Unveiling the gut-pancreas axis: microbial influence on stemness and tumor microenvironment of PDAC.

Introduction

The gut microbiome: implications in disease and cancer
Akin to a fingerprint, each individual’s microbiome is uniquely shaped by genetics, lifestyle, diet, and environmental exposures.1 Among the various microbial niches in the body, the gut microbiome has emerged as a key regulator not only of gastrointestinal health but also of systemic processes through its interactions with distant organs, such as the brain, lungs, and pancreas.2,3 The relationship between the gut microbiome and cancer remains one of the most intricate and challenging areas of study. Barry Marshall and Robin Warren conducted a landmark study in the field. The authors provided compelling evidence for the oncogenic potential of Helicobacter pylori, establishing its role in the pathogenesis of peptic ulcers and its contribution to gastric carcinogenesis.4 Subsequent research has revealed that many cancer patients exhibit dysbiosis, characterized by an altered composition and function of their microbial communities. The impact of the gut microbiome on human health supports critical physiological processes such as nutrient digestion, vitamin biosynthesis, and the maturation and regulation of the immune system. Additionally, it plays key roles in pathogen defense, epithelial repair, the metabolism of dietary and pharmaceutical compounds, immune modulation, and neurobehavioral regulation through the gut-brain axis.2,5,6,7 It produces bioactive compounds such as short-chain fatty acids (SCFAs) and secondary bile acids and is closely involved in the metabolism of xenobiotics.7,8,9 Conversely, disruptions in microbial composition, referred to as gut dysbiosis, have been linked to the onset and progression of numerous diseases.10,11,12 Elucidating the mechanisms by which dysbiosis contributes to systemic pathology remains a critical research priority, with an increase in potential for the development of microbiome-targeted therapeutic interventions.

Gut-pancreas axis-bifurcated entry of pathogens: duodenal reflux and barrier disruption
Gut homeostasis is maintained through a dynamic balance between commensal and pathogenic microorganisms. In the context of cancer, microbial dysbiosis can arise either through direct translocation of bacteria across a compromised epithelial barrier or indirectly via bacterial metabolites and toxins disseminated through the bloodstream or lymphatic system.13,14 Although the colon and oral cavity possess the highest microbial densities, the pancreas, historically considered a sterile organ, is now known to host its distinct microbiome. Due to its anatomical proximity to the gastrointestinal tract, the pancreas is particularly vulnerable to microbial colonization, which may occur through mechanisms such as epithelial barrier disruption or retrograde migration via duodenal reflux.15–17 Gut bacteria can enter the pancreatic duct through the Sphincter of Oddi, a muscular valve, in a phenomenon termed “bacterial reflux,” which disrupts the inherently balanced microbiota of the pancreas by either driving the survival of pathogenic bacteria or depleting the existing commensal bacteria.16–18 Tumors at the head of the pancreas can progress and disrupt the gut-pancreatic epithelia,19 allowing bacteria from the gut to enter the pancreas. In addition to this, metabolites and toxins secreted by the bacteria may penetrate the pancreas through this system of action, colonizing and eliciting changes in the tumor microenvironment (TME), thereby modulating the tumor microbiome.20 The driver-passenger hypothesis, biofilm hypothesis, intestinal microbiota adaptation, alpha bug hypothesis, and bystander effect are among the theories proposed to explain microbe-niche interactions, particularly in colorectal cancer.21 The alpha bug hypothesis suggests that specific bacteria compromise epithelial integrity, increasing tissue exposure to inflammatory signals and carcinogens. According to the driver-passenger hypothesis, certain microbes initiate early tumorigenic changes, creating a favorable niche for other, less aggressive microbes that reach later. The biofilm hypothesis emphasizes the role of dense bacterial communities that sustain inflammation and trigger cancer-related signaling pathways like STAT3 and NFκB. The intestinal microbiota adaptation hypothesis argues that the TME selectively reshapes microbial populations in ways that support cancer growth. Finally, the bystander effect hypothesis points to microbial byproducts such as reactive oxygen species (ROS) that can cause DNA damage or help tumor cells evade the immune system.21,22 Together, these models offer insight into how microbes may influence pancreatic tumor development and may also serve as potential frameworks for understanding microbe-host interactions in pancreatic ductal adenocarcin (PDAC), warranting further investigation into their applicability in this context (Figure 1).

Microbe-mediated mechanisms in pancreatic cancer progression and therapy
Several studies have linked the presence and absence of specific bacterial species to the development of PDAC. For instance, Fusobacterium nucleatum promotes tumor cell migration and growth by elevating cytokines like CXCL1, IL-8, and MIP-3α.23 In the basal-like, more aggressive subtype of PDAC, Pseudomonas, Sphingopyxis, and Acinetobacter were seen to be enriched compared to the classical and hybrid subtypes.24 Families including Kocuria, Streptococcus, Bacillus, Ralstonia, Staphylococcus, Acinetobacter, and Pseudomonas were commonly detected across PDAC datasets.25 In KPC mouse models, the microbiome composition was observed to vary by sex, with dominant phyla being Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Deferribacteres.26 In PDAC patients undergoing surgery, the presence of Enterococcus faecalis in the bile and duodenum worsened outcomes, causing deep infections, more ICU stays, and higher death rates, likely through epithelial disruption and increased TNF-α and IL-6 levels.27 Another study introduced MOCO-GCN, a machine learning model that integrates gut microbiome and exposome data for the diagnosis of PDAC.28 They identified F. nucleatum and F. hwasookii as inflammation drivers acting via LPS-triggered NF-κB signaling. A spatial analysis from CP to late-stage PDAC showed microbial spread from oral regions to the pancreas. Pathogens like P. gingivalis and T. denticola expanded, while Bifidobacterium breve declined, correlating with poorer survival.29,30
In murine studies, transplanting fecal microbiota from PDAC-bearing mice into Kras-mutant recipients led to increased tumor formation.31 This was accompanied by a decline in SCFA-producing bacteria like Roseburia and Butyricicoccus, and an enrichment of Actinobacteriota. These changes were associated with TLR4/NF-κB pathway activation, impaired epithelial regeneration, and potential involvement of IL-6/JAK-STAT3 signaling. A Mendelian randomization study further supported a causal link between certain microbes and PDAC. Protective effects were associated with Clostridium, Romboutsia, mannitol, and methionine, while higher levels of carnitine and 3-methylhistidine were linked to increased risk. Affected pathways included SCFA metabolism, lipid signaling, and one-carbon metabolism, all of which impact DNA methylation and oxidative stress.32 Environmental eustress (EE), a model reflecting reduced psychological stress, was shown to suppress PDAC growth by reshaping the gut microbiota.33 EE elevated Lactobacillus reuteri, which promoted natural killer (NK) cell infiltration and enhanced anti-tumor responses. When NK cells were depleted, this benefit was lost, pointing to TLR2-dependent microbial-immune signaling and SCFA-mediated anti-inflammatory effects. Collectively, these findings support the role of the microbiome as both a diagnostic marker and therapeutic target in PDAC. Emerging human studies have reinforced this connection. Using 16S rRNA sequencing, metagenomic, and metabolomic approaches, researchers have consistently reported microbial imbalances in PDAC patients compared to healthy controls (Supplementary Table 1). Pro-inflammatory and tumor-associated taxa such as Fusobacterium, Enterococcus, Streptococcus, Klebsiella, Veillonella, Escherichia/Shigella, Actinomyces, Lactobacillus, Pseudoxanthomonas, Prevotella, and P. gingivalis were often enriched, while beneficial microbes like Faecalibacterium prausnitzii, Ruminococcus intestinalis, Akkermansia, Anaerostipes, Bifidobacterium, Bacteroidota, and Lactobacillus plantarum were consistently diminished. These beneficial microbes are known for supporting gut integrity, reducing inflammation, and improving therapy outcomes. In addition to these compositional shifts, metabolites such as trimethylamine N-oxide (TMAO), indoleacetic acid, and other indole derivatives are being explored as biomarkers for early PDAC detection and long-term risk assessment.34,35 Bile and tumor-resident microbiota have also been linked to postoperative complications and immune dysfunction. Ongoing clinical trials (Supplementary Table 2) are testing microbiome-modulating strategies in PDAC, including FMT, microbial profiling for stratification, and microbiome-immunotherapy combinations. Together, these findings highlight the growing relevance of microbiome research in PDAC diagnostics and treatment, and the need for broader, more inclusive studies to advance personalized care.

Microbiome-driven regulation of the TME: leveraging key factors in sustaining the cancer stem cell pool in PDAC

Shaping stemness in PDAC
The maintenance of PDAC and its recurrence are highly dependent on a sub-population of cells known as PDAC-stem cells (PDAC-SCs). These PDAC-SCs have high plasticity and self-renewing potential. CSCs have been shown to metabolically reprogram themselves via the influence of the TME and induce aggressiveness.36 As part of the TME, the microbes and their secreted derivatives may play a role in the metabolic reprogramming and maintenance of the cancer stem cell pool in PDAC. The role of bacteria-recruited immune factors such as macrophages and T-cells in renewing intestinal stem cell pools has been well studied.37,38 Prominent levels of macrophage recruitment and increased T cell invasion have been correlated with poor prognosis of PDAC.39–41 The infiltration of microbes can cause increased immune cell infiltration into the tumor stroma and dictate the renewal of the cancer stem cell population in PDAC. The cancer stem cell pool is sustained by various factors, including inflammation, TME dynamics, and fibroblast activity. Therefore, understanding how the gut microbiome influences these factors is crucial to uncovering its role in cancer stem cell maintenance (Figure 1). Hayashi et al. demonstrated that Fusobacterium nucleatum, enriched in the PDAC tumor microbiome, induces secretion of CXCL1.42 This chemokine binds to CXCR2 on tumor cells, activating the MAPK/ERK and NF-κB pathways, both of which are known to support self-renewal and chemoresistance. Dudeja et al. further showed that depleting the gut microbiome in KPC mice reduced tumor-infiltrating Th17 cells and levels of IL-17, both of which are crucial for the maintenance of CSCs.9,43–46 IL-17 acts through the JAK/STAT pathway to enhance the expression of stemness-related factors such as SOX2, CD44, NANOG thereby sustaining an undifferentiated and therapy-resistant tumor cell population.47,48 Additionally, it was identified that intratumoral fungi contribute to a CSC-supportive environment by initiating complement activation and inflammation.49,50 Fungal signals, along with bacterial lipopolysaccharide (LPS), activate TLR4 signaling, which in turn promotes NF-κB-mediated secretion of IL-6 and IL-8.51 These cytokines trigger STAT3 activation, which has been linked to the upregulation of stemness-related genes.48 Collectively, these studies highlight the multiple ways in which the microbiome could influence the CSC niche in PDAC by altering inflammatory, immunological, and signaling pathways that maintain stemness and resistance to therapy (Figure 1).

Microbial remodeling of the tumor microenvironment
The highly variable nature of the PDAC TME supports metabolic shifts that allow tumor cells to adopt more aggressive behaviors by reinforcing stem-like characteristics.36 This dense, fibrotic stroma plays a critical role in shaping tumor growth, invasion, and metastatic potential. Dysbiosis can significantly alter this environment, tipping the scales toward inflammation and dampening the body’s natural anti-tumor defenses. Furthermore over 500 altered metabolites were found to be differentially present in tumor vs normal samples, underscoring the role of microbial metabolism in disease dynamics.52,53Additionally, microbial metabolites are capable of modulating various TME components, including endothelial cells, tumor-infiltrating lymphocytes (TILs), pancreatic stellate cells, myeloid-derived suppressor cells (MDSCs), and regulatory T-cells (Tregs), further contributing to tumor progression.15,52,54 Mirji et al. showed that the microbial metabolite trimethylamine N-oxide (TMAO) influences immune responses in PDAC by enhancing effector T-cell activity, hinting at an immune-stimulating role under certain conditions.35 The hypoxic, nutrient-deprived nature of the PDAC TME also provides a survival niche for anaerobic bacteria, which may contribute to the overall fitness and adaptability of the tumor. Supporting this, a marked drop in microbial α-diversity has been observed in PDAC tissues compared to healthy controls, indicating a disrupted microbial community within the tumor space.19 A key study by Aykut et al. uncovered that the fungal species Malassezia can promote pancreatic cancer progression by activating the mannose-binding lectin (MBL) complement pathway, leading to an immunosuppressive TME.48 These microbial influences may also promote biofilm formation, adaptation to low-oxygen conditions, and increased tumor resilience. In a study conducted by Riquelme et al; showed that pancreatic cancer patients with greater microbial diversity and distinct bacterial profiles within their tumors tend to live longer, likely due to enhanced anti-tumor immune activity. Tumors from PDAC patients who survived long-term showed a notable enrichment of bacterial genera such as Pseudoxanthomonas, Saccharopolyspora, and Streptomyces. Additionally, the species Bacillus clausii was found to be significantly more abundant in these long-term survivors.39 3-indoleacetic acid (3-IAA), a key metabolite produced by gut microbes, has been shown to improve the effectiveness of chemotherapy in pancreatic cancer by influencing myeloperoxidase (MPO) activity, reactive oxygen species (ROS) production, and autophagy-related processes. Elevated levels of 3-IAA were linked to longer progression-free survival and overall survival in patients.55 Taken together, these studies support the emerging view that microbes within the PDAC microenvironment play far more than a passive role. They can act as metabolic regulators and signaling intermediaries that support CSC survival and renewal. Moving forward, a critical question remains: do microbes actively shape CSC phenotypes, or do CSCs construct a niche that favors certain microbes? Clarifying this bidirectional relationship could pave the way for new strategies that combine microbial modulation with anti-CSC therapies.

Microbiota-mediated immune modulation
Existing evidence highlights the role of inflammation in promoting stemness enrichment in multiple GI cancers, including PC.33,56 Microbial-driven inflammation can amplify this effect, contributing to the modulation of the TME. This, in turn, plays a crucial role in supporting the survival and persistence of the CSC pool. For increased activity, the oncogenic K-ras mutation, which is frequently associated with PC, needs to be amplified further.57 Once activated, K-ras engages in a positive feedback loop with inflammation. As a result, oncogenesis can occur from any stimulation that activates Ras or encourages inflammation. Numerous inflammatory factors in inflammation-driven pancreatic cancer progression have been extensively studied.33,58 NFkB, IL6, IL4, IL8, IL1-B, IL1-A, TGF-B, TNF-A, COX2, CXCL2, CXCL2, and TLRs (Toll Like Receptors) have been shown to induce inflammation, pancreatitis, and metastasis.59–61
The general belief is that tumors that lose epithelial and mucosal integrity provide a clear path for microbes to enter the tumor site.62 This leads to the production of pro-inflammatory cytokines and Interleukins that eventually help tumor growth, angiogenesis, and metastasis. Another aspect of microbial-driven inflammation is the activation of TLRs. TLRs play a vital role in the innate immune system, eventually leading to antigen-specific acquired immunity.58,61 TLRs recognize conserved patterns from pathogens known as pathogen-associated molecular patterns (PAMPs), which are distinguishable from host molecules. These transmembrane proteins are best known for recognizing Lipopolysaccharide (LPS), which stimulates the TLR4 receptor; bacterial flagellin, which stimulates TLR5, lipoproteins and lipoteichoic acid, which activate TLR1,2, and 6, ssRNA, which stimulates TLR7 and 8, and dsRNA, which activates the downstream of TLR3. The downstream effectors of TLRs are mainly dependent on the MYD88 signaling pathway, including the anti-apoptotic pathway mediated by NFKB, factors such as COX2 (cyclooxygenase 2) and MMP7 (matrix-metalloprotease 7) which are part of the tumor stroma.58 TLRs shown to promote tumor progression by activating pro-inflammatory signaling pathways like NF-κB and STAT3, which support an immunosuppressive microenvironment.58 TLR4 and TLR9 have been shown to induce pro-inflammatory signaling in transformed epithelial cells.63 Thus, activation of TLRs by different pathogenic bacteria can remodel the immune micro-environment of PDAC and contribute to its progression.

The microbiome-fibroblast dynamics
Cancer-associated fibroblasts (CAFs) contribute to therapy resistance and play a role in cancer progression by establishing a mutually beneficial relationship between themselves and the TME. CAFs can increase the expansion of regulatory T-cells, which can in turn modulate the TME.63,64 Increase in T-regs, CTL, and MDSC recruitment contributes to ECM remodeling through regulation of matrix stiffness and helps support immune cell infiltration, tumor cell migration, and invasion.65 Thus, this mutualistic relationship between CAFs and the TME can contribute to the maintenance of the PDAC-SC population. CAFs change into a secretory phenotype when activated and can influence the extracellular matrix (ECM). ECM makes up the major component of the TME, and its stiffness can lead to immune evasion, metastasis, chemoresistance, and tumor growth. The production of collagen is a major outcome of CAF activation. As mentioned by Chen, Yang et al. in PDAC, COL1 is present in high quantities, along with an enrichment of Bacteriodales.52 This signals that the matrix provides nutrition for the growth and survival of the bacteria, thus establishing the dependence of the bacteria on a fibroblastic TME. In a study conducted by Xu, Zhuoqing et al. Actinomyces in colorectal cancer was seen to be co-localizing with CAFs and activating the TLR2/NF-kB pathway, reducing CD8+ T lymphocyte infiltration in the TME.66 The decrease in CD8+ T cells also supports the role of immune evasion and further tumor progression through a correlation between CAFs and the microbiome. A distinct microbial profile from the already existing ‘healthy’ microbial profile in the TME of PDAC can lead to the onset of a strengthened immune response, which can thereby contribute to CAF activation.

Microbiome-tuned metastatic trajectories
The TME houses certain factors and induces specific bio-signals that help the tumor cells acquire metastatic potential. The niche of the secondary organ plays a leading role in helping invading tumor cells to fraternize and colonize the metastatic organ while adapting to stromal changes and TME interactions. The microbes that have either seeped into the pancreatic duct create a dysbiotic microenvironment that may enable these tumor cells to acquire their metastatic potential. The TME of the secondary metastatic site also needs to be primed to allow the invading tumor cells to colonize, where the microbiome of the secondary site can play a significant role, and the niche is thus maintained for the invading tumor cells to thrive. Bacterial communities can produce bacterial extracellular vesicles or bEVs, which are membrane-bound vesicular structures containing bacterial-derived biomolecules, such as nucleic acids, proteins, and lipids that can interact with the host and other microbes.67 These interactions within bacterial communities or between bacteria and the host can contribute to the formation of a pro-oncogenic TME in metastatic sites.68 These bEVs can course through the lymphatic vessels and blood vessels to reach their desired organ or place of docking and can also mediate inflammation through their route, which again is a driver for PDAC (Figure 1). PDAC tumors and metastatic niches frequently harbor intratumoral bacteria—predominantly Fusobacterium, Proteobacteria, and Firmicutes (Supplementary Table 1). These bacteria can trigger chronic inflammation via TLR4/NFκB signaling, creating an immunosuppressive microenvironment conducive to EMT and tumor cell dissemination. Mechanistically, microbial metabolites such as short-chain fatty acids (SCFAs) engage receptors like FFAR2, a GPCR that can further engage in metastasis induction in Colorectal cancer.69 Although the relationship between them is unknow in PC, mice models show an interaction between SCFAs and FFA2 that can lead to an increase in the production of glucose and a reduction in insulin in the plasma.70 Microbial invasion can disrupt cellular signaling by activating the TGF-β pathway, where microbial products like LPS, flagellin, and muramyl dipeptides (MDP) trigger phosphorylation of the TGF-β receptor. This leads to activation of Smad2 and Smad3, forming a complex that, in turn, engages the Ras-MAPK pathway. Together, these pathways drive key cancer-promoting processes like cell growth, migration, and epithelial-mesenchymal transition (EMT), contributing to tumor progression.71 These results highlight the potential of targeting microbial TLR/NF-κB signaling, SCFA-FFAR2-mediated EMT as strategies to suppress PDAC metastasis. However, clinical validation in patients is still lacking.

The mycobiome’s role
The mycobiome is a term used to describe the fungal community in the human body. Recent evidence has shown that an altered mycobiome exists in certain diseases and tumors. This community is hypothesized to influence metabolism, immune response, metastatic ability, and the TME, similar to the effects of the gut microbiota on cancer. A significant increase in dysbiosis of the mycobiome was observed in PDAC patients, characterized by a distinct mycobiome expression profile enriched for the fungus Malassezia. An accelerated oncogenesis with a parallel increase in Malassezia was observed.81 PDAC patients were also found to have alterations in the duodenal fluid mycobiome. Patients with pancreatic cysts were shown to have a significantly increased fungal community at both phylum and genus levels in comparison to a normal healthy pancreas.82 Kras mutation, which plays a vital role in the prognosis of PDAC, was shown to be influenced via the increased expression of IL33 through the KRAS-MEK-ERK pathway, which is enhanced by the mycobiome of the TME.83 The role of the mycobiome has not been elucidated to the extent that it can be extrapolated to PDAC, and hence, the field requires more input and research to establish its role in cancer prognosis.

Conclusion and perspectives
The gut microbiome represents a promising, non-invasive avenue for improving cancer diagnosis, monitoring, and potentially therapy.16S rRNA sequencing is the sought-after technique used for characterizing bacterial communities and may aid in identifying microbial shifts associated with PDAC. However, while these associations are intriguing, their clinical utility remains to be fully validated. During metastasis, microbial composition may vary across organs depending on local TME factors, and on bEVs in promoting or tracking disease progression is an area of active investigation. Detecting stage-specific microbial patterns using 16rRNA sequencing could eventually support more tailored treatment approaches, but robust evidence for such applications in clinical practice is still lacking due to patient-to-patient variability. The use of chemotherapy and antibiotics, although standard in PDAC management, often comes with significant side effects. Some commensal bacteria may mitigate these effects by producing beneficial metabolites. Longitudinal multi-omics studies and spatial metabolomics provide powerful tools to uncover detailed, dynamic links between the microbiome and the metabolome, particularly in complex diseases like cancer, where they can track changes over time across multiple biological layers (e.g. microbiome, metabolome, transcriptome, immune profile). While probiotics and microbiota-directed therapies are still being explored, their integration into oncologic care requires more rigorous testing. Similarly, microbial therapies aimed at enhancing response or minimizing toxicity are still in the experimental stage. Despite its potential, microbiome research in PDAC is hampered by significant challenges, including a lack of germ-free model systems for in vitro validation, high inter-individual variability due to diet and geography, risks of contamination, and limitations in reproducibility. These issues must be addressed before microbiome-based interventions can be reliably translated into clinical practice. Lifestyle, diet, and industrialization are the most powerful influences shaping the human gut microbiome, often overriding genetic factors and leading to reduced microbial diversity. Migration further alters microbiota by causing rapid shifts such as the loss of Prevotella and plant-fiber-degrading enzymes potentially increasing risks for obesity and chronic disease. Current research urges broader population studies and ecological frameworks to better understand microbiome resilience, adaptation, and long-term health impacts across global communities.72 Metabolic diseases are closely linked to diet, with Western dietary patterns promoting poor metabolic outcomes, while diets like Mediterranean or Japanese offer protective effects. Although host genetics influences how diet shapes gut microbiota, diet consistently plays a stronger role in determining microbial diversity, composition, and related impacts on glucose metabolism and body fat.73 A high-fat diet promotes dysbiosis and cancer progression, while fiber-rich and Mediterranean diets enhance beneficial microbes and improve treatment outcomes. Food like rice and beans or ketogenic regimens also impact cancer risk and therapy response through changes in microbial diversity, SCFA production, and immune modulation, though results vary across cancer types and individuals.74 A study revealed how Western and high-fat diets promote carcinogenesis by altering gut microbial composition, reducing butyrate-producing bacteria and enabling expansion of genotoxin-producing oncomicrobes like Fusobacterium nucleatum and pks+ E. coli. These microbial shifts enhance DNA damage, inflammation, and immune evasion, increasing cancer risk and weakening therapeutic response. Dietary patterns like fiber-rich or prudent diets help maintain microbiome stability, highlighting the potential for personalized dietary strategies in cancer prevention and treatment.75 Taken together, dietary patterns and lifestyle transitions are key drivers of gut microbiome composition, often outweighing genetic influence and contributing to disease risk. Personalized nutrition approaches that restore microbial balance may offer effective strategies for preventing and managing metabolic disorders and cancer. In conclusion, while our understanding of the microbiome’s role in PDAC is in its early stages, continued research may help clarify its contribution to disease mechanisms and therapeutic outcomes. Caution and rigor are essential as the field progresses toward evidence-based applications.

Supplementary Material
Supplementary InformationSupplementary material is available at Stem Cells online.