbed gut microbiome potentially heightens treatment-related toxicity [
61]. Although limited, specific gut microbial taxa were found associated with PNS among adult cancer patients [
13,
16,
24]. Patients with a high PNS cluster burden were more likely to have increased abundance of Bacteroidota and
DTU089 phyla and
Ruminiclostridium-9,
Tyzzerella,
Eubacterium_fissicaten genera, while those with a low PNS cluster burden had higher abundance of
Lactococcus,
Phascolarctobacterium, and
Desulfovibrio genera
. Our study found an increase in the
Akkermansia genus for CWC pre-cycle two chemotherapy and HC, which was negatively linked to PNS in the microbiome–metabolome networks. Findings of this study support that higher abundance of
Akkermansia is associated with lower PNS burden, particularly cognitive dysfunction among CWC post-chemotherapy. Similarly, decreased abundance of
Akkermansia species is associated with various adverse health effects, including metabolic disorders, inflammatory and neurodegenerative diseases, and even cancers [
62,
63]. Having a protective effect,
Akkermansia species can act on host metabolism and metabolites such as SCFAs [
64]. For example, the probiotic
Akkermansia muciniphila is well known as a propionate producer in the presence of vitamin B12 [
65]. Although the mechanism of
Akkermansia species on disease and health outcomes is largely unknown, current key hypothesis is the positive modulation of thickness of intestinal mucosa and intestinal barrier integrity [
66,
67]. For instance, patients with cancer experienced significant epithelial permeability and bacterial translocation [
68]. Thus, therapeutic manipulations (e.g., probiotics and Mediterranean diet) of the
Akkermansia species may maintain the intestinal integrity [
67] and further reduce chemotherapy-related PNS.
In this study, gut microbial genera, such as
Lactobacillus,
Bifidobacterium, and
Roseburia taxa were associated with lower PNS burden [
69,
70]. Decreased abundances of the gut microbial taxa, particularly
Roseburia and
Faecalibacterium were commonly reported among patients with psychiatric disorders [
71]. Although mixed findings using non-experimental study designs (e.g., case–control and observational) were reported about
Bifidobacterium and
Lactobacillus among patients with psychiatric disorders [
71‐
73], probiotic interventions seem to support
Bifidobacterium and
Lactobacillus species (e.g.,
Lactobacillus rhamnosus and
Bifidobacterium breve) as an alternative therapy to alleviate PNS (e.g., anxiety, depression, and cognitive dysfunction [
74‐
77]). Due to methodological shortcomings, further confirmation of these findings is critically needed. Additionally, the gut microbes (e.g.,
Ruminococcaceae_UCG-014) [
78] are associated with lower PNS burden. However, there has also been contradicting evidence regarding probiotics, such as
Alistipes. They may have protective effects in the PNS context but have also been demonstrated to have a pathogenic nature associated with the development of colorectal cancer and depression [
79]. Thus, our findings require further confirmation in a larger cohort of pediatric cancer patients.
Gut microbes metabolize dietary and host-derived molecules to activate or produce functional metabolites with local and systemic effects [
26]. Under the guidance of the MGB axis framework, our previous research identified a group of serum metabolites associated with pain, fatigue, anxiety, depressive symptoms, and the PNS cluster (mean of these symptoms) for CWC (e.g., primarily diagnosed with leukemia and lymphoma) across a chemotherapy cycle [
14]. In particular, the fatty acid pathways were associated with pain, the tryptophan pathway was associated with fatigue, anxiety, and the PNS cluster, and the carnitine shuttle was associated with the PNS cluster [
14]. Furthermore, a dysbiotic gut microbiome was found to potentially modulate PNS through altered lipid metabolism as well as gastrointestinal and neural systems for patients with head and neck cancer [
13]. This study compared microbiome–metabolome–PNS networks among CWC pre-cycle two chemotherapy and post-chemotherapy, and HC and indicated that different patterns of bacteria (e.g.,
Ruminococcus and
Prevotella) linked with metabolites (e.g., fatty acid metabolism, tryptophan, and carnitine shuttle) are associated with PNS by study groups. These network differences may be partially attributed to the effects of chemotherapy and antibiotic use, which can shape the gut microbiome, and in turn further aggravate dysregulations of metabolic pathways, intestinal permeability, and damage to the enteric and peripheral nerves, ultimately leading to physiological and psychological dysfunction [
13,
80,
81]. Specifically,
Ruminococcus has been reported to form secondary bile acid (e.g., ursodeoxycholic acid) that modulates the immune system via reducing cytokine secretion and inhibiting eosinophil activation [
82];
Prevotella has also been reported to produce SCFAs and add in the synthesis of micronutrients (e.g., vitamin K2 and B12) [
83,
84], which can regulate intestinal homeostasis in animal models and human populations. The findings of microbiome–metabolome–PNS networks provide potential targets (e.g., microbes and its functional metabolites) to mitigate PNS for children with cancer receiving chemotherapy. For example, administrating probiotics (e.g.,
Lactobacillus and
Bifidobacterium) can correct microbial dysbiosis and sustain metabolic equilibrium [
85]. Although more work is needed to confirm our findings in CWC, our findings from this study suggest that microbiome–metabolome pathways are associated with PNS among children with cancer receiving chemotherapy.
Single-omics biomarkers (i.e., microbiome or metabolome) are emerging to explain PNS in cancer chemotherapy [
14,
24,
25]. However, there is a paucity of research that integrates both the gut microbiome and metabolome using multi-omics approaches in PNS. Consistent with our previous work [
14], this study found that carnitine shuttle, fatty acid activation and metabolism, and tryptophan metabolism were associated with the gut microbiome and PNS in CWC. Specifically, carnitine is a trimethylated amino acid primarily derived from the diet, essential for the transportation of long-chain fatty acids during fatty acid beta-oxidation for energy support, including cancer [
86‐
88]. An interruption of the carnitine shuttle system during chemotherapy could influence cancer metabolic plasticity and intertwine key metabolic pathways that supply an energetic and biosynthetic demand for cancer cells [
89], ultimately influencing PNS during chemotherapy. Considering the critical role of carnitine-related pathways in cancer care,
l-carnitine supplementation was explored to improve PNS, particularly fatigue [
90,
91].
Additionally, we found that fatty acid activation and metabolism involved in the carnitine shuttle system were associated with PNS. Fatty acids metabolism includes various metabolic processes involving fatty acids, which determine human brain’s integrity and functional performance [
92]. Essential fatty acids, such as omega-3 fatty acids, were found to decrease the symptoms of fatigue and pain in patients during chemotherapy, possibly due to weight maintenance and reduced inflammatory status [
93]. Furthermore, a decrease in bile acid synthesis was reported in patients with chronic fatigue syndrome [
94]. This may be attributed to the role of bile acids in cholesterol homeostasis and microbiome signaling, facilitating excretion, absorption, and transportation of fat and sterols in the liver and intestines [
95]. Together, specific fatty acids, such as omega-3 fatty acids, point towards a precision approach to treat and manage cancer treatment-related symptoms although further investigation is needed to examine the exact benefits of fatty acid-related supplementations or diets rich in omega-3 and omega-6 fatty acids in symptoms among cancer populations, including children with cancer [
93,
96].
Tryptophan, an essential amino acid, is required for structural and functional processes of protein biosynthesis and immunoregulation [
97] and plays a critical role in the MGB axis [
98]. The inflammation activation of tumor cells and cancer treatments can induce the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase, which can convert tryptophan to kynurenine in the gastrointestinal tract and other tissues of the body [
99]. Downstream metabolites of kynurenine include neuroprotective kynurenic acid and neurotoxic quinolinic acid [
18]. Depletion of tryptophan could contribute to serotonin dysregulation and neurobehavioral manifestations [
100,
101]. Meanwhile, the accumulation of downstream metabolites of the kynurenine pathway seems to trigger central nervous system physiology, anxiety, depression, social behavior, cognition, and visceral pain [
18,
102]. Similarly, they were also associated with an increased burden of pain, fatigue, anxiety, and depression [
14,
103,
104], as well as reduced QOL [
105,
106]. This study corroborated previous reports that demonstrate the association of altered tryptophan metabolism during chemotherapy and its adverse association with symptom burden among CWC. Current literature has attempted to identify solutions to inhibit tryptophan breakdown, such as ketogenic diet [
107], Mediterranean and other plant-based diets [
108], probiotics [
109], and physical activity [
110,
111]. Further studies are needed to test the feasibility and efficacy of these promising interventions among pediatric cancer populations.
Utilizing the MGB axis framework, this study confirmed several metabolic pathways, such as carnitine shuttle and tryptophan/kynurenine pathways, associated with psychoneurological toxicities in children [
14,
112] and adults with cancer undergoing chemotherapy [
24,
113,
114]. This is the first study to elucidate microbiome–metabolome pathways linked with PNS in cancer chemotherapy using the multi-omics data integration and analysis approach. This study added to the literature that specific gut microbes (e.g.,
Ruminococcus,
Megasphaera, and
Prevotella), along with carnitine shuttle, fatty acid metabolism/activation, and tryptophan pathways, are associated with PNS burden across cancer chemotherapy. Targeting the gut microbiome through diet, nutritional supplements, probiotics, and exercise [
18,
115] may provide a tractable solution to modulate metabolic pathways, ultimately decreasing PNS burden among CWC. Further validation of these findings is needed in a larger cohort.
There are several limitations to our study. First, the sample size is small, and all cases were recruited from Children’s Healthcare of Atlanta, resulting in limited generalizability into other clinical settings. This pilot study analyzed CWC who completed T
0 and some of them did not complete T
1 yet when we analyzed the data. The unbalanced sample size between T
0 and T
1 for CWC may cause bias. Second, as a preliminary analysis with a smaller sample size, we did not adjust the multi-omics integration for multiple testing. This approach has certainly resulted in some false positive findings, furthering the importance of future replication. However, clustering and pathway analyses are two ways to mitigate the effects false positives in omics research [
116], and our prior research suggests that these approaches might continue to do so in multi-omics research [
117]. Future work should confirm our findings in a larger cohort with multiple testing correction. Third, we were unable to determine whether the fecal metabolites were produced by the microbiome or by the host, and whether these identified metabolites were being absorbed to affect the MGB axis or alternatively being eliminated. Our metabolomics analysis was limited to summaries of metabolic pathways and thus need detailed examination of specific metabolites in future work to determine the magnitude and direction. Lastly, our study could not control for the use of antibiotics and chemotherapy drugs across the cancer treatment trajectory. Therefore, this study cannot determine the impact of specific chemotherapy on PNS and the antibiotic vs. chemotherapy effects on microbiome–metabolome pathways. We cannot discern baseline differences in the fecal microbiome and metabolome due to treatments or cancer per se. Future research should examine the relationships of multi-omics pathways in the chemotherapy-induced PNS context with a larger sample cohort using metagenomic sequencing to elucidate species- and strain-level microbial data, as well as targeted metabolomics that focus on the most salient pathways (e.g., tryptophan), while controlling for covariates such as chemotherapy drugs.