Inflammation promotes stomach epithelial defense by stimulating the secretion of antimicrobial peptides in the mucus.

The mucus serves as a protective barrier in the gastrointestinal tract against microbial attacks. While its role extends beyond merely being a physical barrier, the extent of its active bactericidal properties remains unclear, and the mechanisms regulating these properties are not yet understood. We propose that inflammation induces epithelial cells to secrete antimicrobial peptides, transforming mucus into an active bactericidal agent. To investigate the properties of mucus, we previously developed mucosoid culture models that mimic the healthy human stomach epithelium. Similar to organoids, mucosoids are stem cell-driven cultures; however, the cells are cultivated on transwells at air-liquid interface. The epithelial cells of mucosoids form a polarized monolayer, allowing differentiation into all stomach lineages, including mucus-secreting cells. This setup facilitates the secretion and accumulation of mucus on the apical side of the mucosoids, enabling analysis of its bactericidal effects and protein composition, including antimicrobial peptides. Our findings show that TNFα, IL1ß, and IFNγ induce the secretion of antimicrobials such as lactotransferrin, lipocalin2, complement component 3, and CXCL9 into the mucus. This antimicrobial-enriched mucus can partially eliminate Helicobacter pylori, a key stomach pathogen. The bactericidal activity depends on the concentration of each antimicrobial and their gene expression is higher in patients with inflammation and H.pylori-associated chronic gastritis. However, we also find that H. pylori infection can reduce the expression of antimicrobial encoding genes promoted by inflammation. These findings suggest that controlling antimicrobial secretion in the mucus is a critical component of epithelial immunity. However, pathogens like H. pylori can overcome these defenses and survive in the mucosa.

Correction: Pessolano et al. Annexin A1 May Induce Pancreatic Cancer Progression as a Key Player of Extracellular Vesicles Effects as Evidenced in the In Vitro MIA PaCa-2 Model System. Int. J. Mol. Sci. 2018, 19, 3878.

In the original publication [...].

H3K56 acetylation affects Candida albicans morphology and secreted soluble factors interacting with the host.

In recent years, epigenetics has been revealed as a mechanism able to modulate the expression of virulence traits in diverse pathogens, including Candida albicans. Indeed, epigenetic regulation can sense environmental changes, leading to the rapid and reversible modulation of gene expression with consequent adaptation to novel environments. How epigenetic changes can impact expression and signalling output, including events associated with mechanisms of morphological transition and virulence, is still poorly studied. Here, using nicotinamide as a sirtuin inhibitor, we explored how the accumulation of the H3K56 acetylation, the most prominent histone acetylation in C. albicans, might affect its interaction with the host. Our experiments demonstrate that H3K56 acetylation profoundly affects the production and/or secretion of soluble factors compromising actin remodelling and cytokine production. ChIP- and RNA-seq analyses highlighted a direct impact of H3K56 acetylation on genes related to phenotypic switching, biofilm formation and cell aggregation. Direct and indirect regulation also involves genes related to cell wall protein biosynthesis, ß-glucan and mannan exposure, and hydrolytic secreted enzymes, supporting the hypothesis that the fluctuations of H3K56 acetylation in C. albicans might impair the macrophage response to the yeast and thus promote the host-immune escaping.

High levels of soluble CD73 unveil resistance to BRAF inhibitors in melanoma cells.

Melanoma cells express high levels of CD73 that produce extracellular immunosuppressive adenosine. Changes in the CD73 expression occur in response to tumor environmental factors, contributing to tumor phenotype plasticity and therapeutic resistance. Previously, we have observed that CD73 expression can be up-regulated on the surface of melanoma cells in response to nutritional stress. Here, we explore the mechanism by which melanoma cells release soluble CD73 under low nutrient availability and whether this might be affected by agents targeting the proto-oncogene B-Raf (BRAF). We found that starved melanoma cells can release high levels of CD73, able to convert AMP into adenosine, and this activity is abrogated by selective CD73 inhibitors, APCP or PSB-12489. The release of CD73 from melanoma cells is mediated by the matrix metalloproteinase MMP-9. Indeed, MMP-9 inhibitors significantly reduce the levels of CD73 released from the cells, while its surface levels increase. Of relevance, melanoma cells, harboring an activating BRAF mutation, upon treatment with dabrafenib or vemurafenib, show a strong reduction of CD73 cell expression and reduced levels of CD73 released into the extracellular space. Conversely, melanoma cells resistant to dabrafenib show high expression of membrane-bound CD73 and soluble CD73 released into the culture medium. In summary, our data indicate that CD73 is released from melanoma cells. The expression of CD73 is associated with response to BRAF inhibitors. Melanoma cells developing resistance to dabrafenib show increased expression of CD73, including soluble CD73 released from cells, suggesting that CD73 is involved in acquiring resistance to treatment.

Transcriptome analysis of macrophages during Brucella abortus infection clarifies the survival mechanisms of the bacteria.

Brucellosis is a critical zoonotic disease impacting humans and animals globally, causing symptoms like fever and arthritis in humans and reproductive issues in animals. The disease stems from the Brucella genus, adept at evading the immune system and proliferating within host cells. This study explores how Brucella abortus manipulates host cellular mechanisms to sustain infection, focusing on the interaction with murine macrophages over 24 h. Initial host defenses involve innate immune responses, while Brucella's survival strategies include evading lysosomal degradation and modulating host cell functions through various pathways. The research identified significant transcriptional changes in macrophages post-infection, highlighting pathways such as cytokine storm, pyroptosis signaling, Toll-like receptor pathways, and LXRs/RXRs signaling. The findings shed light on Brucella's complex mechanisms to undermine host defenses and underscore the need for further investigation into therapeutic targets to combat brucellosis.

Chemoproteomics Reveals USP5 (Ubiquitin Carboxyl-Terminal Hydrolase 5) as Promising Target of the Marine Polyketide Gracilioether A.

Mass spectrometry-based chemical proteomic approaches using limited proteolysis have become a powerful tool for the identification and analysis of the interactions between a small molecule (SM) and its protein target(s). Gracilioether A (GeA) is a polyketide isolated from a marine sponge, for which we aimed to trace the interactome using this strategy. DARTS (Drug Affinity Responsive Target Stability) and t-LiP-MS (targeted-Limited Proteolysis-Mass Spectrometry) represented the main techniques used in this study. DARTS was applied on HeLa cell lysate for the identification of the GeA target proteins, and t-LiP-MS was employed to investigate the protein's regions involved in the binding with GeA. The results were complemented through the use of binding studies using Surface Plasmon Resonance (SPR) and in silico molecular docking experiments. Ubiquitin carboxyl-terminal hydrolase 5 (USP5) was identified as a promising target of GeA, and the interaction profile of the USP5-GeA complex was explained. USP5 is an enzyme involved in the pathway of protein metabolism through the disassembly of the polyubiquitin chains on degraded proteins into ubiquitin monomers. This activity is connected to different cellular functions concerning the maintenance of chromatin structure and receptors and the degradation of abnormal proteins and cancerogenic progression. On this basis, this structural information opens the way to following studies focused on the definition of the biological potential of Gracilioether A and the rational development of novel USP5 inhibitors based on a new structural skeleton.

Targeting phosphoglycerate kinases by tatridin A, a natural sesquiterpenoid endowed with anti-cancer activity, using a proteomic platform.

Tatridin A (TatA) is a germacrane sesquiterpenoid containing one E-double bond and one Z-double bond in its 10-membered ring, which is fused to a 3-methylene-dihydrofuran-2-one moiety. Tatridin A bioactivity has been poorly investigated despite its interesting chemical structure. Here, a functional proteomic platform was adapted to disclose its most reliable targets in leukemia monocytic cells, and phosphoglycerate kinases were recognized as the most affine enzymes. Through a combination of limited proteolysis and molecular docking, it has been discovered that tatridin A interacts with the active domains of phosphoglycerate kinase 1, altering its hinge region, and it can be accountable for tatridin A inhibition potency on enzyme activity. A more detailed tatridin A biological profile showed that it is also fully active against gastric cancer cells, downregulating the mRNA levels of chemokine receptor 4 and ß-catenin and inhibiting the invasiveness of living KATO III cells as a direct consequence of phosphoglycerate kinase 1 antagonism.

SARS-CoV-2 RNA in Wastewater and Bivalve Mollusk Samples of Campania, Southern Italy.

SARS-CoV-2 can be detected in the feces of infected people, consequently in wastewater, and in bivalve mollusks, that are able to accumulate viruses due to their ability to filter large amounts of water. This study aimed to monitor SARS-CoV-2 RNA presence in 168 raw wastewater samples collected from six wastewater treatment plants (WWTPs) and 57 mollusk samples obtained from eight harvesting sites in Campania, Italy. The monitoring period spanned from October 2021 to April 2022, and the results were compared and correlated with the epidemiological situation. In sewage, the ORF1b region of SARS-CoV-2 was detected using RT-qPCR, while in mollusks, three targets-RdRp, ORF1b, and E-were identified via RT-dPCR. Results showed a 92.3% rate of positive wastewater samples with increased genomic copies (g.c.)/(day*inhabitant) in December-January and March-April 2022. In the entire observation period, 54.4% of mollusks tested positive for at least one SARS-CoV-2 target, and the rate of positive samples showed a trend similar to that of the wastewater samples. The lower SARS-CoV-2 positivity rate in bivalve mollusks compared to sewages is a direct consequence of the seawater dilution effect. Our data confirm that both sample types can be used as sentinels to detect SARS-CoV-2 in the environment and suggest their potential use in obtaining complementary information on SARS-CoV-2.

Design of an Herbal Preparation Composed by a Combination of Ruscus aculeatus L. and Vitis vinifera L. Extracts, Magnolol and Diosmetin to Address Chronic Venous Diseases through an Anti-Inflammatory Effect and AP-1 Modulation.

Chronic venous disease (CVD) is an often underestimated inflammatory pathological condition that can have a serious impact on quality of life. Many therapies have been proposed to deal with CVD, but unfortunately the symptoms recur with increasing frequency and intensity as soon as treatments are stopped. Previous studies have shown that the common inflammatory transcription factor AP-1 (activator protein-1) and nuclear factor kappa-activated B-cell light chain enhancer (NF-kB) play key roles in the initiation and progression of this vascular dysfunction. The aim of this research was to develop a herbal product that acts simultaneously on different aspects of CVD-related inflammation. Based on the evidence that several natural components of plant origin are used to treat venous insufficiency and that magnolol has been suggested as a putative modulator of AP-1, two herbal preparations based on Ruscus aculeatus root extracts, and Vitis vinifera seed extracts, as well as diosmetin and magnolol, were established. A preliminary MTT-based evaluation of the possible cytotoxic effects of these preparations led to the selection of one of them, named DMRV-2, for further investigation. First, the anti-inflammatory efficacy of DMRV-2 was demonstrated by monitoring its ability to reduce cytokine secretion from endothelial cells subjected to LPS-induced inflammation. Furthermore, using a real-time PCR-based protocol, the effect of DMRV-2 on AP-1 expression and activity was also evaluated; the results obtained demonstrated that the incubation of the endothelial cells with this preparation almost completely nullified the effects exerted by the treatment with LPS on AP-1. Similar results were also obtained for NF-kB, whose activation was evaluated by monitoring its distribution between the cytosol and the nucleus of endothelial cells after the different treatments.

Effects of Hst3p inhibition in Candida albicans: a genome-wide H3K56 acetylation analysis.

Candida spp. represent the third most frequent worldwide cause of infection in Intensive Care Units with a mortality rate of almost 40%. The classes of antifungals currently available include azoles, polyenes, echinocandins, pyrimidine derivatives, and allylamines. However, the therapeutical options for the treatment of candidiasis are drastically reduced by the increasing antifungal resistance. The growing need for a more targeted antifungal therapy is limited by the concern of finding molecules that specifically recognize the microbial cell without damaging the host. Epigenetic writers and erasers have emerged as promising targets in different contexts, including the treatment of fungal infections. In C. albicans, Hst3p, a sirtuin that deacetylates H3K56ac, represents an attractive antifungal target as it is essential for the fungus viability and virulence. Although the relevance of such epigenetic regulator is documented for the development of new antifungal therapies, the molecular mechanism behind Hst3p-mediated epigenetic regulation remains unrevealed. Here, we provide the first genome-wide profiling of H3K56ac in C. albicans resulting in H3K56ac enriched regions associated with Candida sp. pathogenicity. Upon Hst3p inhibition, 447 regions gain H3K56ac. Importantly, these genomic areas contain genes encoding for adhesin proteins, degradative enzymes, and white-opaque switching. Moreover, our RNA-seq analysis revealed 1330 upregulated and 1081 downregulated transcripts upon Hst3p inhibition, and among them, we identified 87 genes whose transcriptional increase well correlates with the enrichment of H3K56 acetylation on their promoters, including some well-known regulators of phenotypic switching and virulence. Based on our evidence, Hst3p is an appealing target for the development of new potential antifungal drugs.

Drug affinity-responsive target stability unveils filamins as biological targets for artemetin, an anti-cancer flavonoid.

Artemetin is a valuable 5-hydroxy-3,6,7,3',4'-pentamethoxyflavone present in many different medicinal plants with very good oral bioavailability and drug-likeness values, owing to numerous bioactivities, such as anti-inflammatory and anti-cancer ones. Here, a multi-disciplinary plan has been settled and applied for identifying the artemetin target(s) to inspect its mechanism of action, based on drug affinity-responsive target stability and targeted limited proteolysis. Both approaches point to the disclosure of filamins A and B as direct artemetin targets in HeLa cell lysates, also giving detailed insights into the ligand/protein-binding sites. Interestingly, also 8-prenyl-artemetin, which is an artemetin more permeable semisynthetic analog, directly interacts with filamins A and B. Both compounds alter filamin conformation in living HeLa cells with an effect on cytoskeleton disassembly and on the disorganization of the F-actin filaments. Both the natural compound and its derivative are able to block cell migration, expectantly acting on tumor metastasis occurrence and development.

Comparative Evaluation of Antimicrobial, Antiamoebic, and Antiviral Efficacy of Ophthalmic Formulations.

The extensive use of ophthalmic antibiotics is contributing to the appearance of resistant bacterial strains, which require prolonged and massive treatments with consequent detrimental outcomes and adverse effects. In addition to these issues, antibiotics are not effective against parasites and viruses. In this context, antiseptics could be valuable alternatives. They have nonselective mechanisms of action preventing bacterial resistance and a broad spectrum of action and are also effective against parasites and viruses. Here, we compare the in vitro antibacterial, antiameobic, and antiviral activities of six ophthalmic formulations containing antiseptics such as povidone-iodine, chlorhexidine, and thymol against Gram-positive and Gram-negative bacteria, the amoeba Acanthamoeba castellanii, and two respiratory viruses, HAdV-2 and HCoV-OC43. The results suggest that, among all the tested formulations, Dropsept, consisting of Vitamin E TPGS-based (tocopheryl polyethylene glycol succinate) in combination with the antiseptic chlorhexidine, is the one with the highest range of activities, as it works efficiently against bacteria, amoeba, and viruses. On the other hand, the solution containing PVA (polyvinyl alcohol) and thymol showed a promising inhibitory effect on Pseudomonas aeruginosa, which causes severe keratitis. Given its high efficiency, Dropsept might represent a valuable alternative to the widely used antibiotics for the treatment of ocular infections. In addition to this commercial eye drop solution, thymol-based solutions might be enrolled for their natural antimicrobial and antiamoebic effect.

Helicobacter pylori Pathogen-Associated Molecular Patterns: Friends or Foes?

Microbial infections are sensed by the host immune system by recognizing signature molecules called Pathogen-Associated Molecular Patterns-PAMPs. The binding of these biomolecules to innate immune receptors, called Pattern Recognition Receptors (PRRs), alerts the host cell, activating microbicidal and pro-inflammatory responses. The outcome of the inflammatory cascade depends on the subtle balance between the bacterial burn and the host immune response. The role of PRRs is to promote the clearance of the pathogen and to limit the infection by bumping inflammatory response. However, many bacteria, including Helicobacter pylori, evolved to escape PRRs' recognition through different camouflages in their molecular pattern. This review examines all the different types of H. pylori PAMPs, their roles during the infection, and the mechanisms they evolved to escape the host recognition.

Rapid and sensitive detection of SARS-CoV-2 variants in nasopharyngeal swabs and wastewaters.

The SARS-CoV-2 virus is continuously evolving, with appearance of new variants characterized by multiple genomic mutations, some of which can affect functional properties, including infectivity, interactions with host immunity, and disease severity. The rapid spread of new SARS-CoV-2 variants has highlighted the urgency to trace the virus evolution, to help limit its diffusion, and to assess effectiveness of containment strategies. We propose here a PCR-based rapid, sensitive and low-cost allelic discrimination assay panel for the identification of SARS-CoV-2 genotypes, useful for detection in different sample types, such as nasopharyngeal swabs and wastewater. The tests carried out demonstrate that this in-house assay, whose results were confirmed by SARS-CoV-2 whole-genome sequencing, can detect variations in up to 10 viral genome positions at once and is specific and highly sensitive for identification of all tested SARS-CoV-2 clades, even in the case of samples very diluted and of poor quality, particularly difficult to analyze.

A Novel Three-Polysaccharide Blend In Situ Gelling Powder for Wound Healing Applications.

In this paper, alginate/pectin and alginate/pectin/chitosan blend particles, in the form of an in situ forming hydrogel, intended for wound repair applications, have been successfully developed. Particles have been used to encapsulate doxycycline in order to control the delivery of the drug, enhance its antimicrobial properties, and the ability to inhibit host matrix metalloproteinases. The presence of chitosan in the particles strongly influenced their size, morphology, and fluid uptake properties, as well as drug encapsulation efficiency and release, due to both chemical interactions between the polymers in the blend and interactions with the drug demonstrated by FTIR studies. In vitro antimicrobial studies highlighted an increase in antibacterial activity related to the chitosan amount in the powders. Moreover, in situ gelling powders are able to induce a higher release of IL-8 from the human keratinocytes that could stimulate the wound healing process in difficult-healing. Interestingly, doxycycline-loaded particles are able to increase drug activity against MMPs, with good activity against MMP-9 even at 0.5 µg/mL over 72 h. Such results suggest that such powders rich in chitosan could be a promising dressing for exudating wounds.

ANXA1 Contained in EVs Regulates Macrophage Polarization in Tumor Microenvironment and Promotes Pancreatic Cancer Progression and Metastasis.

The tumor microenvironment (TME) is a dynamic system where nontumor and cancer cells intercommunicate through soluble factors and extracellular vesicles (EVs). The TME in pancreatic cancer (PC) is critical for its aggressiveness and the annexin A1 (ANXA1) has been identified as one of the oncogenic elements. Previously, we demonstrated that the autocrine/paracrine activities of extracellular ANXA1 depend on its presence in EVs. Here, we show that the complex ANXA1/EVs modulates the macrophage polarization further contributing to cancer progression. The EVs isolated from wild type (WT) and ANXA1 knock-out MIA PaCa-2 cells have been administrated to THP-1 macrophages finding that ANXA1 is crucial for the acquisition of a protumor M2 phenotype. The M2 macrophages activate endothelial cells and fibroblasts to induce angiogenesis and matrix degradation, respectively. We have also found a significantly increased presence of M2 macrophage in mice tumor and liver metastasis sections previously obtained by orthotopic xenografts with WT cells. Taken together, our data interestingly suggest the relevance of ANXA1 as potential diagnostic/prognostic and/or therapeutic PC marker.

The promising pro-healing role of the association of mesoglycan and lactoferrin on skin lesions.

Skin wound repair represents an important topic for the therapeutic challenges. Many molecules are commonly used as active principles of topical devices to induce the correct tissue regeneration. Among these molecules, mesoglycan, a mixture of glycosaminoglycans, and the lactoferrin have recently aroused interest. Here, for the first time, we used mesoglycan/lactoferrin to treat the cell populations mainly involved in wound healing. We showed that human keratinocytes, fibroblasts and endothelial cells migrate and invade more rapidly when treated with the association. Moreover, we found that mesoglycan/lactoferrin, are able to trigger the differentiation process of keratinocytes, the switch of the fibroblasts into myofibroblasts, the acquisition of a mesenchymal phenotype for the endothelial cells which, in this way, start to form the capillary-like structures. Additionally, we proved that the well known antimicrobial behavior of lactoferrin encourages the inhibition of S. aureus and P. aeruginosa biofilm formation by the whole association, providing an appealing feature for this formulation. Finally, by the in vivo analysis, we showed that the mesoglycan/lactoferrin favors the closure of skin wounds performed on the mice back. Beside the decrease of the lesion diameters, by a confocal analysis of mice biopsies we found that the use of the association strongly promote cell activation underlying the correct tissue regeneration. These results encourage to further investigation aiming the development of a new topical patch that includes this association.

TFF1 Induces Aggregation and Reduces Motility of Helicobacter pylori.

Gastric cancer is considered one of the most common malignancies in humans and Helicobacter pylori infection is the major environmental risk factor of gastric cancer development. Given the high spread of this bacterium whose infection is mostly asymptomatic, H. pylori colonization persists for a long time, becoming chronic and predisposing to malignant transformation. The first defensive barrier from bacterial infection is constituted by the gastric mucosa that secretes several protective factors, among which is the trefoil factor 1 (TFF1), that, as mucin 5AC, binds the bacterium. Even if the protective role of TFF1 is well-documented, the molecular mechanisms that confer a beneficial function to the interaction among TFF1 and H. pylori remain still unclear. Here we analyze the effects of this interaction on H. pylori at morphological and molecular levels by means of microscopic observation, chemiotaxis and motility assays and real-time PCR analysis. Our results show that TFF1 favors aggregation of H. pylori and significantly slows down the motility of the bacterium across the mucus. Such aggregates significantly reduce both flgE and flaB gene transcription compared with bacteria not incubated with TFF1. Finally, our results suggest that the interaction between TFF1 and the bacterium may explain the frequent persistence of H. pylori in the human host without inducing disease.

Mesoglycan exerts its fibrinolytic effect through the activation of annexin A2.

Mesoglycan is a drug based on a mixture of glycosaminoglycans mainly used for the treatment of blood vessel diseases acting as antithrombotic and profibrinolytic drugs. Besides the numerous clinical studies, there is no information about its function on the fibrinolytic cascade. Here, we have elucidated the mechanism of action by which mesoglycan induces the activation of plasmin from endothelial cells. Surprisingly, by a proteomic analysis, we found that, following mesoglycan treatment, these cells show a notable amount of annexin A2 (ANXA2) at the plasma membrane. This protein has been widely associated with fibrinolysis and appears able to move to the membrane when phosphorylated. In our model, this translocation has proven to enhance cell migration, invasion, and angiogenesis. Furthermore, the interaction of mesoglycan with syndecan 4 (SDC4), a coreceptor belonging to the class of heparan sulfate proteoglycans, represents the upstream event of the ANXA2 behavior. Indeed, the activation of SDC4 triggers the motility of endothelial cells culminating in angiogenesis. Interestingly, mesoglycan can induce the release of plasmin in endothelial cell supernatants only in the presence of ANXA2. This evaluation suggests that mesoglycan triggers the formation of a chain mechanism starting from the activation of SDC4, and the related cascade of events, including src complex and PKCα activation, promoting the phosphorylation of ANXA2 and its translocation to plasma membrane. This indicates a connection among mesoglycan, SDC4-(PKCα-src), and ANXA2 which, in turn, links the tissue plasminogen activator bringing it closer to plasminogen. This latter is so cleaved to release the plasmin and degrade fibrin sleeves.

Annexin A1 Released in Extracellular Vesicles by Pancreatic Cancer Cells Activates Components of the Tumor Microenvironment, through Interaction with the Formyl-Peptide Receptors.

Pancreatic cancer (PC) is one of the most aggressive cancers in the world. Several extracellular factors are involved in its development and metastasis to distant organs. In PC, the protein Annexin A1 (ANXA1) appears to be overexpressed and may be identified as an oncogenic factor, also because it is a component in tumor-deriving extracellular vesicles (EVs). Indeed, these microvesicles are known to nourish the tumor microenvironment. Once we evaluated the autocrine role of ANXA1-containing EVs on PC MIA PaCa-2 cells and their pro-angiogenic action, we investigated the ANXA1 paracrine effect on stromal cells like fibroblasts and endothelial ones. Concerning the analysis of fibroblasts, cell migration/invasion, cytoskeleton remodeling, and the different expression of specific protein markers, all features of the cell switching into myofibroblasts, were assessed after administration of wild type more than ANXA1 Knock-Out EVs. Interestingly, we demonstrated a mechanism by which the ANXA1-EVs complex can stimulate the activation of formyl peptide receptors (FPRs), triggering mesenchymal switches and cell motility on both fibroblasts and endothelial cells. Therefore, we highlighted the importance of ANXA1/EVs-FPR axes in PC progression as a vehicle of intercommunication tumor cells-stroma, suggesting a specific potential prognostic/diagnostic role of ANXA1, whether in soluble form or even if EVs are captured in PC.