Research Progress in Intestinal Microecology in Pancreatic Cancer Diagnosis and Treatment

The intestinal microbiota has an increasingly recognized role in the development of cancer, in which microbial interactions play a more important than expected role. Pancreatic cancer is a highly fatal disease, in which its mortality is closely related to its morbidity. Early detection is the best chance of improving survival. Through an in-depth understanding of the pancreatic cancer microbiota, we could establish screening or early diagnosis methods for pancreatic cancer, implement bacterial treatment, adjust the therapeutic effect, and even reduce adverse reactions. These would lead to new developments and provide hope for patients with pancreatic cancer. Herein, we review the progress in intestinal microbiology research to diagnose and treat pancreatic cancer.


Introduction
Among the most common malignant tumors of the gastrointestinal tract, pancreatic cancer has an extremely high degree of malignancy and one of the worst prognoses among cancers. Te incidence of pancreatic cancer is increasing. Te only available treatment is surgical resection; however, only about 20% of patients are suitable for resection at the time of diagnosis, and the rate of surgical mortality is prohibitive. After surgical removal, the average survival is 10-20 months [1]. Pancreatic cancer, with its insidious and typical clinical symptoms, is a gastrointestinal malignancy that is difcult to diagnose and treat. Te most common form of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC) [2,3]. In recent years, pancreatic cancer's mortality and morbidity rates have increased signifcantly.
Te majority of patients with PDAC present with locally advanced (40%) or metastatic (40%) disease, and standard medical treatment consists primarily of palliative systemic therapy. Despite treatment options such as surgery, chemotherapy, and biotherapy, patients with PDAC at all stages combined have a 10% 5-year survival rate, while for those with distant metastases it is only 3% [4]. Recent studies have demonstrated that intestinal microecology could have an important role in pancreatic cancer development and that bacteria are associated with pancreatic disease pathogenesis, such as PDAC and autoimmune pancreatitis [5][6][7][8][9][10][11]. Tis review examines how intestinal microecology is used to diagnose and treat pancreatic cancer.

The Role of the Microbiome in the Development and Progression of Pancreatic Cancer
Te etiology of pancreatic cancer is unclear, and its specifc mechanism is still being studied. Oral bacterial taxa, including Fusobacterium and Granulicatella Adiacens, are present and enriched in the cystic fuid of intraductal papillary mucinous neoplasms (IPMN) with high atypia. Elevated bacterial DNA and interleukin-1β in pancreatic sacs have a synergistic efect on IPMN and tumor grade [12]. Te salivary microbiome of patients with pancreatic cancer was analyzed using the human oral microbial identifcation microarray (HOMIM). Te results showed that, with a sensitivity and specifcity of 96.4% and 82.1%, respectively, Neisseria elongata and Streptococcus medialis could distinguish patients with pancreatic cancer from healthy individuals [13]. Te study also found that exposure to Porphyromonas gingivalis, a pathogen associated with periodontal disease, might lead to an increased risk of pancreatic cancer because of the high levels of anti-Pseudomonas gingivalis antibodies found in patients with pancreatic cancer [13]. A sequence analysis of the 16SrRNA gene indicated that the presence of Pseudomonas gingivalis and its associated Actinobacillus in the oral cavity was associated with the occurrence of pancreatic cancer. Li et al. found that swallowing or through the circulatory system, oral bacteria could migrate and colonize remote areas ( Figure 1) [14]. A study examined the tongue coating microbiota of 25 healthy controls and patients with 30 PDAC and found that patients with PDAC patients had a signifcantly enriched microbiota of their tongue coating, which difered signifcantly from that of controls. Patients with PDAC could be diferentiated from the healthy controls according to their small number of certain bacteria (Haemophilus, Porphyromonas, leptothrix, and Clostridium) [15].
Geller et al. showed that there are bacteria in pancreatic cancer tissues, and these bacteria can promote the further deterioration of pancreatic cancer in diferent ways [16]. Zitvogel et al. showed that pancreatic cancer was associated with a specifc intestinal microecological composition and metabolite richness [17]. Cancer-related microorganisms found in the tumor environment of the pancreas might reach the pancreas through ventral intestinal metastasis of the pancreatic duct [18]. Tese results suggest that pancreatic cancer is related to duodenal microbiological changes, and some factors that regulate the intestinal microfora are closely associated with pancreatic cancer occurrence and development.
A Helicobacter pylori infection, along with oral pathogens, such as Porphyromonas gingivalis, Neisseria longchain, Streptococcus, etc., contribute to PDAC [19]. In addition, Aykut et al. showed that the fungal microbiota promotes pancreatic cancer [20]. Fungal migration from the intestinal lumen to the pancreas is associated with PDAC pathogenesis. Pancreatic cancer is promoted by the fungal activation of mannose-binding lectin (MBL). A pattern recognition receptor (PRR) from the innate immune system binds polysaccharides in fungal walls to activate the complement cascade [21,22]. In tumor cells, loss of MBL or complement C3 in the equatorial region and deletion of the complement C3a receptor (C3aR) can prevent tumor progression [20,23]. Sethi et al. showed that the growth of pancreatic tumors in mice is afected by the gut microbiota by regulating immune responses, and in a mouse pancreatic cancer model, depletion of the gut microbiota by oral antibiotics signifcantly reduced the tumor burden [24]. By contrast, in Rag1 (encoding recombination activating 1) knockout mice (which lack mature B and T cells), gut microbiome depletion could not prevent tumor development. Depletion of the intestinal microbiota resulted in a signifcant increase in T cells producing interferon gamma (IFNc) and a corresponding decrease in T cells producing interleukin (IL)-17A and IL-10 [24]. Te study also found that reducing the gut microbiota led to pancreatic tumors and efector T-cell infltration [24].
Pancreatic cystic neoplasms (PCN) are being detected at an increasing rate in the general population. Te latest fndings suggest that bacterial abundance might be increased in pancreatic PCN tissues compared with those in the normal pancreas [25]. A retrospective study using endoscopic ultrasound fne needle aspiration (EUS-FNA) revealed that regardless of the cyst type or clinical and biochemical parameters, the sac fuid of PCNs contained a unique microbiome [26].
Terefore, the diagnosis and treatment of pancreatic cancer have yet to be explored in association with the signifcant role played by the microbiome in the occurrence and development of pancreatic cancer.

Pancreatic Cancer Diagnosis via Intestinal Microbiological Monitoring
A study reported that in excess of 75% of cases of pancreatic cancer are at stage III or IV upon diagnosis and are therefore considered as advanced disease [27]. At present, PDAC has a poor prognosis, and radical surgery is still the only treatment option (surgery followed by chemotherapy) [28]. Early detection of PDAC can improve the quality of life and survival chances of patients. At present, pancreatic cancer diagnosis is based mainly on symptoms, signs, imaging manifestations, tumor markers, histopathology, and/or cytology. However, all this evidence was confrmed after the onset of pancreatic cancer, and positive results are found in the middle and late stages. Early diagnosis of pancreatic cancer is still very difcult with no population-level screening tools or biomarkers [29]. Mendez et al. found that for early pancreatic cancer detection, the altered microbiota could be used as a predictive marker [30]. In the early stages of PDASC, large number of Proteus were found. Moreover, the polyamines and nucleotide biosynthetic pathways have been studied to assess their functional importance in tumor progression. Terefore, changes to the microbiota and the release of metabolites that promote host tumorigenesis are closely linked to early PDAC. However, efective initial diagnosis continues to be difcult, and more specialized biomarkers are needed [31]. A study utilizing HOMIM found that patients with pancreatic cancer could be distinguished from healthy people by the presence of Neisseria longate and Streptococcus mitis, with a sensitivity and specifcity of 96.4% and 82.1%, respectively [13]. Te presence of two biomarkers in oral saliva, Neisseria longate and Streptococcus mitis, can be used as diagnostic tools for the early stage of pancreatic cancer. Torres et al. showed that patients with pancreatic cancer have an increased percentage of leptotrichia and a reduced amount of porphyromonas gingivalis in their saliva in comparison with healthy subjects, and that ratio of the two (LP ratio) can serve as a biomarker of pancreatic cancer [32]. One study evaluated the intestinal fora and its metabolites in KPC mice (a pancreatic ductal adenocarcinoma (PDA) model) and patients with PDAC for the early detection of PDAC [30]. Te study found that early in the development of PDAC, the colonic fora was enriched with proteobacteria and frmicutes. Tis was associated with elevated levels of serum polyamines, a product of active metabolic pathways. Tus, the gut microbiota is a potential noninvasive tool for the early detection of PDAC [30,33]. Studies on the relationship between deregulation of the fungal microbiota and the progress of PDAC are limited. To develop a prognostic tool for PDAC, further studies should also consider fungal profles. With the rapid advance of medical science, prompt analysis of the intestinal microfora might form the basis for the prevention and early diagnosis of pancreatic cancer.  (Figure 2(a)).

Role of Intestinal Microecology in the Treatment of Pancreatic Cancer
Corra et al. found that sodium butyrate can promote the diferentiation of human pancreatic cancer cells and induce the expression of certain tumor-associated antigens [34]. Researchers also found that sodium butyrate can inhibit pancreatic cancer cell invasion and metastasis by inhibiting integrity β4 [35]. Additionally, butyrate might inhibit the efciency of anticytotoxic T lymphocyte associated antigen-4 (CTLA-4) immune checkpoint inhibitors (ICIs) by inhibiting the stimulation of tumor-specifc memory T cells and T cells by dendritic cells [36]. One study found that valproic acid signifcantly downregulates pancreatic cancer cell expression of epidermal growth factor receptor (EGFR), ErbB2 receptor tyrosine kinase 2 (ErbB2), and ErbB2 receptor tyrosine kinase 3 (ErbB3) by inducing microRNAs targeting members of the ErbB family. Furthermore, valproic acid's antipancreatic cancer activity was confrmed in a transplanted tumor model [37]. Terefore, valproic acid has selective antitumor activity against pancreatic cancer coexpressing EGFR/ErbB2/ErbB3. Luu et al. showed that the human symbiotic bacterium Mobilicoccus massiliensis is the only bacterium that synthesizes large amounts of the SCFAs, valerate, and butyrate. Te valerate and butyrate produced by M. massiliensis enhanced CD8 + T cell production of efector cytokines [38]. In the presence of valerate, tumor-specifc T cells were better able to fght solid tumor models. Luu   demonstrating the potential of optimizing CAR T-cell generation by valerate and butyric acid to enhance their efcacy after adoptive transfer [38].
Tese results suggest that SCFAs inhibit the development, invasion, and occurrence of pancreatic cancer signifcantly. Tus, regulating the levels of SCFAs through intervention with the colonic fora could positively afect pancreatic cancer prevention and treatment.  a cancer suppressor produced by Aspergillus coryza, is synthesized in the intestine and transported to other organs, including the pancreas, thereby inhibiting pancreatic cancer growth [39]. Te study also showed that hepatic acid enhances Cyclin B1 via the P38 mitogen activated kinase (MAPK) signaling pathway, a key glycolytic enzyme signaling pathway in pancreatic cancer cells. In the G2 phase, the cyclin B1-cyclin dependent kinase 1 (CDK1) complex is formed (the decisive step for progression of the cell cycle into the M phase), which irreversibly inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH), thus inhibiting GAPDH-mediated glycolysis to produce ATP. Terefore, regulating the activity of Cyclin B1-CDK1 can accelerate the cell cycle, thus inducing pancreatic cancer cell apoptosis and playing an inhibitory role in cancer (Figure 2(b)). In addition, Kita et al. showed that probiotic-derived hyperfne pigments inhibit cancer cell progression to the G2-M phase by activating p53 via phosphorylation, which upregulates p53-mediated mRNA transcription and downregulates the amount of secretase inhibitor protein (Securin) and cyclin B1 [40]. At the same time, endoplasmic reticulum stress is upregulated and the JUN Nterminalkinase-DNA damage inducible transcript 3 (JNK-DDIT3) pathway is activated, which promotes the apoptosis of cancer cells and inhibits cancer (Figure 2(c)). Meanwhile, Chen et al. showed that Lactobacillus can inhibit pancreatic cancer growth by inhibiting the transforming growth factor beta (TGF-β) signaling pathway mediated by Porphyromonas gingivalis [41].

Immunotherapy.
Te intestinal fora promotes immunotherapy against pancreatic cancer by regulating immune checkpoints. As an immunosuppressive molecule an immune checkpoint can inhibit lymphocyte function and allow tumor cells to escape the immune system. Te intestinal fora has an important function in the formation of the human immune system and the induction of immune responses. Studies have demonstrated that CTLA-4, as an immune checkpoint inhibitor, is dependent on intestinal fora in the treatment of tumors [42,43]. When the colonic fora is absent, it cannot produce an efective antitumor efect. By regulating dendritic cell function, intestinal bacteria can regulate the antitumor immune response mediated by T cells. Accordingly, it is benefcial for the prevention and treatment of pancreatic cancer to select an immunotherapy based on altered colonic microbiological conditions.
Studies have demonstrated that a variety of bacteria, including Akkermansia, Fusarium, Clostridium, and nitrobacterium are linked to the antitumor efect of targeted therapy using programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PDL1, also known as the CD274 molecule) [44][45][46]. Inoculating germ-free mice with selected interferon gamma-induced microbial strains enhanced the efcacy of anti-PD-1 ICIs, and antitumor T-cell responses were signifcantly promoted [47]. Tese efects are caused by the infuence of microbial metabolites such as butyric acid and propionic acid. In some cases, however, the infuences of probiotic compounds show conficting results. For example, higher SCFA levels in feces were associated with longer progression-free survival or a stronger antitumor response, while higher systemic levels were linked to a poorer treatment response [48].
Other microbial metabolites also afect ICI. For instance, inosine produced by nitrobacterium enhances ICIs by activating A2A receptors on T cells [49]. Te direct stimulation of lymph node dendritic cells by Akkermansia muciniphila can induce microbial-host interactions in cancer immunotherapy to improve the antitumor efects of ICIs, dependent on IL-12 [46] or the induction of antitumor immune responses by T1 and CD8 + T cells [42,46].
Tus, it is advantageous to select immunotherapies depending on the intestinal microbiology conditions to prevent and treat pancreatic cancer.

Fecal Microbiome Transplantation Terapies.
Fecal microbiome transplantation (FMT) is used to replace a disease-associated microbiota with a healthy confguration. In cancer, the transfer of patient stool samples to ICI-treated sterile or antibiotic-treatedtumor-bearing mice demonstrated that a specifc microbiome confguration could drive improved immunotherapy efcacy [50]. Two other studies have shown that FMT administered to germ-freetumorbearing mice from patients who responded well to ICIs could transfer this ICI reactivity to the recipient mice, while mice receiving an "unresponsive" microbiome did not respond to ICIs [51,52]. A study demonstrated for the frst time that the FMT of a recombinant microbiome in tumorbearing mice from feces of healthy control patients (HC), short-term survival patients (STS), or long-term survival patients without evidence of disease (LTS-NED) refected the recruitment or lack of immune cells to the tumor environment in each group, which afected tumor growth [53]. Te gut microbiota is causally involved in shaping the immune response to tumors and promoting PDAC progression. Te success of these trials were partly determined by the choice of donors and how well the donor material was absorbed by the recipient. Despite these preliminary results, there are several quantifable, regulatory, and scientifc uncertainties that should be addressed before FMT can be adopted routinely, e.g., issues associated with the selection of operative donors and recipients, the preparation of the intestines, and reception procedures. Furthermore, the drivers of the clinical efects of FMT, such as microbial metabolites, phages, or bacteria, are mostly unknown and should be tested in large-scale, prospective clinical trials.

Current Problems and Future Prospects
Increasing evidence that microbes are linked to the progression and treatment response of pancreatic cancer warrants more comprehensive research to reveal the mechanism by which the microbiota exerts these efects. Microbial targeting strategies could provide new hope for the treatment of pancreatic cancer. Te products of the intestinal microbiota, such as SCFAs and certain probiotics, can Journal of Oncology

Pentanoic acid
Heptanoic acid Regulating P38 MAPK signaling pathway (Figure 2 (Table 1); however, this kind of research is in its infancy, and most research is still in the preclinical phase. At present, there is no progress in the relevant research on how to distinguish the abnormal bacterial community associated with pancreatic cancer from the normal symbiotic bacterial community by using antibiotics. Currently, the research progress in this feld is temporarily confned to the mouse pancreatic cancer model experiment, and the development of pancreatic cancer can be observed by using broad-spectrum antibiotics to eliminate various intestinal fora. No experiments have been conducted to eliminate one or more types of intestinal fora alone. Patients with pancreatic cancer are generally in poor physical condition, and most patients will undergo chemotherapy, which is associated with its own burden for patients [54,55]. For example, chemotherapy can cause digestive reactions, including diarrhea and vomiting, together with damage to the kidneys and liver. Te most frequent reaction is a digestive reaction, suggesting that chemotherapy might afect the intestinal fora. In mice with pancreatic cancer transplanted tumors, cimetidine treatment altered the colonic microbial composition [56]. Te results revealed a signifcant reduction in Gram-positive bacteria (39% to 17%) and Gram-negative bacteria (38% to 17%) in the intestines of mice with tumors compared with those in the control group. Te proportion of Proteobacteria (Escherichia coli and Aeromonas hydrophila) and Akermania muciniphila increased signifcantly. Control mice with tumors had predominantly Gram-positive and Gram-negative bacteria in the intestinal tract, in which Proteus and vermiform bacteria occupied subordinate positions [57][58][59]. Corty et al. [60] showed that the use of antibiotics in cancer treatment increased the risk of adverse events, such as hematological and gastrointestinal events. Moreover, Vétizou et al. [42] found that in mouse models of sarcoma, melanoma, and colon cancer, CTLA-4 therapy was rendered inefective when ampicillin, colistin, and streptomycin were administered together. It was also reported that cyclophosphamide (CTX) treatment did not activate antitumor immunity in mice treated with vancomycin (targeting Gram-positive bacteria), leading to treatment failure [61]. However, Geller et al. showed that in models of colorectal cancer, cimetidine and ciprofoxacin could efectively eliminate bacteria-induced chemotherapy resistance, thereby boosting its efectiveness [16]. Meanwhile, Chen et al. showed that the probiotic Lactobacillus and gemcitabine synergistically inhibited tumor growth in a transgenic mouse model of pancreatic cancer [62]. Another study [63] observed that adding antibiotics to chemotherapy could improve its efcacy. An analysis of 169 patients with advanced cancer (including pancreatic cancer) treated with cimetidine was conducted retrospectively, dividing the patients into two groups: a no antibiotics group (treated with solutions containing gramicidin but not antibiotics) and an antibiotic treatment group (using the solution containing cimetidine plus antibiotic treatment). Both groups were assessed for efcacy, overall survival (OS), and progressionfree survival (PFS). Te results showed that the median PFS and OS metrics were higher in the antibiotic treatment group than in the no antibiotics group. Tese fndings suggested that regulating intestinal microecology or using probiotics in combination with chemotherapy could help to treat pancreatic cancer. Above all, gut microbes can become a pancreatic cancer diagnosis standard, and by adjusting the intestinal microecology, could become a new paradigm for disease treatment. Intestinal microbial products (e.g., SCFAs), combined with immune therapy and FMT could be used to treat pancreatic cancer, giving new hope to patients. Chemotherapy can afect the intestinal microecology, and the use of probiotics and antibiotics with combination chemotherapy in the treatment of pancreatic cancer has excellent potential [64]. However, these therapies might have as-yet-unknown side efects and adverse reactions, which will be a problem requiring further research. More studies are also required to optimize combination therapies to bring better results and improve the prognosis of patients with pancreatic cancer.

Conclusions
Pancreatic cancer is one of the deadliest malignant tumors. It sufers from poor early diagnosis and a lack of efective treatments. Intestinal microbiology has a vital function in multiple physiological activities. Tere is a close relationship between pancreatic cancer and the intestinal microecology. With the development of technology and science, the correlation between them will be revealed in detail. Although many studies have focused on microbes and how they contribute to pancreatic cancer occurrence and development, only a few studies have discussed how microorganisms infuence pancreatic cancer treatment. In this review, we provide fresh insights into the interactions between the intestinal microbiota and pancreatic cancer and summarize some meaningful perspectives and recommendations for developing innovative treatment approaches and models. Te intestinal fora are a signifcant and complex system, and their regulatory mechanism in pancreatic cancer forms a rich network chain. To date, there has been progress in the early diagnosis of pancreatic cancer; however, we lack additional and efective biomarkers. Intestinal microbiological regulation is a novel concept in disease treatment and ofers new hope for patients sufering from pancreatic cancer via products of intestinal fora (e.g., SCFAs), combined immunotherapy, and fecal bacteria transplantation. Moreover, the majority of the studies discussed were conducted using mouse models. Tus, the results should be interpreted with caution because there might be diferent efects in human applications. Limitations in this feld, such as the impact of sample sizes in human clinical studies, the homogeneity of disease stages when considering case studies, and whether there is a clinical correlation between the microbiome and the ethnicity of patients and control populations, must also be addressed. Terefore, more epidemiological studies, basic experiments, and clinical trials are needed to explore these aspects and strive for rapid application in clinical treatment. Pancreatic cancer can be treated by regulating intestinal microecology and future treatments and drug development will increasingly rely on microorganisms.

Data Availability
Te data supporting this systematic review are from previously reported studies and datasets, which have been cited. Te processed data are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.

Authors' Contributions
Q.W. and K.C. conceived the manuscript. Z.H. and H.Z. wrote the manuscript. L.L., C.Z., J.Z., and C.L. revised the manuscript. Q.W. and K.C. supervised and revised the paper. All authors have read and agreed to the published version of the manuscript. Zetao Han and Haiyan Zhang contributed equally to this study.