MICROBIOME: A DRIVER OF PANCREATIC INFLAMMATION AND TUMORIGENESIS

Abstract

The human microbiome plays a crucial role in regulating various physiological processes, including immune responses, inflammation, gut permeability, and overall homeostasis. Recent studies have identified bacterial and fungal components in the tumor microenvironment of multiple malignancies, including pancreatic ductal adenocarcinoma (PDAC). These intratumoral microbiomes drive tumorigenesis by activating oncogenic signaling cascades and reprogramming both innate and adaptive immune responses, ultimately establishing a protumorigenic niche. In this review, we highlight the advances, limitations, and challenges of studying the intratumoral microbiome and its cellular products in PDAC tumorigenesis, with a specific focus on their modulation of the immune axis. Finally, we evaluate existing and emerging therapeutic strategies, including microbiome-targeted drugs, antibiotics, probiotics, and engineered microbes, to disrupt protumorigenic microbial influences and improve clinical outcomes.

The pancreatic microbiome

Traditionally, the pancreas has been considered a sterile organ despite its proximity to the gastrointestinal (GI) tract, which harbors a diverse microbiome [1]. With the advent of high-throughput next-generation sequencing and improved DNA and RNA isolation protocols, substantial evidence has emerged indicating that healthy individuals, as well as those with pancreatic diseases, harbor a microbial population [2–7]. Notably, the bacterial DNA profile of the normal pancreas is comparable to that of duodenal tissue, suggesting possible bacterial migration from the gut to the healthy pancreas [8]. Despite this growing understanding, the exact nature and impact of the microbiome in a healthy pancreas, as well as the factors that drive the onset of microbiome-mediated pancreatic inflammation and carcinogenesis, remain poorly understood. Notably, recent research on the human microbiome has established that microbe-derived factors, such as acetaldehyde, nitrosamines, lipopolysaccharide, toxins, and other secondary metabolites, can induce an inflammatory immune response and initiate tumorigenesis [9,10]. While certain bacterial, viral, and fungal species are directly associated with pancreatic cancer, the available evidence is less robust and conclusive than that for other cancer types, such as gastric, colon, and lung carcinomas, primarily due to the low microbial biomass in the pancreas [11]. In this review, we summarize current knowledge on the role of the microbiome in pancreatic cancer and conclude with perspectives on the opportunities and challenges of translating current microbiome understanding into clinical applications.

Microbiome colonization in the pancreas

Bacterial colonization in pancreatic cancer

Genomic analysis of resected normal pancreas, pancreatitis, and PDAC samples identified several distinct bacterial genera, such as Acinetobacter, Afipia, Enterobacter, and Pseudomonas [12,13]. Further, Gammaproteobacteria and Bacilli were isolated from patients with intraductal papillary mucinous neoplasm (IPMN), and their pro-tumorigenic characteristics were validated through ex vivo co-culture experiments [14]. These findings were further supported by observations of bacterial colonization in pancreatic cystic fluid and by a pan-cancer analysis of intratumoral microbial composition, which identified Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter freundii, Enterobacter asburiae, and Fusobacterium nucleatum as abundant in PDAC [11,15]. By contrast, the role of various beneficial bacteria in regulating PDAC tumorigenesis is less explored. Studies have reported a relative reduction in the abundance of beneficial bacterial genera, such as Lactobacillus, in PDAC tumors compared to normal pancreatic tissue [8]. Bacterial colonization of the pancreas may enhance tumorigenesis by producing carcinogenic agents, inducing DNA damage and promoting mutagenesis, or by altering the TME’s inflammatory characteristics, thereby fostering conditions conducive to tumor progression [1,16]. Collectively, these studies demonstrate that bacteria and their components are present in PDAC tumors, highlighting their potential implications for disease progression.

Fungal colonization in pancreatic cancer

For decades, fungal infections have been studied as potential complications of preexisting diseases, which can exacerbate the severity of the underlying condition, particularly in immunocompromised patients [17]. Clinicians generally agree that mycobiome abundance in sterile organs, such as the pancreas, is a sign of infection [18]. To date, no studies have considered fungi as commensals or symbionts coexisting in sterile internal organs. However, substantial evidence indicates the presence of fungi in pancreatic inflammatory diseases, including pancreatitis and pancreatic cancer [3,4,7,19,20]. Pancreatitis [Box 1] is an inflammatory disease that can lead to pancreatic necrosis and is often managed with antibiotics and antifungal agents [21]. Several studies indicate Candida as the most common fungal species in patients with severe acute pancreatitis [18,22], and a few studies have reported the presence of other fungal species, such as Torulopsis glabrata and Saccharomyces fungemia. [23–26]. Notably, the fungal mycobiome has been reported in the normal pancreas without clinically diagnosed pancreatitis, as well as in the adjacent non-tumor pancreatic tissues of PDAC patients [3,4,7,27,28]. A seminal study reported a 3000-fold increase in mycobiome abundance in PDAC samples compared with normal pancreas [4].

BOX 1. Pancreatic diseases:

1. Acute Pancreatitis: Acute pancreatitis is an inflammation of the pancreas that can be caused by various factors, including gallstones, alcohol abuse, and infections.

2. Chronic Pancreatitis: A long-term inflammation of the pancreas that can cause parenchymal injury and fibrosis, potentially leading to permanent damage to its function and exocrine insufficiency.

3. Intraductal Papillary Mucinous Neoplasm (IPMN): It is a benign tumor that forms in the pancreatic ducts and produces excess mucus, which can lead to cyst formation. IPMN is considered a significant risk factor for pancreatic cancer.

4. Pancreatic Intraepithelial Neoplasia (PanIN): A precancerous condition characterized by abnormal cellular changes in the pancreatic ducts. It is classified into distinct grades based on the severity of these changes:

   1. PanIN-1: Considered low-grade lesions, with mild dysplasia. The cells exhibit slight architectural and cytological abnormalities.
   2. PanIN-2: Classified as intermediate-grade lesions, showing moderate dysplastic changes. There are more noticeable abnormalities in cell structure and organization.
   3. PanIN-3: High-grade lesions with significant dysplasia are often considered the most advanced precursor to invasive PDAC. Cells display substantial histological alterations.

5. Pancreatic Ductal Adenocarcinoma (PDAC): An aggressive type of pancreatic cancer, originating from the exocrine cells of the pancreatic ducts. Histologically, PDACs are characterized by a dense desmoplastic stroma interspersed with angulated glands and small nests of malignant epithelial cells.

Furthermore, a murine model of pancreatic intraepithelial neoplasia (PanIN) (KC [LSL-Kras^G12D;p48-Cre]) and PDAC patient samples confirmed colonization of the phyla Ascomycota and Basidiomycota in neoplastic PDAC tissue. [4]. The most prevalent genus in the KC mice pancreas was Malassezia, with approximately 20% relative abundance; this represents a marked increase compared with its abundance in the gut [4]. Our group previously reported that the fungal species Alternaria alternata and Malassezia globosa are the dominant populations driving PDAC progression in murine tumor models. [3]. Another pan-cancer study confirmed fungal mycobiome colonization by analyzing 17,401 patient samples from 35 cancer types, including PDAC, and found low levels of fungal DNA across tumor types. However, histological staining showed only 25% positivity, suggesting technical limitations and sensitivity issues in identifying fungal colonization [7]. These studies confirm the presence of a fungal mycobiome in the pancreas; however, its abundance and direct role in promoting pancreatic inflammation and tumor development need further analysis.

Viral colonization in Pancreatic cancer

Viruses, another component of the microbiome known as the virome, may significantly impact pancreatic tumorigenesis [29]. Detecting live viruses in the pancreas is rare due to the strong antiviral (alpha and beta defensins) defense within the pancreas [30]. Nevertheless, the presence of latent viruses and acute viral infections is increasingly recognized as a potential risk factor for PDAC [31]. Viruses such as Human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), and Epstein-Barr virus (EBV) are implicated in pancreatic tumors, suggesting a role in pancreatic tumorigenesis [32]. The Pan-Cancer Analysis of Whole Genomes Consortium (PCAWG) examined genome-wide sequencing data from 38 cancer types, including pancreatic cancer, and found Roseolovirus (human herpesvirus (HHV)-6A, HHV-6B, and HHV-7) and Alphatorquevirus in both cancerous and adjacent healthy tissues [33]. Moreover, other components of the viriome, such as HBV and HCV, which are more prevalent in hepatic cancer, may play a role in PDAC development and progression due to their proximity to the liver and their shared blood vessels and common duct with the pancreas and duodenum [34,35]. The presence of both HBV and HCV was confirmed by histopathological analysis of pancreatic sections and staining for hepatitis B surface antigen (HBsAg), a standard marker for both HBV infection and HCV antigen [34]. HBV and HCV are linked to increased risk of PDAC [36]. However, these studies remain controversial, and there is scope for larger cohort studies in pancreatic cancer patients to establish any conclusion.

Route of pancreatic microbiome colonization

Recent advancements have demonstrated that the microbiome, especially bacteria and fungi, can migrate to the inflamed pancreas [4,37]. However, the exact route of microbial migration from the gut to the pancreas remains unknown. The proximity of the pancreas to the GI tract provides sufficient rationale to support the migration of gut microbiota under pathophysiological conditions; however, it remains unclear how microbes enter the normal pancreas in healthy individuals. Most studies suggest that the retrograde transfer of gut content via the major papilla of Vater, where the pancreatic duct drains digestive enzymes from the pancreas into the duodenum. Another possible route is increased gut permeability, which can facilitate microbial migration; however, experimental evidence for this route is limited [38,39]. The first study to demonstrate bacterial migration to the pancreas assessed intravenous, colonic, pancreatic duct, and bile duct as potential migration routes in cats with or without acute pancreatitis (Figure 1) [40]. The study found that Escherichia coli can translocate to the pancreas via all of the aforementioned routes, and that pancreatitis significantly increases bacterial colonization compared with the healthy feline pancreas [40]. Several recent studies have also demonstrated that fluorescently labeled bacteria and fungi are translocated to the pancreas from the gut within 30 minutes of oral gavage [4,16]. However, neither study confirmed the exact route of migration; however, they suggested that the plausible route was through the sphincter of Oddi, the gatekeeper of the common bile duct that controls bile duct release and prevents intestinal fluid reflux [4,16,41]. However, there is conflicting evidence about gut microbiota migration to the pancreas. While some evidence suggests that mouse chow-derived bacterial species, such as Agrobacterium and Rhizobium, can colonize in the pancreas of both healthy and PDAC mice, another study shows no such effect after bacterial oral gavage in germ-free mice. [13,16]. These conflicting results suggest that pancreatic inflammation may be a necessary event for microbial translocation to the pancreas, thereby facilitating natural colonization. Additionally, pancreatic inflammation may lead to the malfunctioning of the sphincter of Oddi, resulting in reflux from the gut to the pancreas and, consequently, microbial colonization. All this evidence, along with recent advances in the field, indicates that pancreatic pathology creates a permissive environment for the transmural migration of microbiota into the pancreas.

Figure 1: Route of microbiome migration to the pancreas:

I. Pancreatic duct route: The microbiome migrates to the pancreas via the Sphincter of Oddi.

II. Common bile duct route: The microbiome migrates to the pancreas through the bile duct.

III. Intravenous route: The microbiome migrates to the pancreas by way of the connecting portal veins.

IV. Colonic route: The microbiome migrates to the pancreas through a transmural pathway.

Systemic effects initiated by the microbiome at distant sites that facilitate PDAC progression.

The human body serves as a reservoir for the microbiome, with (i) the skin, (ii) the gut, and (iii) the respiratory tract as the primary sites of colonization [42]. It is now well established that the microbiome can regulate various systemic processes, including metabolism and immune responses [3,4,16,19]. Additionally, microbiomes can migrate from the primary colonization site to distant sites in the body (Table 1) [2,4,5,16]. A key question is whether microbiomes at distant non-pancreatic sites contribute to pancreatic cancer through systemic alterations. In PDAC, systemic changes are mediated by microbial components, such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), as well as metabolites produced by the microbiome, including branched-chain amino acids (BCAA) and short-chain fatty acids (SCFA) [43–47]. During disease progression, these microbial components typically induce metabolic changes and systemic inflammation, thereby disrupting local and peripheral homeostasis [1,43,47,48]. Additionally, by activating inflammatory immune cells, they exacerbate pancreatic inflammation and contribute to an immunosuppressive TME that facilitates PDAC progression [19,47–49].

Table 1:

Microbiome colonization: Tumor vs other sites

Features	PDAC Intratumoral Microbiome	Gastrointestinal Tract (Duodenum/Gut)	Oral Cavity	Skin	Ref.
Microbial Biomass	Low, but significantly higher in the tumor (up to 3000-fold) than in the normal, healthy pancreas	Very High (colon has the highest colonization)	High biomass	High biomass	[2,4,16,55,58, 63,64]
Dominant Phyla	Bacteroides, Firmicutes, Actinobacteria, Basidiomycota, and Ascomycota	Firmicutes, Bacteroidetes, Proteobacteria, anaerobic archaea, Ascomycota, and Basidiomycota	Firmicutes, Bacteroidetes, Proteobacteria	Firmicutes, Actinobacteria, Ascomycota, and Basidiomycota	5,7,11,12,17,57,79]
Enriched species	Acinetobacter baumannir, Fusobacterium nucleatum, Klebsiella pneumoniae, Malassezia globosa, and Alternaria alternata.	Alloscardovia omnicolens, Veillonella atypica, Escherichia coli, Candida albicans, and Saccharomyces cerevisiae	Porphyromonas gingivalis, Eubacterium. nodatum, and Parvimonas micra	Cutibacterium acnes, Staphylococcus epidermidis, Streptococcus anginosus, Corynebacterium amycolatum, Candida albicans, and Malassezia globosa	[2–5,7,11,12,16,46,64]
Clinical Significance	Linked to: Chemoresistance (e.g., Gammaproteobacteria metabolizing Gemcitabine), Poor survival, and an Immunosuppressive tumor microenvironment	Linked to systemic inflammation and direct migration to the PDAC tumor site.	Linked to increased PDAC risk (e.g., P. gingivalis and F. nucleatum associated with periodontitis).	Linked to increased PDAC risk	[3–5,7,11,12,16,46,64]
Translocation Route	Pancreatic Duct, bile duct, pancreatic portal veins, and through transmural migration from the colon.	Translocate via the pancreatic duct or through transmural migration.	Translocate to the tumor via the blood vessels and through the gut.	Blood vessels and through the gut.	[4,16,40,63,64]

Association of skin microbiome in pancreatic inflammation and tumor progression

Skin is the largest organ of the human body and serves as the first line of defense against invading pathogens. Skin architecture, made up of multiple invaginations, sweat glands, and sebaceous glands, provides favorable conditions for microbial growth [50,51]. The skin microbiome comprises diverse microbial communities that support protective functions, helping maintain tissue homeostasis and overall health [50]. Several bacterial species, such as Cutibacterium acnes, Staphylococcus epidermidis, and Corynebacterium amycolatum, and fungal species such as Candida albicans and Malassezia globosa, have been identified as colonizing the skin [52–54]. Although the skin-resident microbiome primarily consists of commensal organisms, it frequently encounters pathogenic microbes that can disrupt homeostatic balance and induce pathological conditions [55,56]. Disruption of skin integrity can lead to the migration of microbial populations to distant sites via blood vessels or other migratory routes, such as the skin-gut axis, where they can contribute to pathophysiological conditions [57]. However, there is limited knowledge about the direct migration and colonization of the skin microbiota in relation to the progression of PDAC, although correlative studies suggest that the skin microbiome may play a role in disease progression. A clinical trial investigating the relationship between the skin microbiome and PDAC progression revealed increased abundance of bacterial species, including Streptococcus anginosus ChDC B695, Streptococcus mitis SK642, Snograssella alvi wkB12, and Streptococcus gordonii Challis CH1, in the skin of PDAC patients, suggesting a potential link with PDAC progression [58].

Furthermore, a preclinical study reported significant alterations in the levels of Lactobacillus, Staphylococcus, Anaerotruncus, Blautia, and Devosia in the skin of PDAC tumor-bearing mice, underscoring the importance of the skin microbiome in PDAC tumorigenesis [59]. In addition, infection with the human papillomavirus (HPV), which can cause warts on the skin, has been linked to increased risk of pancreatic cancer [60]. Moreover, HPV-16 was found within a mucinous neoplastic cyst of the pancreas, suggesting a possible association with pancreatic cancer development [61].

Furthermore, Malassezia globosa, a common skin commensal, is strongly associated with PDAC development. Several studies focusing on the role of the fungal mycobiome in PDAC progression identified Malassezia globosa and Malassezia restricta as the most abundant species in the PDAC tumors [3,4,7]. Moreover, transplantation of Malassezia globosa in preclinical mouse models significantly accelerated tumor progression, suggesting a potential link to the skin-PDAC axis [3,4].

Oral microbiome and association with pancreatic cancer.

Research indicates that individuals harboring the bacterial pathogens P. gingivalis and A. actinomycetemcomitans are at increased risk of pancreatic cancer. In contrast, the opposite has been reported for the phylum Fusobacteria and its genus, Leptotrichia [62]. However, the underlying mechanism of tumorigenesis remains unclear. The central question remains whether oral bacteria migrate to the pancreas and, if so, whether systemic changes induced by these bacteria promote tumorigenesis. Supporting this inquiry, an independent study has demonstrated that the oral bacterium P. gingivalis migrates to the pancreas and is associated with pancreatic tumorigenesis [63].

Furthermore, elevated serum antibodies against P. gingivalis are correlated with increased risk of pancreatic cancer. Moreover, a recent epidemiological study showed that three oral bacteria, P. gingivalis, E. nodatum, and P. micra, and the fungal genus Candida were linked to an increased risk of pancreatic cancer, establishing the relationship between the oral bacterial and fungal microbiome in the development of pancreatic cancer [64]. All these studies indicate that the oral microbiome can colonize in the pancreas and can increase the risk of pancreatic cancer.

Gut microbiome and its association with pancreatic cancer

The region beyond the oral cavity, comprising the upper and lower GI tract, harbors the most diverse microbial communities, which play a vital role in the etiology of various diseases, including pancreatic cancer [6]. Over the past decade, several studies have associated the gut microbiome with PDAC development [2–4,16,65]. Interestingly, the microbiome of pancreatic cancer patients is predominantly composed of Proteobacteria, Firmicutes, and Bacteroidetes, mirroring the composition of the normal gut microbiome [16]. This observation suggests retrograde bacterial migration from the duodenum, which drains into the pancreatic duct, as previously discussed. Additionally, similar studies in PDAC mouse models that use fungal microbiome transplantation via oral gavage detect fungi in tumor tissues, further confirming fungal migration from the gut to the pancreas [3,4,16].

Although there is a well-established pathway for the migration of the gut microbiome to the pancreas, the intrapancreatic microbiome differs between healthy and tumor tissues [3,4,6,8,16]. The differential colonization of the gut microbiome in normal versus PDAC tissue can shape the tissue microenvironment, thereby driving PDAC tumorigenesis. A recent study reported that Veillonella, a bacterial species, is highly abundant in PDAC samples and in oral and gut mucosa [6]. However, an Amplicon Sequence Variant (ASV) analysis revealed no intraindividual matches of Veillonella ASVs between tumor and healthy tissue samples [6], indicating intraspecies microbial variability and distinct subpopulations may emerge from different origins, such as oral and gut sources.

Microbiome and immune axis in pancreatic inflammation and PDAC progression

The interplay between the commensal microbiome and the host immune system is necessary for the development of both the innate and adaptive immune system [66,67]. The intratumor microbiome plays a pivotal role in pancreatic inflammation and shaping the immunosuppressive PDAC TME (Figure 2) [3,4,66,68]. For instance, interactions between pancreatic epithelial cells and the microbiome activate the innate immune signaling pathway, leading to the release of chemokines and cytokines. Cytokines and chemokines further activate both innate and adaptive immune cells, thereby eliciting a robust immune response [3,16,47]. In addition, the microbiome drives Th1, Th2, and Th17 immune responses either through direct interactions with their cellular components or by releasing secondary metabolites, thereby shaping the PDAC TME [16].

Figure 2: PDAC microbiome immune axis:

This illustration demonstrates how the intra-tumor microbiome in pancreatic ductal adenocarcinoma (PDAC), which includes bacteria, fungi, and viruses, contributes to tumor development through various signaling pathways that influence the tumor microenvironment (TME). The PDAC intratumor microbiome activates the C3a-MBL axis, leading to oncogenic signaling and promoting tumor cell proliferation. Additionally, the intratumor microbiome facilitates the release of the cytokine IL-33 from PDAC tumor cells. This IL-33 then binds to its specific receptor, ST2, found on ILC2 and Th2 immune cells, triggering their activation. Activated ILC2 and Th2 immune cells release IL-4 and IL-13, which facilitates the polarization of M0 macrophages into M2 macrophages. These M2 macrophages, in turn, secrete TGF-β, which further activates MDSCs. Both M2 macrophages and MDSCs contribute to creating an immunosuppressive tumor microenvironment, ultimately suppressing the CD8 T cell anti-tumor immune response and facilitating PDAC tumor progression.

Furthermore, accumulating evidence suggests that the gut microbiome correlates with patient outcomes and survival in pancreatic cancer. Notably, the gut microbiome’s diversity differentially modulates the immune profile of short-term survivors (STS) and long-term survivors (LTS) of PDAC patients. LTS patients exhibit greater microbiome diversity and higher frequencies of CD4+ and CD8+ T-cell infiltration in the PDAC TME than STS patients [2]. These observations highlight that microbiome diversity not only influences immune infiltration in the TME but also modulates immune cell activation, thereby influencing the overall survival of PDAC patients [2]. Moreover, the immune-microbe interaction and immune modulation by the microbiome were confirmed through the detection of bacterial components, such as LPS and LTA, within CD68⁺ macrophages [11]. In addition to bacteria, fungi, such as Malassezia, Alternaria, Candida, and Aspergillus, are associated with the immunosuppressive PDAC TME [3,4,7]. Malassezia globosa activates Mannose-Binding Lectin (MBL)-C3-C3aR signaling pathway and M2 macrophage-mediated immunosuppressive TME in PDAC (Figure 2)[3,4]. Similarly, Alternaria alternata triggers the release of the alarmin IL-33 from PDAC cells, which activates the immunosuppressive type 2 immune cells, including innate lymphoid cells (ILC2)/Th2, thereby driving PDAC progression [69]. These examples highlight the diversity of the PDAC TME, in which microbiomes become part of the TME, driving tumorigenesis through immune modulation.

Therapeutic strategy to target PDAC by modulating the intra-tumor microbiome

Ablation of the bacterial and fungal microbiomes, or repopulation with FMT or probiotics, significantly alters the PDAC TME (Table 2) [2–4,16,70,71]. This alteration is characterized by a reduction in myeloid-derived suppressor cells (MDSCs), an increase in M1 macrophage polarization, Th1 differentiation of CD4⁺ T cells, activation of CD8⁺ T cells, and a significant improvement in immune checkpoint inhibitor efficacy in preclinical studies [16,72,73].

Table 2:

Targeting microbiome for PDAC therapy:

	Microbiota Alteration Therapy	Clinical or pre-clinical Studies	Mechanism/significance of Treatment	Ref.
1	Ampicillin, Vancomycin, Neomycin, Metronidazole, Amphotericin, and αPD-1 mAb	Pre-clinical: Animals used - C57BL/6J, KPC (KrasG12D/+; Trp53R172H/+; Pdx-1cre) and KC (KrasG12D/+; Pdx-1cre) mice. Cell line used: KPC cells.	Bacterial antibiotics reprogram the immunogenic microenvironment of PDA tumor, i.e., decreased in MDSC, increased macrophage (M1) polarization, TH1 differentiation of CD4 + T cells, and T-cell activation of CD8 + cells. Bacterial depletion increases the efficacy of checkpoint-targeted immunotherapy through upregulation of PD-1 expression.	[16]
2	Streptomycin, Gentamicin, Bacitracin, and Ciprofloxacin	Pre-clinical: Animal used-NOD.CB17-Prkdcscid/J(‘Nod-SCID’) Cell line used-L3.6pl derived from COLO-357 and BxPC3 (human origin)	PDAC xenograft tumor progression was decreased upon gut microbial ablation. The presence of the gut microbiota is associated with immune suppression.	[13]
3	Vancomycin, Neomycin, Metronidazole, Ampicillin, and Amphotericin B	Pre-Clinical: Animal used-C57BL/6J, KPC mice Cell line used: KPC cells	Microbiome depletion in the gut significantly decreases tumor growth in pancreatic cancer by increasing the infiltration of effector T cells inside the pancreatic tumor. Decreases liver metastasis of pancreatic cancer cells.	[74]
4	Fecal Microbiota Transplantation (FMT) from long-term survival (LTS) or short-term survival (STS) PDAC patient or healthy control (HC) donor to mice	Clinical: Human fecal sample from LTS (n=21), STS (n=21), and HC Preclinical: Mouse model-antibiotics (ATBx) pretreated C57BL/6 mice Cell line- KPC pancreatic adenocarcinoma cell lines.	PDAC tumor growth in mice was decreased upon FMT from LTS compared to STS or HC. FTM-LTS increases recruitment of the CD8+ T-cell population and its activation. FTM-STS increases MDSC infiltration.	[2]
5	Metformin in diet induced obesity associated PDAC	Preclinical: Mice- KC mice	Metformin alters the microbiome of the duodenum and correlates with a decrease in PDAC incidence in KC mice	[80]
6	Amphotericin B with Gemcitabine or Fluconazole alone	Preclinical: Mouse mode- KC mice, C57BL/6, Cell line- FC1242 cell derived from KPC mice	Ablation of fungal mycobiome through Amphotericin B potentiates the efficacy of gemcitabine in PDAC. Single treatment of Fluconazole was able to restrict PDAC tumor progression	[4]
7	Amphotericin B	Preclinical: Mouse mode-Cell line-	Fungal depletion with amphotericin B decreases PDAC tumor progression through inhibition of IL-33-mediated Th2 and ILC2 infiltration, leading to increased survival.	[3]
8	Probiotics (Lactobacillus paracasei GMNL-133 and Lactobacillus reuteri GMNL-89) along with gemcitabine	Preclinical: Mice used-KC (LSL-KrasG12D/−-Pdx-1-Cre) transgenic mice	The combination treatment of probiotics and gemcitabine reduces pancreatic intraepithelial neoplasia (PanIN) formation and decreases the expression of Ki67 and vimentin.	[71]
9	Antibiotics (Atb): quinolones (n = 141), beta-lactams (n = 135), glycopeptides (n = 59), nitroimidazoles (n = 51), macrolides (n = 31), tetracyclines (n = 27), and sulfa drugs (n = 21)	Clinical: Metastatic PDAC patient (n=238)	Modulation of microbiome through antibiotics intake increases the overall survival (OS) (13.3 months vs. 9.0 months) and progression-free survival (PFS) (4.4 months vs. 2 months) in metastatic PDAC patients.	[73]
11	Probiotic Escherichia coli Nissle 1917 (EcN)	Preclinical: Mice used: KPC (KrasLSL.G12D/+; P53LSL.R172H/+; Pdx1-Cre), KC (KrasLSL.G12D/+; Pdx1-Cre), and PC (P53LSL.R172H/+; Pdx1-Cre) mice	Engineered probiotic administration selectively colonized tumor tissue, enhancing overall survival compared to standard chemotherapy in preclinical model	[75]

Enhancing immunotherapy efficacy

Although clinical studies targeting PDAC with immune checkpoint inhibitors (ICIs) have demonstrated limited success due to desmoplastic stroma, the combination of microbiome modulation with ICIs holds significant promise to enhance the effectiveness of these treatments. Notably, following bacterial depletion, the efficacy of checkpoint-targeted immunotherapy improves due to upregulation of PD-1 expression by both CD4⁺ and CD8⁺ T cells [16]. Furthermore, eliminating the gut microbiota has been shown to significantly inhibit PDAC progression by promoting effector T-cell infiltration into pancreatic tumors and reducing liver metastasis [13,74]. In addition, the gut microbiome is involved in the production of secondary metabolites and essential macro and micronutrients; some of these can trigger systemic inflammation and contribute to immune therapy response in cancer patients. One such gut microbe-derived metabolite, trimethylamine N-oxide (TMAO), enhances antitumor immunity in PDAC. Additionally, combining TMAO with immune ICI therapy (anti-PD-1 and/or anti-TIM-3) in a murine model of PDAC significantly reduced tumor burden and improved survival compared with TMAO or ICI alone [48]. Another promising approach in PDAC treatment involves targeting the microbiome by using beneficial or engineered probiotics to modulate the intratumoral microbiota, thereby enhancing the antitumor immune response [70,72,75]. Recent studies show that the use of engineered probiotics Lactobacillus rhamnosus GG, functionalized with a gallium-polyphenol network (LGG@Ga-poly), enhances PDAC immunotherapy by modulating microbiota-immune interactions [70]. Although the effectiveness of microbial modulation in PDAC patients is still in the early stages. In this regard, several clinical trials are underway to evaluate the efficacy of microbial ablation combined with antibiotics and ICI in chemotherapy-treated, surgically resectable pancreatic cancer [76–78]. Additionally, multiple ongoing clinical trials are studying the efficacy of microbiome ablation in combination with chemotherapy (Table 3).

Table 3:

Microbiome and PDAC Clinical trials:

	Study Title	Status	Participant and Location/Country of Study	Intervention (Mode of Diagnosis/Treatment/Drug Used)	ClinicalTrials.gov Identifier (NTC No)
1	Oral Microbiome and Pancreatic Cancer: A Prospective Case-Control Study	Completed (December 1, 1992-December 1, 2010)	Participants: 732 (Case: 361 & Control: 371) Location: New York, United States	16S rRNA gene sequencing assay	NCT03302637
2	The Microbiome of Pancreatic Cancer:”PANDEMIC” Study	Unknown (February 1, 2020-December 31, 2020)	Participants: 20 Location: Verona, Italy	Microbiome evaluation	NCT04274972
3	Pancreatic Cancer and Oral Microbiome	Recruiting (July 26, 2021-December 31, 2023)	Participants: 120 Location: Italy	Dental plaque sampling and qPCR	NCT04993846
4	Pilot Study of Gut Microbiome Modulation to Enable Efficacy of Neoadjuvant Checkpoint-based Immunotherapy Following Chemotherapy in Pancreatic Adenocarcinoma	Not yet recruiting (August 2022-April 2029)	Participants: 25 Location: New York, United States	Biopsy / Surgical Resection. Drugs: FOLFIRINOX, Ciprofloxacin, Metronidazole, Pembrolizumab	NCT05462496
5	Pancreatic Ductal Adenocarcinoma - Microbiome as Predictor of Subtypes	Recruiting (December 31, 2022-December 31, 2023)	Participants: 200 Location: Göttingen, Germany	Oral and rectal swabs for microbiome sequencing	NCT04922515
6	GutMicrobiota Impact for Prognosis and Treatment Outcomes in Early Luminal Breast Cancer and Pancreatic Cancer Patients	Recruiting (May 12, 2022August 2925)	Participants: 35 Location: Moscow, Russian Federation	mFOLFIRINOX, Doxorubicin, Cyclophosphamid, Paclitaxel, Carboplatin	NCT05580887
7	Correlation Between Complications After Pancreaticoduodenect omy and Biliary, Pancreatic, Blood, Oral, Intestinal and Fecal Microbiota	Recruiting (August 6, 2021-December 31, 2023)	Participants: 30 Location: Toulouse, France	Mircobiota analysis on blood, stool and saliva and bile, pancreatic and intestinal mucosa samples	NCT04931069

Reprogramming antitumor innate and adaptive immunity

In addition to immunotherapy, the microbiota-derived metabolite indole-3-acetic acid (3-IAA) potentiates the chemotherapeutic combination of 5-fluorouracil, irinotecan, and oxaliplatin by increasing CD8⁺ T cells and decreasing suppressive neutrophils in the FMT humanized mouse model [79]. In addition, the ablation of the fungal mycobiome using Amphotericin B enhanced the effectiveness of gemcitabine in treating PDAC [4]. Depleting fungi with Amphotericin B reduced tumor progression by inhibiting IL-33-mediated Th2 and ILC2 infiltration, resulting in increased survival in preclinical models [3]. In another study, Escherichia coli Nissle 1917 (EcN) probiotics, which produce a pore-forming theta toxin (Nis-theta), were locally injected to inhibit PDAC tumor progression. Bacterial presence in the tumors coincided with an antitumor immune response, characterized by the influx of CD8+ T cells, while simultaneously reducing immunosuppressive myeloid cells [75]. Additionally, a simple cocktail of Lactobacillus casei and Lactobacillus reuteri in a preclinical setting was effective in suppressing pancreatic cancer cell proliferation, migration, and invasion, and in inhibiting the polarization of immunosuppressive M2 macrophages [72].

Although there are many conceptual advances in microbiome research to improve the efficacy of the standard of care for pancreatic cancer patients, several caveats remain when implementing these concepts in clinical practice, due to microbial diversity in PDAC patients. Moreover, the use of broad-spectrum antibiotics and antifungals eliminates both beneficial and pathogenic microbial populations, thereby disrupting immune homeostasis, promoting antibiotic resistance, and worsening systemic inflammation. Therefore, there is a need to precisely target the pathogenic microbiome and repopulate the beneficial microbiome with probiotics to improve the clinical management of PDAC patients. Furthermore, the lack of a clinical microbiome biomarker signature and the durability of FMT transplantation create a significant clinical problem. Therefore, patient stratification must be considered in future clinical trials to achieve maximum therapeutic advancement.

Concluding remarks

In conclusion, pancreatic inflammation and tumorigenesis pose significant challenges due to their complex biology and limited effective therapeutic options. The intrapancreatic microbiome has emerged as a key contributor to pancreatic cancer. Recent research has highlighted the critical role of the microbiome, especially the migration of gut microbiota to the pancreas, which may impact pancreatic inflammation. This gut-to-pancreas migration can modify the local inflammatory immune response, creating an environment more conducive to PDAC tumorigenesis. Moreover, recent advances in the field have identified specific bacterial, viral, and fungal species associated with PDAC progression, providing a unique opportunity to target them. Beyond its association with risk factors, the microbiome holds therapeutic potential for the prognosis and treatment of pancreatic pathogenesis, but several questions remain to realize its full impact in the PDACmicrobiome field (see outstanding questions).

OUTSTANDING QUESTIONS.

• How can we overcome the challenges in detecting and assessing the presence and functionality of low-biomass live microbiomes in human PDAC patient samples?

• How can we eliminate potential contamination during sample collection and processing, which further clouds the validity of findings related to the microbiome?

• How can the route of migration of microbial populations from the oral cavity, gut, and skin to the pancreas during PDAC tumorigenesis in human subjects be mapped accurately?

• How do immune cells, such as macrophages, help in the migration of microbial populations from distant sites to the pancreas?

• How does the interkingdom interaction between bacterial, fungal, and viral species drive pancreatic inflammation and oncogenesis?

• How can the complexity of functional interactions between the microbiome, tumor cells, and the surrounding PDAC tumor microenvironment be delineated?

HIGHLIGHTS.

• The gut and intratumor microbiome are significant risk factors for pancreatic inflammation and tumorigenesis.

• The microbiome has the potential to translocate from the gastrointestinal tract to the pancreas in the context of dysbiosis.

• Microbial components or metabolites modulate the host immune response.

• Targeting a specific microbiome might significantly enhance patients’ response to anti-cancer therapy.