Slovenian Veterinary Research 2025 | Vol 62 Suppl 27 | 13 Gut Microbiome in Cancer: the Next big Opportunity for Better Patient Outcomes? Key words gut microbiome; cancer; treatment outcome; tumor models; glioma Jure Povšin1,2#, Timotej Sotošek1,2#, Metka Novak1, Barbara Breznik1* 1National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, 121 Večna pot, 1000 Ljubljana, 2Faculty of Chemistry and Chemical Engineering, University of Ljubljana, 113 Večna pot, 1000 Ljubljana, Slovenia #Shared first authorship *Corresponding author: barbara.breznik@nib.si Abstract: The gut microbiome, a diverse community of microorganisms in the human body, plays an important role in maintaining health and influences various processes such as digestion, immunity, and protection against pathogens. A person's unique gut microbiome, shaped by factors such as birth method, diet, antibiotics, and lifestyle, contributes to bodily functions such as nutrient metabolism, drug processing, and im- mune regulation. Changes in the gut microbiome are associated with a predisposition to cancer and can influence the effectiveness of cancer treatments. Dysbiosis in the gut microbiome can lead to inflammation, tumor development, and metastasis, highlight- ing its importance in cancer research and prevention. The gut microbiota significantly influences cancer development and treatment outcomes. Certain bacteria enhance the effects of therapies such as cyclophosphamide and contribute to the body's im- mune response against tumors. Microbes produce anti-cancer molecules and probiotic compounds, making them potential tools in cancer prevention and treatment. Future research aims to develop targeted antibiotics and explore fecal microbiota transfer to selectively manipulate the microbiota for improved cancer treatment. Due to genetic and physiological similarities, mouse models are invaluable in biomedical research. However, because the gut microbiome of humans and mice and the composition of the tumor microenvironment differ, direct comparison between these two models can be challenging in research. Bridging these gaps is crucial for comparative medicine, especially in cancer research where the microbiome plays an important role in treat- ment outcomes. One important area where the gut microbiome could offer potential new treatment options is in primary brain tumors such as gliomas. To date, there are no long-lasting effective treatments for this type of cancer, but research in mouse models shows a link between tumor progression and response to treatment with changes in the gut microbiome. Overall, the gut microbiome and its modulation represent an opportu- nity for more efficient future cancer treatment. Received: 5 April 2024 Accepted: 21 October 2024 Slov Vet Res DOI 10.26873/SVR-2029-2024 UDC 543.95:576.36:591.434:616-006:616-052:636.09 Pages: 13–26 Review Article Introduction The gut microbiome is known to play a crucial role in main- taining normal homeostasis in humans. The microbiome is a collection of all microorganisms, such as bacteria, fungi, viruses, and their genes, that naturally reside on and in our bodies. Often referred to as the "forgotten organ", the mi- crobiome contains a metagenome that is 100 times larger than our own genome and performs key functions that are vital to human health (1). For instance, the human body comprises approximately 40-100 trillion microbial cells, which is ten times more than the number of human somat- ic cells. A healthy individual's gut contains around 300-500 different species of bacteria (2), although some sources mention up to 3500 bacterial species (3). Over time, the microbiome and the host have evolved into a complex "superorganism" with their symbiosis benefiting 14 | Slovenian Veterinary Research 2025 | Vol 62 Suppl 27 the host in numerous ways, such as food metabolism, protection against pathogens, and assistance in the devel- opment of the immune system. The majority (99%) of mi- crobial mass resides in the gastrointestinal tract (GI) and functions both locally and over long distances. As a result, the gastrointestinal microbiome not only has the most substantial impact on overall health and metabolic status among all microbiomes but is also the most extensively studied microbiome, serving as a model for understanding interactions between the host, microorganisms, and dis- eases (4, 5). The role of a normal gut microbiome A hypothesis suggests that the GI's microbiome plays a significant role in maintaining an individual's gut health and is crucial for overall human health (6, 7). Everyone has a unique gut microbiome profile, which serves specific func- tions in host nutrient metabolism, xenobiotic metabolism, maintenance of the structural integrity of the intestinal mu- cosal barrier, immunomodulation, and protection against pathogens. Under normal circumstances, the host's immune system recognizes markers that are specific to pathogenic mi- croorganisms, making it easier to eliminate them. The fact is that the majority of the host's microbiome is non- pathogenic and lives in symbiosis with the host's immune system. Intestinal bacteria play an important role in this, as they inhibit the growth and spread of pathogens, provide essential nutrients, and assist in nutrient and drug metabo- lism. In the meantime, the host's immune system must pre- vent the invasion of both pathogenic and non-pathogenic microbes. Immune cells, including macrophages, phago- cytes, and dendritic cells, closely interact with the intestinal microbiome and its metabolites thus maintaining intestinal homeostasis and recognizing bacteria that might be patho- genic (8). The gut microbiome of each individual is formed early in life, and several factors play a role in its development. These factors include in what way a baby is born (vaginal or cesarean section), childhood diet (breast milk or for- mula), adult diet (vegan or meat-based), as well as the use of antibiotics or antibiotic-like molecules derived from the environment or the gut's commensal community (7). The gut microbiome of an adult host is relatively stable. Still, it varies from person to person, mainly due to differences in lifestyle, including frequency of physical activity and cultur- al practices, as well as enterotype, body mass index (BMI), and dietary habits. Alpha and beta diversity are two metrics used to look at microbiome diversity. Alpha diversity mea- sures how many types of species live in a given area in a person or a single sample and beta diversity measures the differences in the microbiome composition between body sites or people (9). As a result, there is no unique optimal composition of the gut microbiome, as it differs for everyone. However, despite this diverse composition, statistics show that dominant types of gut microbes include Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia, with Firmicutes and Bacteroidetes comprising 90% of gut microbes (10, 11). The gut microbiome and cancer Microbiota-induced tumors are estimated to represent about 20% of all tumors worldwide (12). In the past de- cade alone, the number of cancer patients in the USA has increased from 13.8 to 18.1 million, and it is projected to continue rising in the upcoming years. Additionally, a diet high in fat and heavily processed foods is believed to in- fluence the diversity of gut microbiomes. A recent study showed that higher fat consumption in healthy young adults is associated with unfavorable changes in gut micro- biomes, which could impact the overall health of the host (13). Numerous studies point out that changes in the gut microbiome can lead to a predisposition to various types of cancer. Moreover, bacteria and their metabolites have been found not only to contribute to cancer development, such as colorectal cancer but also to alter the pharmacodynam- ics of cancer drugs (14, 15). When the balance in the gut mi- crobiome is disrupted, bacteria can penetrate the intestinal mucosa and surrounding tissues, causing inflammation. As the inflammatory process promotes tumor develop- ment and progression, it accelerates the invasion of tumor cells and may eventually lead to metastasis. Increased lev- els of inflammatory cytokines can directly damage the DNA of epithelial cells, triggering inflammation-associated can- cer (16, 17). For instance, the presence of the bacterium Helicobacter pylori can promote an immune response and chronic in- flammation, which can cause stomach cancer. Many products from this bacterium disrupt the regulation of nor- mal cell homeostasis, resulting in a build-up of cytokines and other signaling molecules that cause stomach and esophageal cancer. In healthy individuals, the esophagus is densely populated with Firmicutes and Streptococcus (mostly Gram-positive bacteria). In cases of dysbiosis, Gram-negative bacteria (anaerobic and microaerophilic) replace Gram-positive bacteria, which can later lead to esophagitis or inflammation of the esophagus. Among other gut microbes associated with esophageal cancer are Bacteroidetes, Firmicutes, and Proteobacteria (18). One of the more significant health problems across the world is colorectal cancer. Many studies have proven that several bacterial species play a role in colorectal carcino- genesis, including Escherichia coli, Bacteroides fragilis, Fusobacterium spp., Enterococcus faecalis, Streptococcus bovis, H. pylori and Clostridium septicum. The colon con- tains ten times more bacteria than the small intestine, Slovenian Veterinary Research 2025 | Vol 62 Suppl 27 | 15 which means that the likelihood of developing colorectal cancer is about 12 times higher in the colon than in the small intestine, emphasizing the role of commensal bacte- ria (19, 20). Other research has indicated that obesity may be associated with the gut microbiome composition and increased liver cancer. In obese individuals, the epithelium junctions are damaged, allowing gut bacteria to enter the bloodstream and cause systemic infections. Additionally, the gut microbiota––driven COX2 pathway secretes sec- ondary metabolites and other small molecules such as li- poteichoic acid (LTA), lipopolysaccharides (LPS), and bile acids, which cause inactivation of the immune system in the liver, possibly leading to liver cancer (21–24). The role of the gut microbiome in tumorigenesis The symbiotic microbiome is recognized for its vital func- tion in upholding human well-being and fortifying the host's immune defenses. Nevertheless, a group of bacteria re- mains associated, either directly or indirectly, with the ad- vancement and progression of cancer (25). In an imbal- anced gut ecosystem, harmful microbes can inflict various damages upon the host's organism in several ways (Figure 1). Research has proven that the intestinal flora can infil- trate deep within bodily tissues, instigating tumor formation in mouse models deficient in IL-10, a critical cytokine pivotal in the host's anti-cancer immunity (26–29). When there is a microbial imbalance in the gut microbiota, pathogenic bacteria can generate and discharge an array of toxins. These toxins can make the genome unstable by instigating breaks in the host's DNA, triggering the devel- opment of tumor in predisposed tissues. One example of such a toxin is cytotoxin-associated gene A (CagA), synthe- sized by H. pylori. Through this cytokine, CagA can degrade the tumor suppressor protein p53 in gastric cells, disrupting the host's serine/threonine kinase pathway which leads to inhibition of cell apoptosis, survival of damaged cells and stomach cancer development (Figure 2). Similarly, E. coli produces colibactin, and CDT+ strains produce a cytotoxic toxin (CDT) with DNase activity that leads to host cell apop- tosis. When these toxins are released near the intestinal epi- thelium, they create double-strand breaks in the host's DNA, culminating in genetic mutations and the development of tumors. By producing enzymes like Virulence A (VirA) and inositol phosphate phosphatases D (IpgD), Shigella flexneri can induce degradation of the p53 protein in host cells and disrupt DNA damage and repair pathways (30, 31). Pathogenic bacteria are capable of indirectly influencing tumorigenesis in host cells through various mechanisms. For instance, they can produce molecules such as differ- ent bile acid metabolites that inhibit the host's immune response and enhance inflammation and with this help cancer cells evade the immune system (32). Bacteria can also generate oxidative stress, which subsequently drives genetic mutations in host cells (33, 34). For example, fla- voenzyme (spermine oxidase) can be activated in host cells by Helicobacter pylori and Bacteroides fragilis, creat- ing hydrogen peroxide (H2O2) and reactive oxygen species (ROS), which adds to DNA damage accumulation. Bacteria Enterococcus faecalis can infiltrate host cells by producing reactive oxygen species, accelerating host DNA damage (35–37). Fusobacterium nucleatum can block the cytotoxic Figure 1: The interaction between the gut microbiota and the immune system. In an imbalanced gut ecosystem, harmful microbes can inflict various damages upon the host's organism and affect local and systemic immune responses. Adapted from (30). The Scheme was created using Biorender.com Abbreviations: interleukin (IL), short-chain fatty acids (SCFAs), tumor necrosis factor (TNF), nuclear factor kappa B (NF-κB), T helper 17 cell (Th17) Figure 2: The role of different bacteria in cancer progression. An illustration of how numerous bacteria can contribute to or even outright cause cancer progression and their mechanism of action. The Scheme was created using Biorender.com Abbreviations: B. fragilis toxin (BFT), cytotoxin-associated gene A (CagA), Cytolethal distending toxins (CDT), fibroblast activation protein-2 (Fap2), inositol phosphate phosphatases D (IpgD), natural killer (NK), reactive oxygen species (ROS), spermine oxidase (SMO), virulence A (VirA) 16 | Slovenian Veterinary Research 2025 | Vol 62 Suppl 27 activity of natural killer cells (NK) by producing a virulence factor (Fap2), which can bind to the NK inhibitory receptor TGIT and ITIM domain, preventing NK cells from attacking cancer cells (25, 38). Anti-cancer properties of intestinal microbiota Microbiota plays a crucial role in tumor formation and the outcomes of anticancer therapies (39, 40). The interaction between the host's immune system and gut microbiota en- ables immune cells to recognize and eliminate opportunis- tic bacteria before they can invade the host's body. Apart from that, the interaction also impacts food digestion and the elimination of metabolites against gastrointestinal anti- gens (41). Moreover, the microbiota influences both innate and acquired immune systems systemically. In studies with germ-free mouse models lacking gut microbiota, scientists have observed a lack of the mucus layer, altered immuno- globulin A (IgA) levels, and mesenteric lymphadenitis (41, 42). Additionally, microbiota deficiency has been shown to negatively affect the efficacy of therapeutic interventions (43). Cyclophosphamide (CTX) is a popular chemotherapeutic drug that stimulates the host's T-cell immune response. It is used as a treatment for different types of cancers, and it works by crossing the small intestine and breaching the ep- ithelial membrane. In this way commensal gut microbiota is transferred to the spleen and mesenteric lymph nodes, thus stimulating helper T cells (TH17 cells) and further in- ducing anti-cancer effects against tumor development (25, 44). Studies on mouse models have shown that sev- eral bacterial species such as Branesiella intestinihominis, Enterococcus hirae, and Lactobacillus johnsonii enhance the anti-cancer activity of CTX (44, 45). On the other hand, anti- biotic-treated and germ-free mouse models have shown re- duced immune responses due to the lack of Gram-positive bacteria in their intestines. Based on these studies, it has been found that commensal bacteria can alter the efficacy of immunotherapy and chemotherapy drugs (44). Bacteria Bifidobacterium longum and B. breve have been shown to enhance the dendritic cell function. Active den- dritic cells can then trigger the recruitment of cytotoxic T cells in the tumor microenvironment. Cytotoxic T cells and NK cells are the main components of the immune system responsible for eliminating cancer cells (25). Numerous studies analyzing 1000 patients with sarcoma have found out that heat-killed bacteria Serratia and Streptococcus pyogenes can increase the survival rate of patients by ap- proximately 80% over five years. Additionally, heat-killed mi- croorganisms may activate a sustained immune response and potentially exhibit anti-cancer effects against sarco- mas. It is presumed that CD8+ cells can effectively infiltrate infected cells or tissues in solid tumors because of the gut microbiota (46). For example, it was shown that CTLA-4 inhibitors have anti-cancer effects and depend on the gut microbiota, especially Gram-negative obligate anaerobic bacteria. The CTLA-4 inhibitor had no anti-cancer effects in germ-free mice, but when Gram-negative obligate anaero- bic bacteria were introduced into sterile mice, the inhibitor's anti-cancer efficiency was restored (47). Molecules or components derived from microorganisms have potent anti-cancer properties. Short-chain fatty acids (SCFA), produced by the gut microbiota, play a crucial role in suppressing tumors/cancer (48–50). Common SCFAs produced by commensal gut bacteria like butyrate and propionate have effective anti-cancer effects (51). They in- hibit the histone deacetylases of cancer cells and induce the programmed cell death known as apoptosis. In patients with colorectal cancer there were lower levels of bacteria producing butyrate found. Butyrate activates the GPR109A receptor, which then induces IL-18 production in epithelial cells of the intestinal mucosa, which may trigger the repair mechanisms of the mucous layer (52). Metabolites obtained from probiotics can initiate an indi- rect immune response against tumor formation by modi- fying the host's immune system. For instance, lipopoly- saccharides (LPS) can activate Toll-like receptor 4 (TLR 4), which further enhances the T cell immune response against tumor cells (53). Likewise, monophosphoryl lipid A from the bacterium Salmonella enterica which has a high efficacy against cervical cancer is used as an adjuvant in vaccine development (54). Some gut bacteria can produce probiotic molecules with anti-tumor effects. For instance, Lactobacillus casei produces ferrichrome, which activates the c-Jun N-terminal kinase signaling pathway and ulti- mately induces programmed cell death in cancer cells (55). Lactobacilli are also believed to play a role in an anti-tumor response by stimulating the host's immune system such as dendritic cells, NK cells, and TH1 cells (38). Bacteria and viruses in the intestines influence the ef- fects of chemotherapy, immunotherapy, and the immune response. Studies have shown that mice with tumors that do not typically respond to immunotherapy drugs can start responding if they receive specific gut bacteria from mice that have positively responded to the drugs (56). The same phenomenon has been observed in humans, where alter- ing the composition of bacteria in the gut microbiota using fecal transplants can improve the condition of some pa- tients with tumors who did not respond to immunotherapy or drugs. The most well-known donor of his feces is Zion Levy, who was diagnosed with melanoma but showed very good responsiveness to immune treatment with nivolum- ab. Doctors concluded that with his feces, he could also help other patients who do not respond to immunotherapy as well. This led to the first such research studies at Sheba Medical Center in Israeli study and a study led by scientists at Memorial Sloan Kettering Cancer Center in New York City and scientists at the University of Minnesota Medical School in the USA in which the transfer of microbes from Slovenian Veterinary Research 2025 | Vol 62 Suppl 27 | 17 feces (FMT) improved the response to immunotherapy in patients. The results of these two studies, published in the journal Science (56, 57), were modest - out of 26 people who did not previously respond to immunotherapy, about one in three responded after FMT. However, they attracted a lot of attention. In the Israeli study, ten individuals with advanced melanoma were included, whose cancer had progressed despite treatment with checkpoint inhibitors. Only three out of ten overcame treatment resistance, and only two of them partially. Interestingly, all three respond- ers received FMT from donor Levy. None of the five partici- pants who received material from another donor who also survived cancer responded. Due to encouraging results, there are now at least 30 clinical studies of fecal microbiota transplantation being conducted (58). The role of probiotics in cancer prevention Probiotics are generally considered safe live microorgan- isms and can play a key role in preventing various diseases, including different types of tumors (59). Besides live pro- biotics, it has also been proven that dead probiotics, such as bacterial components (cell wall), have numerous ben- efits in managing various diseases, including cancer (60). Although probiotics are generally considered safe, they should be carefully considered before giving them to can- cer patients since they are immunocompromised. There may be a risk of antibiotic resistance transfer and the de- velopment of opportunistic infections (61). Nonetheless, probiotics have many positive effects in cancer patients, as they can prevent diarrhea and other gastrointestinal is- sues and increase the populations of beneficial bacteria in the gut, thereby enabling the establishment of a healthy gut microbiota (62). For example, in a study where a patient received the probiotic strains Lactobacillus johnsonii and Bifidobacterium longum, the strain successfully adhered to the intestinal mucosa and eliminated pathogenic bacteria by triggering a local immune response (63). A similar study showed that Bifidobacterium longum and Lactobacillus acidophilus significantly reduced severe diarrhea during pelvic radiotherapy indicating their probiotic efficacy (64). Likewise, a blend of ten probiotic strains not only alleviated diarrhea but also demonstrated decreased chemotherapy- induced cytotoxicity in the treatment of certain patients battling metastatic colorectal cancer (65). Probiotic bacte- ria can indirectly prevent colorectal cancer by attenuating the activity of intestinal enzymes responsible for convert- ing amines and complex aromatic hydrocarbons into active carcinogens (66). Furthermore, research has established that Bifidobacterium animalis subspecies Lactis and other probiotic strains modulate the host's immune response by activating phagocytic cells, which subsequently target and eliminate cancer cells in their early developmental stages (67–69). The direct consumption of probiotics by colorec- tal cancer patients has been associated with proapop- totic effects on cancer cells. For example, Lactobacillus delbrueckii significantly upregulates the expression of cas- pase 3, thereby triggering programmed cell death in human colorectal cancer cells. Consequently, probiotic bacteria stand as promising biotherapeutic agents capable of pre- venting intestinal dysbiosis, enhancing the host's immune response, and eliminating various curable and difficult-to- cure diseases, including different types of cancers (70). Microbiota of the gut interacts not only with the immune system but also engages gut epithelial cells via inflamma- some activation. These inflammasomes, expressed by both intestinal and immune cells, enable the distinction between toxic and non-toxic molecules produced by pathogenic and non-pathogenic microorganisms based on the nucleotide- binding oligomerization domain-like receptors (NOD-like receptors) (71). When homeostasis in the body is disrupted for any reason, inflammasomes become active and medi- ate a strong immune response. They activate caspase 1, which in turn triggers the secretion of pro-inflammatory cy- tokines (IL-1β), IL-18, and ultimately leads to apoptosis (72). Dysregulation of inflammasomes is implicated in various diseases, including autoimmune conditions, neurodegen- erative and metabolic disorders, and cancer, with the gut microbiome emerging as a pivotal factor in their activation (73). Comparative studies of microbiome between human and animal models Scientists have been using animals to study human diseas- es for over a hundred years. In this regard, mice have been particularly useful, as they share many biological character- istics with humans. Moreover, they share over 80% of their genome with humans. Due to their phylogenetic similar- ity, physiological resemblance to humans, ease of mainte- nance and breeding in laboratories, and the availability of numerous inbred strains, domestic mice (Mus musculus) have long served as models for human biology and diseas- es, including cancer (74). However, despite the anatomical, histological, and physi- ological similarities between mouse and human intes- tines, there are significant differences in size, metabolism rate, and dietary habits. The use of mice as model organ- isms for studying human biology is based on genetic and physiological similarities between them. It is important to be aware that despite their phylogenetic closeness, mice, and humans have evolved and adapted to different environ- ments, leading to significant differences in their character- istics. This is also why mice often respond to experimental interventions in ways that differ considerably from humans. Mice models in the laboratory are 'specific-pathogen-free' (SPF) mice. These differences are also reflected in the de- velopment of the gut microbiome compared to humans. Instead of SPF mice, microbially exposed, or ‘dirty’ mice model that better mimics the diverse infectious history that is typical of most humans, can be used (75). For example, 18 | Slovenian Veterinary Research 2025 | Vol 62 Suppl 27 a study done by Sjaastad et al found the potential limitation of exclusive use of SPF mice when testing vaccine efficacy compared with "dirty" mice, which may also be one of the reasons that some new therapies work in experimental mice and then the efficacy is lost when transferred to hu- mans (76). The anatomy of the gastrointestinal tract plays a crucial role in these differences, with significant variations between the two species. The ratio of the length of the small and large intestines is greater in mice than in humans, mice have a distinct cecum, and they lack an appendix. An important site for microbial fermentation of undigested food in mice is the cecum. Thus, both species provide different environ- ments that support the growth of different gastrointestinal microbiota (77). An example where the impact of anatomy can be observed is in mice with "cecal lymphoid patches, " which can be syn- onymous with the human appendix. Here, the flora of these two compartments differs, with Firmicutes, Proteobacteria, Bacteroidetes, and Fusobacteria being predominant in the human appendix in terms of abundance, whereas the mouse cecal lymphoid patches consist of Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria (78, 79). Previous studies have shown that human and mouse gut flora share 90% and 89% similarity in species and genera, respectively. However, more recent research has shown that the differences are much larger than previously thought (80). A study from 2021 revealed that more than half of the species in both human and mouse microbiomes belonged to the Firmicutes_A species. Firmicutes_A and Bacteroidota (Bacteroidetes) were the most common species in both human and mouse microbiomes. Firmicutes_B was more common in mice than in humans, while Firmicutes_C was less represented. In general, 16 species were common to both human and mouse microbiomes, with 5 species found only in humans and not in mice. In contrast, species such as Deferribacterota, Thermoto gota and two species Chlamydia muridarum and Chlamydophila psittaci were specific to mice. No archaea were reconstructed from the mouse gut metagenome, whereas 0.4% of genomes in the human gut were attributed to this domain. At the family level, humans and mice shared 88 out of 109 taxa, and their average abundances in human and mouse microbiota were strongly correlated. Two families Lachnospiraceae and Oscillospiraceae which dominate Firmicutes_A were highly present in both humans and mice. In mice, the Muribacula ceae family was more than 30 times more abundant than in humans, while the Bacteroidaceae family was 14 times smaller. While 255 out of 412 taxa were shared at the ge- nus level, the abundance of genera showed a moderate correlation (r = 0.44), consistent with the results of 16S rDNA sequencing (81). Interestingly, the genus Collinsella (phylum Actinobacteria), associated with atherosclerosis and rheumatoid arthritis, was represented by 579 species in humans but was not found in the mouse metagenome. Surprisingly, out of 1573 CMMG (comprehensive mouse microbiota genome) species, only 170 (10.8%) were iden- tified in the human gut microbiota. Common species, on average, represented 13% of the composition of the mouse gut microbiome. Mapping mouse metagenome samples to the human reference database and vice versa achieved only a 30% mapping rate (82). While these numbers may initially suggest a high degree of similarity in the gut microbiota, a closer look reveals key de- viations, especially in terms of microbial composition and abundance. This demonstrates that mice and the human microbiome are significantly different. These results chal- lenge our analogy between the human and mouse micro- biota. Such changes in the microbiome composition can have a significant impact on experimental plans and re- search approaches to studying the human gut microbiome using mice as intermediaries. Considering the significant influence that the microbiome can have on the efficacy of various drugs, the differences between the human and mouse gut microbiomes present a considerable challenge. Establishing a humanized gnotobi- otic mouse model by transplanting human fecal microbiota into mice without their own microbiota represents an inno- vative and powerful tool for mimicking the human microbial system in mice. However, creating such a model requires careful consideration of various factors, from aspects relat- ed to human donors to the genetic background of the mice, all of which can influence the final research outcomes (83). It is also important to question how much of the human mi- crobiome mice can retain, given the anatomical differences in the gastrointestinal tract between humans and mice. While mice models are the most prevalent models to study the gut microbiome (84), there are also other animal candi- dates for these studies. For example, non-human primates have the most similar microbiome to human primates than to any other animal (85), therefore various studies have been conducted using them to explore the influence of dif- ferent diets, for example, Western and Mediterranean diets on the gut microbiome (86, 87). We need to note here that of course there are differences in abundance of certain bacterial taxa between species. It was found that in non- human primates and in rats there is a higher abundance of Prevotella compared to humans and mice (88). As many diseases, disorders, and cancer progression have been linked to an abnormal gut microbiome in humans, research has now expanded to other animal models such as horses (89, 90) and dogs (91) to name a few. Researchers are also trying to create pig models that resemble the human gut microbiome to make future experiments easier and more reliable (92, 93). For now, using mouse models to study the gut microbiome seems to be the optimal option, and the potential of opti- mized humanized gnotobiotic mouse models is something to look forward to in the future. Slovenian Veterinary Research 2025 | Vol 62 Suppl 27 | 19 Studying the microbiome using alternative in vitro and ex vivo models Despite great progress being made in deciphering the role of the gut microbiota in connection with various diseases using different animal models, especially mouse models, there are still several limitations with those models. They are time-consuming, under ethical considerations, and of- ten fail to copy real human conditions because of inter-spe- cies differences and the complexity of the gut microbiome in general (94). Advances in three-dimensional cell biology and bioengineering enabled researchers to come up with alternative in vitro and ex vivo cellular and tissue models to study the microbiome. These models could decrease the number of animal experiments in the future. Organoids are gaining more and more attraction since they have been proven to be valuable in vitro systems for model- ing different human diseases (95). They are 3D self-assem- bled tissue constructs that contain highly polarized cells that mimic the in vivo organization and architecture of the tissue of origin (96). Organoids can be derived either from pluripotent stem cells (PCSs) or adult stem cells (ASCs). In the intestines, ACSs are located at the bottom of the intes- tinal crypts which can then be grown in extracellular ma- trix Matrigel to make organoid models which contain fully mature goblets cells, enteroendocrine cells, enterocytes and Paneth cells (97). When using PCSs to form organoids, cells are often either derived from embryonic stem cells or from induced pluripotent stem cells which are then treated with specific growth factors that direct the tissue-specific development of the cells (98). The PCS-derived organoids can contain also mesenchymal cells in comparison to in- testinal epithelial cell types seen in ASC-derived organoids (99). Gastrointestinal organoids either from ASC or PCS-derived cells have the basal membrane displayed outwards and the lumen in the center of the construct. The most popular method to deliver the bacteria or their metabolites to form a relevant microbiome organoid model is through microin- jection into the lumen. This method mimics bacteria that normally also infects the host from the lumen but has also downsides since it is a difficult method to perform, and or- ganoid damage often happens during the whole process. There is also a method where organoids in suspensions are mixed with microbes and then cultivated to reform 3D organoids but again this method does not appropriately capture the mechanisms by which microbes infect the cells (100, 101). One very important limitation researchers should be aware of includes the lack of cellular components of the microenvironment particularly in ASC-derived organoids (102). Among cellular components, organoids lack mes- enchymal cell heterogeneity and architecture, vasculature, neuronal connections, and interaction with immune cells and the intestinal microbial flora (103). Because of the emerging connection of microbiome influ- ence on tumor development and progression, scientists also try to use different cancer organoid models to study this correlation. Traditional 2D cell cultures fail to mimic the complex tumor microenvironment and the interaction of tumor and non-tumor cells as well as the interaction with the extracellular matrix. The results so far show that tumor cells in organoids react differently to chemotherapy than 2D cell cultures or just tumor cells embedded in 3D gels, showing a promising future for the use of organoids (104). Another very promising in vitro model is organs-on-chips, which can mimic the physiology, structure, function, and pathology of human organs. Scientists have invented organ chip models of the human intestine which are novel cell cul- ture devices that offer greater control over important bio- logical parameters such as oxygen availability and pH lev- els using different micro-fluidic channels (105). The chips complexity over the last years has increased and can now even mimic intestinal peristaltic-like motions and flow, us- ing different mechanical forces (106). The advancements also include a variety of channels that are surrounded by commensal microbes, pathogens, immune cells, and hu- man microvascular endothelium and can also enable villus- crypt formation and added mucus layer (105). For example, researchers have made an intestine chip that contains epi- thelial cells from human intestinal biopsies that success- fully mimics real human physiological conditions (107). Apart from primary cells, other types of cells can be grown on such chips for example ASC or PCS-derived organoids (108, 109) or immortalized cell lines (110). In the end, these models could serve as a great option in addition to animal models to study the influence of the gut microbiome as well as being a valuable tool to study different kinds of tissues. Alternative cell and tissue models in the laboratory will al- low us to study the interactions between tissue, tumor, and microbiome by mimicking microbiome-host-cancer inter- actions as a function of species. This will allow us to un- derstand the molecular mechanisms and develop alterna- tive treatment approaches for various diseases, including cancer. Gut microbiota in patients with aggressive primary brain tumors We know that gut microbiota influences tumor growth and progression. Since gliomas are the most aggressive prima- ry brain tumors in adults and are challenging to treat there is ongoing research about the connection to the gut micro- biome trying to find new ways to improve current treatment outcomes (111). Researchers have found out that glioma tumor growth leads to dysbiosis in the gut microbiome in mice before weight loss occurs. They found a significant decrease in the Firmicutes to Bacteroides (F/B) ratio follow- ing tumor growth. They observed a decrease in Firmicutes and an increase in Verrucomicrobia phyla. Interestingly, 20 | Slovenian Veterinary Research 2025 | Vol 62 Suppl 27 after treating the mice with the oral chemotherapy drug te- mozolomide (TMZ) there was no glioma-induced dysbiosis seen since there was no significant difference in the F/B ratio. They later showed that TMZ administration in healthy mice causes dysbiosis but with no significant change in Verrucomicrobia. When they analyzed fecal samples from human glioma patients and healthy controls they found a correlation with their mice results, with similar microbiome changes observed in both species (112). We mentioned in previous sections the effects of short- chain fatty acids (SCFAs) like butyrate, acetate, and pro- pionate have anti-cancer properties. In a study made by A. Dono, et al. they show that the short fatty acids were all decreased in mouse models after glioma growth after an analysis of fecal metabolites. Apart from that, they saw a decrease in important neurotransmitters such as norepi- nephrine and 5-hydroxyindoleacetic acid (5-HIAA) after tumor development and an increase in serotonin, 3-methyl valerate, caproate, and acetylcholine. This study also found the same increases in the Verrucomicrobia phylum and a decrease in Bacteroidetes which coincides with the find- ings of the study mentioned in the previous paragraph since Bacteroides belong to the Bacteroidetes phylum of Gram-negative bacteria. After treating the mice with TMZ there was no significant change in SCFAs which is consis- tent with the findings that glioma growth impacts the gut microbiome. Similar data was also obtained from human samples which means that we can draw parallels between the mouse model and actual human samples (113). Another study found conflicting results with the previously mentioned studies. After the growth of glioma in mouse models they observed a decrease in the abundance of Bacteroidia (new name for Bacteroidetes) and an increase in the abundance of Firmicutes. However, the results still suggest that glioma growth contributed to dysbiosis in the gut. They also found that after administering antibiotics to mice, the glioma progression is worse than in non-antibiot- ic treated mice. These results confirm the hypothesis that gut dysbiosis can worsen glioma progression. After detect- ing expression levels of CD8 and Foxp3 in mouse brain tissues in antibiotic and non-antibiotic-treated mice, they found out that CD8 expression levels were not significantly different between the two groups. On the other hand, there was a decrease in Foxp3 expression in antibiotic-treated mice compared to the control group suggesting that gut microbiome dysbiosis downregulated Foxp3 expression in glioma tissue and Foxp3 may act as a tumor suppressor protein (114). The importance of gut microbiota in glioma development was highlighted in another study where they also found that glioma growth increases in mice treated with antibiotics. They also observed changes in microglia phenotype and a reduction in CD27+/CD11b+ NK cells that are involved in tumor cell lysis which could explain the tumor size increase in antibiotic-treated mice (115). The Bifidobacterium genus was shown to have antitumor effects (116) and in a study where mixtures of Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium lactis, and Bifidobacterium bifidum were administered into an orthotopic mouse model of glio- ma via gavage treatment, the tumor volume was reduced, and the lifespan of the mice was prolonged (117). This ex- periment gives us promising new therapeutic options for future glioma treatments. Glioblastoma is the most aggressive type of glioma with a median survival of 12-15 months (118). The most effec- tive treatment so far is the combination of surgical removal of the tumor, radiotherapy, and chemotherapy with TMZ. Immunotherapy is successful only in preclinical mouse models however, it is not effective in humans. The reason could be in the difference between the gut microbiome in humans and mouse models since studies report that 85% of bacterial genera found in the mouse gut microbiota are not present in human (77). A study using humanized mouse models, where they transplanted the human gut mi- crobiome into mice, found that mice with different human gut donors responded differently to immunotherapy using the checkpoint inhibitor anti-PD-1 drug. Out of five tested human mouse models only 2 responded to the treatment and displayed a significant increase in survival compared to the control groups. The two responsive mouse models had an abundance of Bacteroides cellulosilyticus, while the non- responsive models had an abundance of Bacteroides in- testinalis and Bacteroides uniformis. They also found an in- crease in cytotoxic CD8+ and CD4+ T-cells producing IFN-γ after anti-PD-1 treatment in a humanized mouse model that was responsive to immunotherapy. The same results were not observed in the humanized mouse model that did not respond to immunotherapy (119). A recent study com- paring healthy individuals with glioblastoma brain tumor (GBM) patients has shown that the GBM patients had a higher gut microbial diversity compared to the healthy indi- viduals. The GBM group had a decrease in Firmicutes and an increase in the Proteobacteria phylum (120). The gut microbiota also plays a role in the metabolism of various amino acids, which has different effects on the progression of gliomas. For example, the gut microbiome plays an important role in tryptophan metabolism, the prod- uct of which are AHR agonists. AHR stands for the aryl hy- drocarbon receptor, a transcription factor that is activated by various ligands and is involved in cell proliferation, differ- entiation, cell death, and cell adhesion (121). The receptor is expressed in gliomas with the highest expression seen in glioblastomas (122). The AHR agonists produced by gut bacteria can activate the AHR receptor which then increas- es FoxP3+ regulatory T cells through different mechanisms. It also regulates other T cell function as well as the differ- entiation and function of dendritic cells (123). In the case of arginine metabolism, the gut microbiota can turn dietary arginine into polyamines and nitric oxide which arrive to the brain through the blood-brain barrier (124, 125). Polyamine can affect tumor growth by up-regulating the expression of Slovenian Veterinary Research 2025 | Vol 62 Suppl 27 | 21 ornithine decarboxylase, spermidine, spermine acetyltrans- ferase, and Akt1 which can induce tumor cell proliferation and metastasis (126). The effects of nitric oxide on glioma are still not fully known but since nitric oxide can interfere with T cell function by promoting T cell apoptosis it can be speculated that it could promote glioma development (127, 128). These studies suggest that the gut microbiome is also cru- cial for distant tumors, such as primary brain tumors, and that the microbiome is also found in the microenvironment of brain tumors (129), where its role remains to be explored. In this regard, the microbiome should be exploited to in- crease the efficacy of current therapies and/or to develop new, more efficient treatments. Future outlook and conclusions The gut microbiome has been shown to influence the suc- cess of various cancer therapies (Figure 3). Some bacteria have a tumor-inhibiting effect and help the individual fight cancer, while others have the opposite effect and worsen the state of health. Ideally, it would be desirable to eliminate the harmful bacteria while promoting the proliferation of the beneficial ones. However, current antibiotics have a broad spectrum of action that affects both types of bacteria. For this reason, research is being carried out into the synthe- sis of specific antibiotics that only eliminate the bad bac- teria and leave the good bacteria untouched. Another pos- sible solution would be the use of bacteriophages, which only infect and destroy certain bacteria, thus creating a microbiome that benefits the patient. Research on fecal mi- crobiome transfer seems promising, but scientists have yet to determine why some donors' feces are more successful in increasing the effectiveness of immunotherapy than oth- ers. Nonetheless, this area of research is gaining increasing attention and could prove to be a crucial approach to can- cer treatment in the future. The gut microbiome is a dynamic and influential compo- nent of human health. While it offers many benefits, dis- turbances in its balance can contribute to the development and progression of cancer. It plays an important role in the anti-tumor response, as demonstrated by successful can- cer remission after fecal transplantation in mice and hu- mans. Understanding the intricate relationship between the gut microbiome and cancer is crucial for the development of new strategies to prevent and treat this complex disease. Further research is needed to explore the full extent of these interactions and their potential therapeutic implications so that we can improve treatment outcomes for difficult ma- lignancies such as gliomas in the future. Because mouse models have genetic and physiological similarities to hu- mans, they are invaluable for biomedical research, includ- ing research into human disease. However, it is important to recognize the microbiome-related differences between these two species, as this is the only way we can achieve more relevant results. New in vitro models such as organ- oids on chip organs offer a promising alternative to animal models to study the gut microbiome in a more controlled and species-specific way. This emerging field of research could help us fight cancer and other diseases in a novel way that could significantly improve our overall well-being. Figure 3: The influence of a healthy gut microbiome on chemotherapy treatment. On the left we can see that without chemotherapy the tumor microenvironment can suppress the immune system and hence hindering adequate immune response to eliminate the tumor. In the middle we can see that the absence of a gut microbiome, in gnotobiotic mice for instance, after administration of a chemotherapeutic drug CTX we can see no improvement in immune response. On the right where we have a healthy gut microbiome, the administration of CTX increases immune response and therefore impacts the natural immune system and increases the anti-tumor immune response. The Scheme was created using Biorender.com Abbreviations: major histocompatibility complex 1 (MHC1), short-chain fatty acids (SCFAs), cyclophosphamide (CTX), pathogen-associated molecular pattern molecules (PAMPs). 22 | Slovenian Veterinary Research 2025 | Vol 62 Suppl 27 Acknowledgement This work was supported by the Slovenian Research and Innovation Agency (Programme and research grants P1- 0245, J3-4504, J3-2526 and NC-0023) and by the European Union’s Horizon project Twinning for excellence to strate- gically advance research in carcinogenesis and cancer (CutCancer; 101079113) References 1. Schwabe RF, Jobin C. The microbiome and cancer. Nat Rev Cancer 2013; 13(11): 800–12. doi: 10.1038/nrc3610 2. Gomaa EZ. Human gut microbiota/microbiome in health and diseas- es: a review. Antonie Van Leeuwenhoek 2020; 113(12): 2019–40. doi: 10.1007/s10482-020-01474-7 3. Frank DN, St. Amand AL, Feldman RA, Boedeker EC, Harpaz N, Pace NR. 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Edinstven črevesni mikrobiom posameznika, ki ga oblikujejo dejavniki, kot so način rojstva, prehrana, vnos antibiotikov in življenjski slog, prispeva k različnim telesnim funkcijam. Te funkcije so presnova hranil, metabolizem zdravil in uravnavanje imunskega sistema. Spremembe v črevesnem mikro- biomu so povezane s predispozicijo za nastanek raka in lahko vplivajo na učinkovitost njegovega zdravljenja. Porušeno črevesno ravnovesje oziroma disbioza v črevesnem mikrobiomu lahko vodi do vnetja, razvoja tumorjev in metastaz, kar poudarja njegov pomen v raziskavah raka. Črevesna mikrobiota pomembno vpliva na razvoj raka in rezultate zdrav- ljenja. Nekatere bakterije povečajo učinke terapij, kot je ciklofosfamid, in prispevajo k boljšemu imunskemu odzivu proti raku. Mikroorganizmi proizvajajo protirakave molekule in probiotične spojine, ki so pomembno orodje pri preprečevanju in zdravljenju raka. Z nadaljnjimi raziskavami si znanstveniki želijo razviti ciljne antibiotike in raziskati prenos fekalne mikrobiote za selektivno manipulacijo mikrobiote. Zaradi genetskih in fizioloških podobnosti so mišji modeli neprecen- ljivi v biomedicinskih raziskavah, vendar pa zaradi razlik v črevesnem mikrobiomu ljudi in miši ter sestavi tumorske- ga mikrookolja neposredna primerjava med tema dvema modeloma lahko predstavlja izziv. Premostitev teh vrzeli je ključna za primerjalno medicino zlasti pri raziskavah raka, kjer mikrobiom igra pomembno vlogo pri izidih zdravljen- ja. Pri možganskih tumorjih gliomih lahko črevesni mikrobiom izkoristimo za potencialne nove možnosti zdravljenja. Dolgoročnega učinkovitega zdravljenja za to vrsto raka še ni, vendar raziskave na mišjih modelih kažejo povezavo med napredovanjem tumorja in odzivom na zdravljenje ter spremembami v črevesnem mikrobiomu. Črevesni mikrobiom in njegova modulacija predstavljata priložnost za učinkovitejše zdravljenje raka v prihodnosti. Ključne besede: črevesni mikrobiom; rak; izid zdravljenja; tumorski modeli; gliom