Immunonutrition is an integral part of complex nutritional support in cancer patients. Arginine, glutamine, omega-3 fatty acids, and nucleic acids are nutritional immunomodulatory substances which can be used in oncology (1–3). Patients with medium/high risk of malnutrition should receive immunonutrition orally/enterally in the perioperative period. This recommendation refers in particular to patients who will undergo surgery due to a tumor located in the upper part of the gastrointestinal tract (3–5). Immunonutrition provides multiple benefits. Among others, it enhances immune response, reduces the over-production of pro-inflammatory mediators, maintains CD4/CD8 lymphocyte balance, and contributes to a better clinical outcome (6, 7). It was meta-analytically demonstrated that such preoperative intervention decreases the prevalence of infectious complications, including wound infection, respiratory and urinary tract infections, and anastomotic leakage, by around 30% (8, 9). However, the data regarding the impact of immunonutrition on gut microbiome is still limited. Notably, the gut microbiome plays a significant role in the human body through maintaining intestinal barrier integrity (for instance, by producing short-chain fatty acids), reduction of secondary bile acids, increasing the level of anti-inflammatory cytokines, and carcinogen binding (10). Gut microbiome imbalance is observed in gastrointestinal cancers; however, the gut microenvironment imbalance depends on the type of cancer. In gastric cancer patients, an increased amount of Helicobacter pylori, Lactobacillus, Escherichia-Shigella, Nitrospirae, Burkholderia fungorum, and Lachnospiraceae is noted (11, 12). In the case of colorectal cancer patients, the abundance of Escherichia coli, Enterococcus faecalis, Helicobacter hepaticus, Fusobacterium nucleatum, Porphyromonas gingivalis, Bacteroides fragilis, Peptostreptococcus anaerobius, Helicobacter pylori, and Streptococcus bovis is observed (13). Additionally, in these patients, an imbalance of mycobiota (fungal part of microbiota) also occurs (10). Interestingly, in 2024, it was reported that overall insight into untargeted metabolomics in gastric and colorectal cancer patients showed that there is a link between the gut metabolome and both local and distal metastasis (14). The positive modulation of gut microbiota may restore microbial homeostasis, improve gut barrier integrity, and consequently contribute to the reduction of side effects of anti-cancer treatments (15).

Currently, as was mentioned above, there is a significant lack of data with regard the impact of immunonutrition in the perioperative period on gut microbiota composition (both bacteria and fungi). Additionally, in ClinicalTrials.gov system, there is only one trial registered (until February 5, 2025; keywords: cancer, immunonutrition, gut microbiota), except our trial (ClinicalTrials.gov system, identifier: NCT04980950), which assesses preoperative nutritional intervention and gut microbiota-related aspects. It actually concerns the case of head and neck cancer. Therefore, the aim of the present randomized trial is to assess the impact of enteral immunonutrition on gut microbiota in gastric/colorectal cancer patients in the preoperative period.

The participants (n = 14, n = 9 colorectal cancer, and n = 5 gastric cancer) were recruited in the Unit of Surgery with the Unit of Oncological Surgery in Specialist Hospital in Koscierzyna, Koscierzyna Poland, Department of Surgical Oncology with the Subunit of Breast Cancer, Skin and Soft Tissue Surgery in Maritime Hospital PCK, Gdynia Poland, and Department of Surgical Oncology at Medical University of Gdansk, Poland. The patients were included to this study if they met all of the following inclusion criteria: age 18–80 years, presence of gastric/colorectal cancer, preparing for surgery due to tumor occurrence, and with written consent to take part in the study. The exclusion criteria included the following: age <18 years, presence of any type of cancer other than gastric/colorectal cancer, presence of any of inflammatory bowel diseases, pregnancy, or postpartum period. Randomization was performed using a random number generator. This study was conducted from 2021 to 2024. The study protocol has been approved by the Independent Bioethics Committee for Scientific Research at the Medical University of Gdansk (identifiers: NKBBN/129/2021, NKBBN/129-647,703/2021, NKBBN/129-281/2022, NKBBN/129-422/2022, NKBBN/129-126/2023, KB/129-378/2023).

The analysis was conducted using R version 4.4.2. These are the following analysis: alpha diversity analysis, taxonomic data processing, beta diversity analysis, and differential abundance analysis (DAA).

This study included 14 patients (n = 9 colorectal cancer and n = 5 gastric cancer). The analysis is based on two groups, i.e., patients who received immunonutrition (I) and patients consuming standard products (N). The participants from both groups received products within 7 days in the preoperative period.

The summary statistics for alpha diversity indices grouped by diet indicate similar distributions across the two groups. The richness index, representing the number of unique taxa, showed a mean of 392.51 (SD: 56.14) for the immunodiet group (I) and 366.64 (SD: 84.73) for the standard nutridrink group (N). Evenness, which measures the uniformity of species distribution, had mean values of 0.674 (SD: 0.041) for group I and 0.686 (SD: 0.042) for group N. Simpson’s index, reflecting species dominance, remained high for both groups (mean 0.969 for I and 0.971 for N), suggesting stable community structures. Shannon’s index, incorporating both richness and evenness, showed comparable means (4.02 for I and 4.04 for N). There was no significant differences between the two dietary groups for any of the alpha diversity indices as visualized in the boxplot representation ( Figure 1 ).

The PERMANOVA analysis indicated that there were no statistically significant differences in beta diversity between the dietary groups (F = 0.92, p = 0.37). This suggests that microbial community compositions were largely similar between the immunodiet and standard nutridrink groups. The PCoA plot further supports this finding, as the points corresponding to both groups exhibit a substantial overlap, indicating that the dietary interventions did not induce distinct clustering of microbial communities ( Figures 2 , 3 ).

Differential abundance analysis (DAA) using the Wilcoxon test identified several taxa with nominal p-values <0.05, suggesting potential differences in abundance between groups. However, after applying the Bonferroni correction for multiple comparisons, none of these findings remained statistically significant. The taxa with nominal significance included Bilophila, CAG-56, Clostridium sensu stricto 1, Coprobacter, Holdemania, Fusicatenibacter, Ruminococcus, and [Eubacterium] eligens group.

In the current study, the impact of immunonutrition given orally on the composition of gut microbiota in gastric/colorectal cancer patients in the preoperative period was analyzed. Enteral immunonutrition may provide many benefits to gastrointestinal cancer patients, such as boosting the immune system, promoting tissue repair (especially arginine) as well as improving the nutritional status (16). Arginine, which is known as a nutritional immunomodulatory substance, takes part in wound healing process. Thus, its administration in the perioperative period may be significant (1). It should be mentioned that arginine is a non-essential amino acid; however, it can be conditionally essential in cases of cancer/trauma. In systematic review and meta-analysis, it was noted that perioperative immunonutrition in patients with gastrointestinal cancers reduces the complications after surgery and the length of hospital stay (17). In another systematic review and meta-analysis (seven studies, n = 583), Cheng et al. (18) have shown that enteral immunonutrition modulates inflammatory reaction and reduces postoperative complications in gastric cancer patients undergoing total gastrectomy. In colorectal cancer patients undergoing surgery, enteral immunonutrition also shortened the length of hospital stay (pooled mean difference, 2.53; 95% CI, 1.29–3.41) and reduced infectious complications (pooled odd risk, 0.33; 95% CI, 0.21–0.53) (9). Immunonutrition stimulates the immune system. In a randomized, double-blind, controlled trial, Li et al. (19) proved that early postoperative enteral immunonutrition enhances the function of the immune system in gastric cancer patients. Similar results were obtained in another study in which it was noted that early enteral immunonutrition promotes the immune function in gastric cancer patients (20). The level of CD4+/CD8+ was significantly higher in the group receiving immunonutrition than in control subjects (P < 0.05) (20).

The gut microbiome affects the anti-cancer treatment efficacy. Among others, it has an impact on the effectiveness of the surgical treatment. Anastomotic leak is a complication in colorectal surgery with multifactorial etiology including the interaction between host genetics, inflammation, and gut microbiome (21–24). Bacteria, such as Enterococcus faecalis and Pseudomonas aeruginosa, participate in the development of an anastomotic leak (25, 26). Low gut microbial diversity and the abundance of mucin-degrading bacteria (Lachnospiraceae and Bacteroidaceae families) also contribute to anastomotic leak occurrence (27). Moreover, damage of tight junction and increased intestinal permeability in gastrointestinal cancer patients are observed (28). As was mentioned above, previously published studies concentrated mainly on the influence of immunonutrition on surgical-related complications. However, the data regarding its impact on gut microbiota in cancer patients is limited. Therefore, in the current study, we focused on immunonutrition and gut microbiome in gastric and colorectal cancer patients. Nevertheless, the summary statistics for alpha diversity indices grouped by diet indicate similar distributions across the two groups, i.e., I and N. Therefore, statistical analysis did not indicate any significant differences (p > 0.05) between the two dietary groups for any of the alpha diversity indices. Similarly, this was also true in the case of beta diversity. However, DAA using the Wilcoxon test identified several taxa with nominal p-values below 0.05, suggesting potential differences in abundance between groups. The taxa with nominal significance included Bilophila, CAG-56, Clostridium sensu stricto 1, Coprobacter, Holdemania, Fusicatenibacter, Ruminococcus, and [Eubacterium] eligens group. In the further analysis, it was noted that none of these findings remained statistically significant. It should be emphasized that some of the listed microbes act with pro-inflammatory properties—for instance, Bilophila increases the risk of intestinal permeability. Ruminococcus gnavus presents pro-inflammatory activity and promotes the development of gut dysbiosis (29, 30). Nevertheless, some strains of Ruminococcus are known as a SCFA producers; thus, they can exert beneficial effects (31).

The analysis of gut microbiota in the context of immunonutrition is a new area in oncology. Restoration of the gut microbiota balance is strongly needed in gastrointestinal cancer patients. Therefore, modern methods of gut microbiota modulation should be found. Notably, immunonutrients, immunometabolism-related aspects, mucosal immune system, and its interactions with gut microbiota seem to be crucial, and it is a new perspective for cancer patients. In the current study, microbial community compositions were largely similar between the immunodiet and standard nutridrink groups. However, differential abundance analysis identified several taxa with nominal p-values below 0.05, suggesting potential differences in abundance between the studied groups. Nevertheless, despite some initial microbial alterations, it was not finally confirmed that immunonutrition has a beneficial effect on gut microbiota in gastric and colorectal cancer patients in the preoperative period.

There are possible limitations in the design/conduct of the study, such as the following:

• small sample size (n = 14), limiting the statistical power

• short duration of intervention (7 days)

At the beginning, the post-allocation gut microbiota assessment was planned at day 0 and after 7 and 10 days, respectively, of treatment in all participants. The surgery was planned at day 8. Therefore, the study products’ administration was planned to be interrupted after the first 7 days, and then it was planned to be continued 1 day after the surgical procedure. It could (1) provide the impact of surgical treatment on gut microbiota in a short time and (2) show the changes of microbiota caused by the consumption of immunonutrients for a longer time (10 days instead of 7 days).

• Uncertainty regarding compliance with supplementation.

Next, the patients received the study products at home. Therefore, the researchers cannot be completely sure if they consumed the products. However, the participants were asked about compliance at follow-up visits.

The future direction is with regard the assessment of the impact of immunonutrition on microbiome-derived metabolites in cancer patients. In the current study, it was not finally confirmed that immunonutrition has a beneficial influence on overall gut microbiota. However, the alterations could be observed in the level and proportion of gut-microbiome-related metabolites (for instance, short-chain fatty acids). The aspects of untargeted metabolomics also seem to be crucial in this context.