Abstract Risk stratification in localized prostate cancer (PCa) relies on clinical and pathological parameters, as well as molecular assays that are often limited in accessibility. While circulating myeloid cells are associated with poor prognosis in advanced disease, their role in primary PCa remains unclear. This study aimed to determine whether circulating CD11b+CD33+ myeloid cells could serve as a minimally invasive, cost effective prognostic biomarker in localized PCa. We analyzed a prospective cohort of 79 patients with localized PCa undergoing radical prostatectomy. Circulating CD11b+CD33+ myeloid cells were quantified by flow cytometry. Associations between CD11b+CD33+ frequency and clinical parameters were evaluated, including EAU risk group, postoperative pathological features and biochemical recurrence (BCR). RNA sequencing was performed on FACS sorted circulating CD11b+CD33+ cells from 39 patients, including 15 from this prostatectomy cohort and 24 from an independent biopsy cohort. Circulating CD11b+CD33+ cell frequency was significantly higher in patients classified as high risk at diagnosis, versus intermediate (p = 0.0419) and low risk (p = 0.0087), and associated with adverse pathological features such as ISUP grade 4 and 5 (p = 0.0034) and perineural invasion (p = 0.0303). Transcriptomic profiling of these cells revealed distinct transcriptional programs in high risk versus intermediate risk patients, with enrichment of tumor promoting and immunosuppressive gene signatures. Higher circulating CD11b+CD33+ frequency associated with shorter BCR free survival (p = 0.028), particularly in high risk patients, after a median follow up of 31.8 months. Notably, the frequency of these cells outperformed the EAU risk classification, identifying a subset of high risk PCa patients at highest risk of early recurrence.To validate these findings, we analyzed the TCGA cohort of localized PCa patients (n = 329); although only tumor transcriptomic data were available, high intratumoral CD11b+CD33+ gene expression was associated with shorter disease free survival, especially in high risk patients. A custom four gene myeloid signature derived from genes upregulated in our cohort further refined risk stratification, predicting early relapse in high risk cases. Higher frequencies of circulating CD11b+CD33+ myeloid cells are observed in high risk localized PCa and associate with shorter BCR free survival. Remarkably, this myeloid expansion occurs even in organ confined disease, revealing an early systemic immune response previously unrecognized in localized PCa. Transcriptomic profiling confirmed enrichment of immunosuppressive and tumor promoting programs in these cells. Circulating CD11b+CD33+ frequency and associated gene signatures represent a novel prognostic biomarker, with potential to improve risk stratification in localized PCa, warranting validation in larger cohorts. Citation Format: Viola Moscarda, Sara Merler, Daniele Braga, Bianca Calì, Federica Cetti, Giuseppe Reitano, Filippo Carletti, Gianmarco Randazzo, Davide Minardi, Sara Zumerle, Mirko Minini, Anna Sordo, Giovanna Pecoraro, Nicola Fossati, Andrea Gallina, Ricardo Pereira Mestre, Silke Gillessen, Alessandro Morlacco, Fabrizio Dal Moro, Andrea Alimonti. Prognostic value of circulating CD11b+CD33+myeloid cells in localized prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1195.

Hepcidin is the key hyposideremic hormone produced primarily by the liver. However, recent reports reveal extrahepatic functional sources of hepcidin, including the intestine, the site of dietary iron absorption. To determine whether intestinal hepcidin may play a role in plasma iron lowering, we generated transgenic mice overexpressing the peptide specifically in this tissue. At 1 month of age, transgenic mice exhibited severe iron deficiency along with decreased hematologic indices and a drastic suppression of liver hepcidin in response to hyposideremia. Mechanistically, we showed that intestinal hepcidin was produced in the intestine lumen, inducing a striking downregulation of divalent metal transporter 1 (DMT1) protein at the enterocyte. To confirm the capacity of hepcidin to decrease DMT1, we developed food-grade recombinant lactic acid bacteria (recLAB) genetically modified to deliver hepcidin directly into the intestinal lumen. These recLAB induced a rapid decrease of duodenal DMT1 and, most importantly, when daily orally administrated, protected against iron overload in a mouse model of hemochromatosis. Taken together, our data reveal a previously unrecognized role of intestinal hepcidin as a regulator of systemic iron homeostasis, acting on DMT1 on the apical side of enterocytes, with potential therapeutic relevance for hematologic or iron disorders.

Cellular senescence can exert dual effects in tumors, either suppressing or promoting tumor progression. The senescence-associated secretory phenotype (SASP), released by senescent cells, plays a crucial role in this dichotomy. Consequently, the clinical challenge lies in developing therapies that safely enhance senescence in cancer, favoring tumor-suppressive SASP factors over tumor-promoting ones. Here, we identify the retinoic-acid-receptor (RAR) agonist adapalene as an effective pro-senescence compound in prostate cancer (PCa). Reactivation of RARs triggers a robust senescence response and a tumor-suppressive SASP. In preclinical mouse models of PCa, the combination of adapalene and docetaxel promotes a tumor-suppressive SASP that enhances natural killer (NK) cell-mediated tumor clearance more effectively than either agent alone. This approach increases the efficacy of the allogenic infusion of human NK cells in mice injected with human PCa cells, suggesting an alternative therapeutic strategy to stimulate the anti-tumor immune response in "immunologically cold" tumors.

Accumulating senescent cells within tissues contribute to the progression of aging and age-related diseases. Botanical extracts, rich in phytoconstituents, present a useful resource for discovering therapies that could target senescence and thus improve healthspan. Here, we show that daily oral administration of a standardized extract of Salvia haenkei (Haenkenium (HK)) extended lifespan and healthspan of naturally aged mice. HK treatment inhibited age-induced inflammation, fibrosis and senescence markers across several tissues, as well as increased muscle strength and fur thickness compared with age-matched controls. We also found that HK treatment reduced acutely induced senescence by the chemotherapeutic agent doxorubicin, using p16LUC reporter mice. We profiled the constituent components of HK by mass spectrometry, and identified luteolin-the most concentrated flavonoid in HK-as a senomorphic compound. Mechanistically, by performing surface plasmon resonance and in situ proximity ligation assay, we found that luteolin disrupted the p16-CDK6 interaction. This work demonstrates that administration of HK promotes longevity in mice, possibly by modulating cellular senescence and by disrupting the p16-CDK6 interaction.

Although hypercoagulability is commonly associated with malignancies, whether coagulation factors directly affect tumor cell proliferation remains unclear. Herein, by performing single-cell RNA sequencing (scRNA-seq) of the prostate tumor microenvironment (TME) of mouse models of castration-resistant prostate cancer (CRPC), we report that immunosuppressive neutrophils (PMN-MDSCs) are a key extra-hepatic source of coagulation factor X (FX). FX activation within the TME enhances androgen-independent tumor growth by activating the protease-activated receptor 2 (PAR2) and the phosphorylation of ERK1/2 in tumor cells. Genetic and pharmacological inhibition of factor Xa (FXa) antagonizes the oncogenic activity of PMN-MDSCs, reduces tumor progression, and synergizes with enzalutamide therapy. Intriguingly, F10high PMN-MDSCs express the surface marker CD84 and CD84 ligation enhances F10 expression. Elevated levels of FX, CD84, and PAR2 in prostate tumors associate with worse survival in CRPC patients. This study provides evidence that FXa directly promotes cancer and highlights additional targets for PMN-MDSCs for cancer therapies.

Serine-Threonine kinase CK2 supports malignant B-lymphocyte growth but its role in B-cell development and activation is largely unknown. Here, we describe the first B-cell specific knockout (KO) mouse model of the β regulatory subunit of CK2. CK2β KO mice present an increase in marginal zone (MZ) and a reduction in follicular B cells, suggesting a role for CK2 in the regulation of the B cell receptor (BCR) and NOTCH2 signaling pathways. Biochemical analyses demonstrate an increased activation of the NOTCH2 pathway in CK2β KO animals, which sustains MZ B-cell development. Transcriptomic analyses indicate alterations in biological processes involved in immune response and B-cell activation. Upon sheep red blood cells (SRBC) immunization CK2β KO mice exhibit enlarged germinal centers (GCs) but display a limited capacity to generate class-switched GC B cells and immunoglobulins. In vitro assays highlight that B cells lacking CK2β have an impaired signaling downstream of BCR, Toll-like receptor, CD40, and IL-4R all crucial for B-cell activation and antigen presenting efficiency. Somatic hypermutations analysis upon 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Chicken Gamma Globulin (NP-CGG) evidences a reduced NP-specific W33L mutation frequency in CK2β KO mice suggesting the importance of the β subunit in sustaining antibody affinity maturation. Lastly, since diffuse large B cell lymphoma (DLBCL) cells derive from GC or post-GC B cells and rely on CK2 for their survival, we sought to investigate the consequences of CK2 inhibition on B cell signaling in DLBCL cells. In line with the observations in our murine model, CK2 inactivation leads to signaling defects in pathways that are essential for malignant B-lymphocyte activation.

Recent proteomic, metabolomic, and transcriptomic studies have highlighted a connection between changes in mitochondria physiology and cellular pathophysiological mechanisms. Secondary assays to assess the function of these organelles appear fundamental to validate these -omics findings. Although mitochondrial membrane potential is widely recognized as an indicator of mitochondrial activity, high-content imaging-based approaches coupled to multiparametric to measure it have not been established yet. In this paper, we describe a methodology for the unbiased high-throughput quantification of mitochondrial membrane potential in vitro, which is suitable for 2D to 3D models. We successfully used our method to analyze mitochondrial membrane potential in monolayers of human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers. Moreover, by combining automated image analysis and machine learning, we were able to discriminate melanoma cells from macrophages in co-culture and to analyze the subpopulations separately. Our data demonstrated that our method is a widely applicable strategy for large-scale profiling of mitochondrial activity.

The tumor microenvironment (TME) impacts different phases of tumor progression and therapy resistance. Zhang et al. show that senescent stromal cells activate an epigenetic program that controls the senescence-associated secretory phenotype and can be targeted to boost responses to chemotherapy.

Cellular senescence has been widely recognised for decades as a stable arrest of cell proliferation. A recent study identifies senescence establishment and maintenance as a dynamic and reversible process regulated by (in)activation of a predetermined enhancer landscape controlled by the pioneer transcription factor AP-1.

Bleeding and altered iron distribution occur in multiple gastrointestinal diseases, but the importance and regulation of these changes remain unclear. We found that hepcidin, the master regulator of systemic iron homeostasis, is required for tissue repair in the mouse intestine after experimental damage. This effect was independent of hepatocyte-derived hepcidin or systemic iron levels. Rather, we identified conventional dendritic cells (cDCs) as a source of hepcidin that is induced by microbial stimulation in mice, prominent in the inflamed intestine of humans, and essential for tissue repair. cDC-derived hepcidin acted on ferroportin-expressing phagocytes to promote local iron sequestration, which regulated the microbiota and consequently facilitated intestinal repair. Collectively, these results identify a pathway whereby cDC-derived hepcidin promotes mucosal healing in the intestine through means of nutritional immunity.

Recently, there has been a great effort to develop tests based on non-invasive urinary biomarkers (NMIBCs). These tests are based on the fact that NMIBCs are heterogeneous at the molecular level and can be divided into different molecular groups useful to predict prognosis and response to treatment. The assessment of epigenetic alterations, such as DNA methylation, represents a promising cancer biomarker. DNA methylation is an epigenetic modification that affects gene expression without modifying the DNA sequence. Several studies have highlighted the presence of methylated loci in the context of bladder cancer, indicating its potential application as a diagnostic and prognostic biomarker. One of the novel assays based on a DNA methylation profile, the Bladder EpiCheck, analyzes DNA from spontaneous urine, detecting disease-specific DNA methylation patterns in bladder cancer patients. This test, due to its non-invasive nature and highly promising performance could, in future, become an invaluable tool in the follow-up of bladder cancer patients. Potential new applications could include diagnosis and surveillance of upper-tract disease, for the replacement of invasive testing and ureteroscopy.

Extracellular ATP is a signaling molecule exploited by the immune cells for both autocrine regulation and paracrine communication. By performing live calcium imaging experiments, we show that triggered mouse macrophages are able to propagate calcium signals to resting bystander cells by releasing ATP. ATP-based intercellular communication is mediated by P2x4 and P2x7 receptors and is a feature of pro-inflammatory macrophages. In terms of functional significance, ATP signaling is required for efficient phagocytosis of pathogen-derived molecules and apoptotic cells and may represent a target for macrophage regulation by CD39-expressing cells. These results highlight a cell-to-cell communication mechanism tuning innate immunity.

Pulmonary iron excess is deleterious and contributes to a range of chronic and acute inflammatory diseases. Optimal lung iron concentration is maintained through dynamic regulation of iron transport and storage proteins. The iron-regulatory hormone hepcidin is also expressed in the lung. In order to better understand the interactions between iron-associated molecules and the hepcidin-ferroportin axis in lung iron balance, we examined lung physiology and inflammatory responses in two murine models of systemic iron-loading, either hepcidin knock-out (Hepc KO) or liver-specific hepcidin KO mice (Hepc KOliv), which do (Hepc KOliv) or do not (Hepc KO) express lung hepcidin. We have found that increased plasma iron in Hepc KO mice is associated with increased pulmonary iron levels, consistent with increased cellular iron uptake by pulmonary epithelial cells, together with an increase at the apical membrane of the cells of the iron exporter ferroportin, consistent with increased iron export in the alveoli. Subsequently, alveolar macrophages (AM) accumulate iron in a non-toxic form and this is associated with elevated production of ferritin. The accumulation of iron in the lung macrophages of hepcidin KO mice contrasts with splenic and hepatic macrophages which contain low iron levels as we have previously reported. Hepc KOliv mice with liver-specific hepcidin deficiency demonstrated same pulmonary iron overload profile as the Hepc KO mice, suggesting that pulmonary hepcidin is not critical in maintaining local iron homeostasis. In addition, the high iron load in the lung of Hepc KO mice does not appear to enhance acute lung inflammation or injury. Lastly, we have shown that intraperitoneal LPS injection is not associated with pulmonary hepcidin induction, despite high levels of inflammatory cytokines. However, intranasal LPS injection stimulates a hepcidin response, likely derived from AM, and alters pulmonary iron content in Hepc KO mice.

Spatiotemporal compartmentalization of signaling pathways and second messengers is pivotal for cell biology and membrane rafts are, therefore, required for several lymphocyte functions. On the other hand, T cells have the specific necessity of tuning signaling amplification depending on the context in which the antigen is presented. In this review, we discuss of membrane rafts in the context of T cell signaling, focusing on CD28-mediated costimulation.

La presente invention concerne des antogonistes de l'hepcidine destines a etre utilises dans le traitement de maladies inflammatoires.

Iron is fundamental for the organism, but can be toxic if in excess. The major regulator of iron homeostasis is hepcidin, first identified as an antimicrobial peptide. Hepcidin deficiency is associated with hereditary hemochromatosis, a human genetic disease characterized by iron overload. Conversely, abnormal hepcidin induction is the cause of some types of anemia. Hepcidin is produced and secreted mainly by the liver, but many cells and tissues express low levels of the hormone. To investigate the contribution of hepcidin-producing tissues in iron homeostasis, we generated a new mouse model in which the hepcidin gene can be conditionally inactivated: the hepcidin floxed mice. The main aim of my thesis was to evaluate the contribution of different tissues in hepcidin production and iron homeostasis regulation. I generated hepcidin liver-specific knockout mice and compared them to total knockouts: hepatic hepcidin ablation fully recapitulates the severe iron overload phenotype observed in the total knockout. Moreover, hepcidin was undetectable in the plasma of liver-specific knockouts, showing that hepatocytes are the main source of the peptide in the bloodstream. These results demonstrate that hepatic hepcidin is the major regulator of iron homeostasis and that tissues other than the liver are not able to compensate. The hepcidin floxed mice provide a useful tool for the investigation of hepcidin role in extra-hepatic tissues in physiological and pathological conditions.

Most of the iron required for erythropoiesis is acquired by heme iron recycling of senescent erythrocytes by tissue macrophages (mainly spleen, bone marrow and liver macrophages). This process, called erythrophagocytosis (EP), is essential for mammalian iron homeostasis. Perturbations in EP occur in

Hepcidin is a 25-amino-acid peptide demonstrated to be the iron regulatory hormone capable of blocking iron absorption from the duodenum and iron release from macrophages. Mutations affecting hepcidin regulators or the hepcidin gene itself cause hemochromatosis, a common genetic disorder. Hepcidin is produced mainly by the liver, but many cells and tissues express low levels of the hormone. To determine the contribution of these hepcidin-producing tissues in body iron homeostasis, we have developed a new mouse model in which the hepcidin gene can be conditionally inactivated. Here we compare a liver-specific knockout (KO) mouse model with total KO mice. We show that the liver-specific KO mice fully recapitulate the severe iron overload phenotype observed in the total KO mice, with increased plasma iron and massive parenchymal iron accumulation. This result demonstrates that the hepatocyte constitutes the predominant reservoir for systemic hepcidin and that the other tissues are unable to compensate.