Gynecological Cancers and Microbiota Dynamics: Insights into Pathogenesis and Therapy.

In recent years, the relationship between the microbiota and various aspects of health has become a focal point of scientific investigation. Although the most studied microbiota concern the gastrointestinal tract, recently, the interest has also been extended to other body districts. Female genital tract dysbiosis and its possible impact on pathologies such as endometriosis, polycystic ovary syndrome (PCOS), pelvic inflammatory disease (PID), and gynecological cancers have been unveiled. The incursion of pathogenic microbes alters the ecological equilibrium of the vagina, triggering inflammation and compromising immune defense, potentially fostering an environment conducive to cancer development. The most common types of gynecological cancer include cervical, endometrial, and ovarian cancer, which occur in women of any age but especially in postmenopausal women. Several studies highlighted that a low presence of lactobacilli at the vaginal level, and consequently, in related areas (such as the endometrium and ovary), correlates with a higher risk of gynecological pathology and likely contributes to increased incidence and worse prognosis of gynecological cancers. The complex interplay between microbial communities and the development, progression, and treatment of gynecologic malignancies is a burgeoning field not yet fully understood. The intricate crosstalk between the gut microbiota and systemic inflammation introduces a new dimension to our understanding of gynecologic cancers. The objective of this review is to focus attention on the association between vaginal microbiota and gynecological malignancies and provide detailed knowledge for future diagnostic and therapeutic strategies.

Insights into the role of gut and intratumor microbiota in pancreatic ductal adenocarcinoma as new key players in preventive, diagnostic and therapeutic perspective.

Microbiota consists of a dynamic organization of bacteria, viruses, archaea, and fungal species involved in a number of vital functions spanning from the digestion of carbohydrates, vitamin synthesis, involvement in immune system to drug metabolism. More than 95 % of microbiota resides within the gut and it is essential for maintaining gut homeostasis. Dysregulation of gut microbiota contributes to the onset of several non-communicable diseases including cancer. Among the latter, pancreatic cancer is catching the attention of scientists around the globe being one of the most aggressive and resistant to therapies positioning the pancreatic cancer as one of the leading causes of death from cancer worldwide. In recent years, several studies have shown that the gut and tumor microbiota play a key role in the development, progression and prognosis of PDAC, mainly due to microbial ability to modulate host immune system and metabolize drugs. This review will focus on the new insights into the role of the microbiota as a new key player in pancreatic cancer PDAC development and prognosis by enlightening the microbial potential to interact with chemo/immunotherapeutic drugs and to modulate tumor microenvironment, thus impacting on cancer therapy success with the aim to pave the way to new integrative and interventional diagnostics or therapeutics approaches to prevent, diagnose and treat pancreatic cancer.

Modulation of the Tumor Microenvironment by Microbiota-Derived Short-Chain Fatty Acids: Impact in Colorectal Cancer Therapy.

Finding new therapeutic approaches towards colorectal cancer (CRC) is of increased relevance, as CRC is one of the most common cancers worldwide. CRC standard therapy includes surgery, chemotherapy, and radiotherapy, which may be used alone or in combination. The reported side effects and acquired resistance associated with these strategies lead to an increasing need to search for new therapies with better efficacy and less toxicity. Several studies have demonstrated the antitumorigenic properties of microbiota-derived short-chain fatty acids (SCFAs). The tumor microenvironment is composed by non-cellular components, microbiota, and a great diversity of cells, such as immune cells. The influence of SCFAs on the different constituents of the tumor microenvironment is an important issue that should be taken into consideration, and to the best of our knowledge there is a lack of reviews on this subject. The tumor microenvironment is not only closely related to the growth and development of CRC but also affects the treatment and prognosis of the patients. Immunotherapy has emerged as a new hope, but, in CRC, it was found that only a small percentage of patients benefit from this treatment being closely dependent on the genetic background of the tumors. The aim of this review was to perform an up-to-date critical literature review on current knowledge regarding the effects of microbiota-derived SCFAs in the tumor microenvironment, particularly in the context of CRC and its impact in CRC therapeutic strategies. SCFAs, namely acetate, butyrate, and propionate, have the ability to modulate the tumor microenvironment in distinct ways. SCFAs promote immune cell differentiation, downregulate the expression of pro-inflammatory mediators, and restrict the tumor-induced angiogenesis. SCFAs also sustain the integrity of basement membranes and modulate the intestinal pH. CRC patients have lower concentrations of SCFAs than healthy individuals. Increasing the production of SCFAs through the manipulation of the gut microbiota could constitute an important therapeutic strategy towards CRC due to their antitumorigenic effect and ability of modulating tumor microenvironment.

Targeting Gut Microbiota in Cancer Cachexia: Towards New Treatment Options.

Cancer cachexia is a complex multifactorial syndrome whose hallmarks are weight loss due to the wasting of muscle tissue with or without the loss of adipose tissue, anorexia, systemic inflammation, and multi-organ metabolic alterations, which negatively impact patients' response to anticancer treatments, quality of life, and overall survival. Despite its clinical relevance, cancer cachexia often remains an underestimated complication due to the lack of rigorous diagnostic and therapeutic pathways. A number of studies have shown alterations in gut microbiota diversity and composition in association with cancer cachexia markers and symptoms, thus supporting a central role for dysbiosis in the pathogenesis of this syndrome. Different tools of microbiota manipulation, including probiotics, prebiotics, synbiotics, and fecal microbiota transplantation, have been investigated, demonstrating encouraging improvements in cachexia outcomes. Albeit pioneering, these studies pave the way for future research with the aim of exploring the role of gut microbiota in cancer cachexia more deeply and setting up effective microbiota-targeting interventions to be translated into clinical practice.

A Pilot Interaction Analysis of Gut Microbiota and Peripheral Markers of Aging in Severe Psychiatric Disorders: A Role for Lachnoclostridium?

Excessive predominance of pathological species in the gut microbiota could increase the production of inflammatory mediators at the gut level and, via modification of the gut-blood barrier, at the systemic level. This pro-inflammatory state could, in turn, increase biological aging that is generally proxied by telomere shortening. In this study, we present findings from a secondary interaction analysis of gut microbiota, aging, and inflammatory marker data from a cohort of patients with different diagnoses of severe mental disorders. We analyzed 15 controls, 35 patients with schizophrenia (SCZ), and 31 patients with major depressive disorder (MDD) recruited among those attending a community mental health center (50 males and 31 females, mean and median age 46.8 and 46.3 years, respectively). We performed 16S rRNA sequencing as well as measurement of telomere length via quantitative fluorescence in situ hybridization and high-sensitivity C-reactive protein. We applied statistical modeling with logistic regression to test for interaction between gut microbiota and these markers. Our results showed statistically significant interactions between telomere length and gut microbiota pointing to the genus Lachnostridium, which remained significantly associated with a reduced likelihood of MDD even after adjustment for a series of covariates. Although exploratory, these findings show that specific gut microbiota signatures overexpressing Lachnoclostridium and interacting with biological aging could modulate the liability for MDD.

Immunotherapy for Biliary Tract Cancer in the Era of Precision Medicine: Current Knowledge and Future Perspectives.

Biliary tract cancers (BTC) represent a heterogeneous and aggressive group of tumors with dismal prognosis. For a long time, BTC has been considered an orphan disease with very limited therapeutic options. In recent years a better understanding of the complex molecular landscape of biology is rapidly changing the therapeutic armamentarium. However, while 40-50% of patients there are molecular drivers susceptible to target therapy, for the remaining population new therapeutic options represent an unsatisfied clinical need. The role of immunotherapy in the continuum of treatment of patients with BTC is still debated. Despite initial signs of antitumor-activity, single-agent immune checkpoint inhibitors (ICIs) demonstrated limited efficacy in an unselected population. Therefore, identifying the best partner to combine ICIs and predictive biomarkers represents a key challenge to optimize the efficacy of immunotherapy. This review provides a critical analysis of completed trials, with an eye on future perspectives and possible biomarkers of response.

Body site-dependent variations of microbiota in pancreatic cancer pathophysiology.

Lack of specific symptoms and reliable biomarkers, along with aggressive nature and resistance to therapies makes pancreatic cancer (PC) one of the leading causes of death from cancer worldwide. The search for new diagnostic, prognostic, predictive, and therapeutic tools that could improve clinical outcomes of patients has led, in recent years, to the investigation of potential roles for the microbiota in the pathogenesis of this disease. The human microbiota encompasses trillions of microorganisms residing within several body tissues and organs, where they provide beneficial functions for host homeostasis and health. Derangements of the microbial ecology in different anatomic districts have been described in PC, as in many other diseases, both in patients and in animal models. In detail, infection from the gastric pathogen Helicobacter pylori and changes in composition and diversity of oral, intestinal, and pancreatic microbiota have been found to associate with PC. Future research should assess how to potentially exploit such differences in microbiota composition as diagnostic, prognostic, or predictive biomarkers, and as targets for therapeutic interventions, in the hope of improving the dismal prognosis of this insidious cancer.

Impact of Different Types of Diet on Gut Microbiota Profiles and Cancer Prevention and Treatment.

: Diet is frequently considered as a food regimen focused on weight loss, while it is actually the sum of food consumed by the organism. Western diets, modern lifestyle, sedentary behaviors, smoking habits, and drug consumption have led to a significant reduction of gut microbial diversity, which is linked to many non-communicable diseases (NCDs). The latter kill 40 million people each year, equivalent to more than 70% of all deaths globally. Among NCDs, tumors play a major role, being responsible for 29% of deaths from NCDs. A link between diet, microbiota, and cancer prevention and treatment has recently been unveiled, underlining the importance of a new food culture based on limiting dietary surplus and on preferring healthier foods. Here, we review the effects of some of the most popular "cancer-specific" diets on microbiota composition and their potential impact on cancer prevention and treatment.

Fasting inhibits hepatic stellate cells activation and potentiates anti-cancer activity of Sorafenib in hepatocellular cancer cells.

Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short-term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi-tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short-lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown. A 24 hr fasting (2% serum, 0.1% glucose)-induced changes on human hepatic stellate cells (HSC) LX-2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)-induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. A 24 hr fasting-induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation, and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib. Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells. Fasting or fasting-mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use.

Exploring the microbiota to better understand gastrointestinal cancers physiology.

Gastrointestinal cancers account for around 40% of cancer-related deaths worldwide, representing a global health burden. There is a growing body of evidence highlighting the link between microbiota and gastrointestinal tumorigenesis and/or resistance to therapy. In the present manuscript, we reviewed the published studies on the relationship between the microbiota and the different gastrointestinal tumors, namely, gastric, colorectal and esophageal, including also the cancer of accessory organs such as liver and pancreas. There is an emergent interest in the manipulation of gastrointestinal microflora in order to understand the gastrointestinal tumorigenesis' processes and the establishment of chemoresistance mechanisms.

Analysis of Gut Microbiota in Rheumatoid Arthritis Patients: Disease-Related Dysbiosis and Modifications Induced by Etanercept.

A certain number of studies were carried out to address the question of how dysbiosis could affect the onset and development of rheumatoid arthritis (RA), but little is known about the reciprocal influence between microbiota composition and immunosuppressive drugs, and how this interaction may have an impact on the clinical outcome. The aim of this study was to characterize the intestinal microbiota in a groups of RA patients treatment-naïve, under methotrexate, and/or etanercept (ETN). Correlations between the gut microbiota composition and validated immunological and clinical parameters of disease activity were also evaluated. In the current study, a 16S analysis was employed to explore the gut microbiota of 42 patients affected by RA and 10 healthy controls. Disease activity score on 28 joints (DAS-28), erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, anti-cyclic citrullinated peptides, and dietary and smoking habits were assessed. The composition of the gut microbiota in RA patients free of therapy is characterized by several abnormalities compared to healthy controls. Gut dysbiosis in RA patients is associated with different serological and clinical parameters; in particular, the phylum of Euryarchaeota was directly correlated to DAS and emerged as an independent risk factor. Patients under treatment with ETN present a partial restoration of a beneficial microbiota. The results of our study confirm that gut dysbiosis is a hallmark of the disease, and shows, for the first time, that the anti-tumor necrosis factor alpha (TNF-α) ETN is able to modify microbial communities, at least partially restoring a beneficial microbiota.

Clock genes-dependent acetylation of complex I sets rhythmic activity of mitochondrial OxPhos.

Physiology of living beings show circadian rhythms entrained by a central timekeeper present in the hypothalamic suprachiasmatic nuclei. Nevertheless, virtually all peripheral tissues hold autonomous molecular oscillators constituted essentially by circuits of gene expression that are organized in negative and positive feed-back loops. Accumulating evidence reveals that cell metabolism is rhythmically controlled by cell-intrinsic molecular clocks and the specific pathways involved are being elucidated. Here, we show that in vitro-synchronized cultured cells exhibit BMAL1-dependent oscillation in mitochondrial respiratory activity, which occurs irrespective of the cell type tested, the protocol of synchronization used and the carbon source in the medium. We demonstrate that the rhythmic respiratory activity is associated to oscillation in cellular NAD content and clock-genes-dependent expression of NAMPT and Sirtuins 1/3 and is traceable back to the reversible acetylation of a single subunit of the mitochondrial respiratory chain Complex I. Our findings provide evidence for a new interlocked transcriptional-enzymatic feedback loop controlling the molecular interplay between cellular bioenergetics and the molecular clockwork.

Cancer sniffer dogs: how can we translate this peculiarity in laboratory medicine? Results of a pilot study on gastrointestinal cancers.

BACKGROUND: Identification of cancer biomarkers to allow early diagnosis is an urgent need for many types of tumors, whose prognosis strongly depends on the stage of the disease. Canine olfactory testing for detecting cancer is an emerging field of investigation. As an alternative, here we propose to use GC-Olfactometry (GC/O), which enables the speeding up of targeted biomarker identification and analysis. A pilot study was conducted in order to determine odor-active compounds in urine that discriminate patients with gastrointestinal cancers from control samples (healthy people). METHODS: Headspace solid phase microextraction (HS-SPME)-GC/MS and GC-olfactometry (GC/O) analysis were performed on urine samples obtained from gastrointestinal cancer patients and healthy controls. RESULTS: In total, 91 key odor-active compounds were found in the urine samples. Although no odor-active biomarkers present were found in cancer carrier's urine, significant differences were discovered in the odor activities of 11 compounds in the urine of healthy and diseased people. Seven of above mentioned compounds were identified: thiophene, 2-methoxythiophene, dimethyl disulphide, 3-methyl-2-pentanone, 4-(or 5-)methyl-3-hexanone, 4-ethyl guaiacol and phenylacetic acid. The other four compounds remained unknown. CONCLUSIONS: GC/O has a big potential to identify compounds not detectable using untargeted GC/MS approach. This paves the way for further research aimed at improving and validating the performance of this technique so that the identified cancer-associated compounds may be introduced as biomarkers in clinical practice to support early cancer diagnosis.

Deregulated expression of cryptochrome genes in human colorectal cancer.

BACKGROUND: Circadian disruption and deranged molecular clockworks are involved in carcinogenesis. The cryptochrome genes (CRY1 and CRY2) encode circadian proteins important for the functioning of biological oscillators. Their expression in human colorectal cancer (CRC) and in colon cancer cell lines has not been evaluated so far. METHODS: We investigated CRY1 and CRY2 expression in fifty CRCs and in the CaCo2, HCT116, HT29, SW480 cell lines. RESULTS: CRY1 (p = 0.01) and CRY2 (p < 0.0001) expression was significantly changed in tumour tissue, as confirmed in a large independent CRC dataset. In addition, lower CRY1 mRNA levels were observed in patients in the age range of 62-74 years (p = 0.018), in female patients (p = 0.003) and in cancers located at the transverse colon (p = 0.008). Lower CRY2 levels were also associated with cancer location at the transverse colon (p = 0.007). CRC patients displaying CRY1 (p = 0.042) and CRY2 (p = 0.043) expression levels over the median were hallmarked by a poorer survival rate. Survey of selected colon cancer cell lines evidenced variable levels of cryptochrome genes expression and time-dependent changes in their mRNA levels. Moreover, they showed reduced apoptosis, increased proliferation and different response to 5-fluorouracil and oxaliplatin upon CRY1 and CRY2 ectopic expression. The relationship with p53 status came out as an additional layer of regulation: higher CRY1 and CRY2 protein levels coincided with a wild type p53 as in HCT116 cells and this condition only marginally affected the apoptotic and cell proliferation characteristics of the cells upon CRY ectopic expression. Conversely, lower CRY and CRY2 levels as in HT29 and SW480 cells coincided with a mutated p53 and a more robust apoptosis and proliferation upon CRY transfection. Besides, an heterogeneous pattern of ARNTL, WEE and c-MYC expression hallmarked the chosen colon cancer cell lines and likely influenced their phenotypic changes. CONCLUSION: Cryptochrome gene expression is altered in CRC, particularly in elderly subjects, female patients and cancers located at the transverse colon, affecting overall survival. Altered CRY1 and CRY2 expression patterns and the interplay with the genetic landscape in colon cancer cells may underlie phenotypic divergence that could influence disease behavior as well as CRC patients survival and response to chemotherapy.

Interplay between SOX9, ß-catenin and PPARγ activation in colorectal cancer.

Colorectal carcinogenesis relies on loss of homeostasic mechanisms regulating cell proliferation, differentiation and survival. These cell processes have been reported to be influenced independently by transcription factors activated downstream of the Wnt pathway, such as SOX9 and ß-catenin, and by the nuclear receptor PPARγ. The purpose of this study was to explore the expression levels and functional link between SOX9, ß-catenin and PPARγ in the pathogenesis of colorectal cancer (CRC). We evaluated SOX9, ß-catenin and PPARγ expression levels on human CRC specimens by qPCR and immunoblot detection. We tested the hypothesis that PPARγ activation might affect SOX9 and ß-catenin expression using four colon cancer cell lines (CaCo2, SW480, HCT116, and HT29 cells). In CRC tissues SOX9 resulted up-regulated at both mRNA and protein levels when compared to matched normal mucosa, ß-catenin resulted up-regulated at protein levels, while PPARG mRNA and PPARγ protein levels were down-regulated. A significant relationship was observed between high PPARG and SOX9 expression levels in the tumor tissue and female gender (p=0.005 and p=0.04, respectively), and between high SOX9 expression in the tumor tissue and age (p=0.04) and microsatellite instability (MSI), in particular with MSI-H (p=0.0002). Moreover, treatment with the synthetic PPARγ ligand rosiglitazone induced different changes of SOX9 and ß-catenin expression and subcellular localization in the colon cancer cell lines examined. In conclusion, SOX9, ß-catenin and PPARγ expression levels are deregulated in the CRC tissue, and in colon cancer cell lines ligand-dependent PPARγ activation unevenly influences SOX9 and ß-catenin expression and subcellular localization, suggesting a variable mechanistic role in colon carcinogenesis.

The TRPA1 channel is a cardiac target of mIGF-1/SIRT1 signaling.

Cardiac overexpression of locally acting muscle-restricted (m)IGF-1 and the consequent downstream activation of NAD(+)-dependent protein deacetylase sirtuin 1 (SIRT1) trigger potent cardiac antioxidative and antihypertrophic effects. Transient receptor potential (TRP) cation channel A1 (TRPA1) belongs to the TRP ion channel family of molecular detectors of thermal and chemical stimuli that activate sensory neurons to produce pain. Recently, it has been shown that TRPA1 activity influences blood pressure, but the significance of TRPA1 in the cardiovascular system remains elusive. In the present work, using genomic screening in mouse hearts, we found that TRPA1 is a target of mIGF-1/SIRT1 signaling. TRPA1 expression is increased in the heart of cardiac-restricted mIGF-1 transgenic (Tg) mice, both in cardiomyocytes and noncardiomyocytes. In wild-type mice, SIRT1 occupied the TRPA1 promoter, inhibiting its expression, whereas in the presence of the cardiac mIGF-1 transgene, SIRT1 was displaced from the TRPA1 promoter, leading to an increase in its expression. Cardiac-specific ablation of SIRT1 (cardiac-specific knockout) in mIGF-1 Tg mice paradoxically did not increase TRPA1 expression. We have recently reported a systemic "hormetic" effect in mIGF-1 Tg mice, mild hypertension, which was depleted upon cardiac-specific knockout of SIRT1. Administration of the selective TRPA1 antagonist HC-030031 to mIGF-1 Tg mice restored blood pressure to basal levels. We identified TRPA1 as a functional target of the cardiac mIGF-1/SIRT1 signaling pathway, which may have pharmacological implications for the management of cardiovascular stress.

Modeling interactions between Human Equilibrative Nucleoside Transporter-1 and other factors involved in the response to gemcitabine treatment to predict clinical outcomes in pancreatic ductal adenocarcinoma patients.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy, characterized by largely unsatisfactory responses to the currently available therapeutic strategies. In this study we evaluated the expression of genes involved in gemcitabine uptake in a selected cohort of patients with PDAC, with well-defined clinical-pathological features. METHODS: mRNA levels of hENT1, CHOP, MRP1 and DCK were evaluated by means of qRT-PCR in matched pairs of tumor and adjacent normal tissue samples collected from PDAC patients treated with gemcitabine after surgical tumor resection. To detect possible interaction between gene expression levels and to identify subgroups of patients at different mortality/progression risk, the RECursive Partitioning and Amalgamation (RECPAM) method was used. RESULTS: RECPAM analysis showed that DCK and CHOP were most relevant variables for the identification of patients with different mortality risk, while hENT1 and CHOP were able to identify subgroups of patients with different disease progression risk. CONCLUSION: hENT1, CHOP, MRP1 and DCK appear correlated to PDAC, and this interaction might influence disease behavior.

Histone variants and lipid metabolism.

Within nucleosomes, canonical histones package the genome, but they can be opportunely replaced with histone variants. The incorporation of histone variants into the nucleosome is a chief cellular strategy to regulate transcription and cellular metabolism. In pathological terms, cellular steatosis is an abnormal accumulation of lipids, which reflects impairment in the turnover of triacylglycerols, affecting any organ but mainly the liver. The present review aims to summarize the experimental evidence for histone variant functions in lipid metabolism.

Epithelial-mesenchymal transition: molecular pathways of hepatitis viruses-induced hepatocellular carcinoma progression.

Hepatocellular carcinoma is the fifth most common tumor and the third cause of death for cancer in the world. Among the main causative agents of this tumor is the chronic infection by hepatitis viruses B and C, which establish a context of chronic inflammation degenerating in fibrosis, cirrhosis, and, finally, cancer. Recent findings, however, indicate that hepatitis viruses are not only responsible for cancer onset but also for its progression towards metastasis. Indeed, they are able to promote epithelial-mesenchymal transition, a process of cellular reprogramming underlying tumor spread. In this manuscript, we review the currently known molecular mechanisms by which hepatitis viruses induce epithelial-mesenchymal transition and, thus, hepatocellular carcinoma progression.

A powerful machine learning approach to identify interactions of differentially abundant gut microbial subsets in patients with metastatic and non-metastatic pancreatic cancer.

Pancreatic cancer has a dismal prognosis, as it is often diagnosed at stage IV of the disease and is characterized by metastatic spread. Gut microbiota and its metabolites have been suggested to influence the metastatic spread by modulating the host immune system or by promoting angiogenesis. To date, the gut microbial profiles of metastatic and non-metastatic patients need to be explored. Taking advantage of the 16S metagenomic sequencing and the PEnalized LOgistic Regression Analysis (PELORA) we identified clusters of bacteria with differential abundances between metastatic and non-metastatic patients. An overall increase in Gram-negative bacteria in metastatic patients compared to non-metastatic ones was identified using this method. Furthermore, to gain more insight into how gut microbes can predict metastases, a machine learning approach (iterative Random Forest) was performed. Iterative Random Forest analysis revealed which microorganisms were characterized by a different level of relative abundance between metastatic and non-metastatic patients and established a functional relationship between the relative abundance and the probability of having metastases. At the species level, the following bacteria were found to have the highest discriminatory power: Anaerostipes hadrus, Coprobacter secundus, Clostridium sp. 619, Roseburia inulinivorans, Porphyromonas and Odoribacter at the genus level, and Rhodospirillaceae, Clostridiaceae and Peptococcaceae at the family level. Finally, these data were intertwined with those from a metabolomics analysis on fecal samples of patients with or without metastasis to better understand the role of gut microbiota in the metastatic process. Artificial intelligence has been applied in different areas of the medical field. Translating its application in the field of gut microbiota analysis may help fully exploit the potential information contained in such a large amount of data aiming to open up new supportive areas of intervention in the management of cancer.