IRCCS San Raffaele Scientific Institute Showcase

Despite recent progress in cancer treatment, liver metastases persist as an unmet clinical need. Here, we show that arming liver and tumor-associated macrophages in vivo to co-express tumor antigens (TAs), IFNα, and IL-12 unleashes robust anti-tumor immune responses, leading to the regression of liver metastases. Mechanistically, in vivo armed macrophages expand tumor reactive CD8+ T cells, which acquire features of progenitor exhausted T cells and kill cancer cells independently of CD4+ T cell help. IFNα and IL-12 produced by armed macrophages reprogram antigen presenting cells and rewire cellular interactions, rescuing tumor reactive T cell functions. In vivo armed macrophages trigger anti-tumor immunity in distinct liver metastasis mouse models of colorectal cancer and melanoma, expressing either surrogate tumor antigens, naturally occurring neoantigens or tumor-associated antigens. Altogether, our findings support the translational potential of in vivo armed liver macrophages to expand and rejuvenate tumor reactive T cells for the treatment of liver metastases. Data availability: The MERFISH, single-cell RNA sequencing and bulk RNA sequencing data have been deposited in the GEO repository under the accession number GSE273615. Additionally, the WES data have been uploaded to the ENA portal with the accession number PRJEB78386. Code availability: Code is available at the following link: http://www.bioinfotiget.it/gitlab/custom/notaro_mouse_lm_2025.

Upon metastatic seeding in the liver, liver macrophages, including Kupffer cells, acquire a transcriptional profile typical of tumor-associated macrophages (TAMs), which support tumor progression. MicroRNAs (miRNAs) fine-tune TAM pro-tumoral functions, making their modulation a promising strategy for macrophage reprogramming into an anti-tumoral phenotype. Here, we analyze the transcriptomic profiles of liver and splenic macrophages, identifying miR-342-3p as a key regulator of liver macrophage function. miR-342-3p is highly active in healthy liver macrophages but significantly downregulated in colorectal cancer liver metastases (CRLMs). Lentiviral vector-engineered liver macrophages enforcing miR-342-3p expression acquire a pro-inflammatory phenotype and reduce CRLM growth. We identify Slc7a11, a cysteine-glutamate antiporter linked to pro-tumoral activity, as a direct miR-342-3p target, which may be at least partially responsible for TAM phenotypic reprogramming. Our findings highlight the potential of in vivo miRNA modulation as a therapeutic strategy for TAM reprogramming, offering an approach to enhance cancer immunotherapy. Data and code availability: Next-generation sequencing data are deposited at the Gene Expression Omnibus (GEO) with the following accession numbers: GEO: GSE274043 (scRNA-seq), GSE274044 (RNA-seq on iKCs), GSE274045 (small RNA-seq on splenic and hepatic cell populations), and GSE274046 (bulk RNA-seq on splenic and hepatic cell populations). The code is available at GitLab: http://www.bioinfotiget.it/gitlab/custom/Bresesti_Cell_Reports_2025.

The dataset includes the raw data of graphs shown in figures 1, 2, 3, 5, 6, and 7 and in supplementary figures 1, 2, 3, 4, 5, 7, and 8 of the paper: Human iPSC-derived neural stem cells displaying radial glia signature exhibit long-term safety in mice. Luciani M, Garsia C, Beretta S, Cifola I, Peano C, Merelli I, Petiti L, Miccio A, Meneghini V, Gritti A. Nat Commun. 2024 Nov 1;15(1):9433. doi: 10.1038/s41467-024-53613-7. PMID: 39487141 The bulk RNA-seq, SREBF1-deficient RNA-seq, and ChIP-seq data generated in this study have been deposited at GEO under accession number GSE239446. The single-cell RNA-seq data generated in this study have been deposited at GEO under accession number GSE238206. The processed RNA-seq, ChIP-seq, and scRNA-seq data (list of DEGs and GO terms) are available in Supplementary Data 1-4 files. Human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NSCs) hold promise for treating neurodegenerative and demyelinating disorders. However, comprehensive studies on their identity and safety remain limited. In this study, we demonstrate that hiPSC-NSCs adopt a radial glia-associated signature, sharing key epigenetic and transcriptional characteristics with human fetal neural stem cells (hfNSCs) while exhibiting divergent profiles from glioblastoma stem cells. Long-term transplantation studies in mice showed robust and stable engraftment of hiPSC-NSCs, with predominant differentiation into glial cells and no evidence of tumor formation. Additionally, we identified the Sterol Regulatory Element Binding Transcription Factor 1 (SREBF1) as a regulator of astroglial differentiation in hiPSC-NSCs. These findings provide valuable transcriptional and epigenetic reference datasets to prospectively define the maturation stage of NSCs derived from different hiPSC sources and demonstrate the long-term safety of hiPSC-NSCs, reinforcing their potential as a viable alternative to hfNSCs for clinical applications.

Clonal dominance characterizes hematopoiesis during aging and increases susceptibility to blood cancers and common non-malignant disorders. VEXAS syndrome is a recently discovered adult-onset autoinflammatory disease burdened by a high mortality rate and caused by dominant hematopoietic clones bearing somatic mutations in the UBA1 gene. However, pathogenic mechanisms driving clonal dominance are unknown. Moreover, the lack of disease models hampers the development of disease-modifying therapies. Here, we performed immunophenotypic characterization of hematopoiesis and single-cell transcriptomics in a cohort of 9 male patients with VEXAS syndrome, revealing pervasive inflammation across all lineages. Hematopoietic stem/progenitor cells (HSPCs) in patients are skewed toward myelopoiesis and acquire senescence-like programs. Humanized models of VEXAS syndrome, generated by inserting the causative mutation in healthy HSPCs through base editing, recapitulated proteostatic defects, cytologic alterations, and senescence signatures of patients’ cells, as well as disease hematologic and inflammatory hallmarks. Competitive transplants of human UBA1-mutant and wild-type HSPCs showed that while mutant cells are more resilient to the inflammatory milieu, likely through the acquisition of the senescence-like state, wild-type ones are progressively exhausted and overwhelmed by VEXAS clones, overall impairing functional hematopoiesis and leading to bone marrow failure. Our study unveils the mechanism of clonal dominance, provides models for preclinical investigation of therapeutic strategies, and might have implications for clinical management of VEXAS syndrome.

The dataset includes data collected at the Division of Pancreatic Surgery of San Raffaele Scientific Institute, Milan, Italy and at HPB Disease Unit of Karolinska Hospital in Stockholm, Sweden. Demographics, symptoms, clinical history, diagnostic work-up (including laboratory, radiologic and endoscopic data), type of surgery, and pathology data were prospectively collected. All patients were preoperatively discussed in conference [14] . High-risk stigmata (HRS) and worrisome features (WF) of IG and absolute indications (AI) and relative indications (RI) of EG were retrospectively applied. HRS include jaundice, solid mass, main pancreatic duct (MPD) ≥10 mm, enhancing nodules ≥5 mm and cytology positive for high-grade dysplasia or adeno- carcinoma. WF include cyst size ≥30 mm, MPD size of 5–9 mm, pancreatitis, enhancing mural nodule < 5 mm, thickened/enhancing cyst walls, abrupt change in MPD caliber, lymphadenopathy, serum levels of CA 19.9 > 37 U/mL and cyst growth rate > 5 mm/2 years. AI include jaundice, MPD ≥10 mm, enhancing nodules ≥5 mm, solid mass and cytology positive for high-grade dysplasia or adenocar- cinoma. RI include cyst size ≥40 mm, MPD size of 5–9.9 mm, pan- creatitis, enhancing mural nodule < 5 mm, serum levels of CA-19.9 > 37 U/mL, new onset of diabetes and cyst growth rate > 5 mm/year.

CD4+ T cells play a critical role in antiviral humoral and cellular immune responses. We have previously reported that subcutaneous lymphocytic choriomeningitis virus (s.c. LCMV) infection is characterized by a stark compartmentalization of CD4+ T cells, leading to strong TH1 polarization but virtually absent T follicular helper (TFH) cells, a key driver of humoral immunity. Here, we investigated the mechanisms responsible for this impaired TFH differentiation. We found that T-bet+ cells induced by s.c. LCMV infection encompass a TH1 subset expressing Granzyme-B (GzmB) and a Tcf-1+ subset that retains the potential for TFH differentiation without expressing mature TFH markers. Interestingly, IFN-γ blockade enables full differentiation of Tcf-1+ cells into TFH, formation of germinal centers and increased antibody production. Of note, the suppression of TFH cells by IFN-γ is not directly mediated through CD4+ T cells but rather involves another cell type, likely dendritic cells (DCs). Our study provides novel insights into the mechanisms directing early CD4+ T cell polarization and affecting humoral responses to viruses, laying a foundation for the development of effective vaccine strategies.

Several factors may affect response to treatment in Major Depressive Disorder (MDD) including immune/inflammatory alterations and regional brain volumes, particularly in hippocampal regions which have shown to be influenced by inflammatory status. Neutrophil-to-lymphocyte ratio (NLR) is an inflammatory marker found to be elevated in depressed women in large population studies. Here we investigate the effect of NLR on treatment response in MDD patients, and the role of sex and hippocampal volume on influencing this relationship. A sample of 124 MDD depressed inpatients (F = 80) underwent MRI acquisition, admission NLR was calculated by dividing absolute neutrophil by absolute lymphocyte counts and depression severity was assessed at admission and discharge via the Hamilton Depression Rating Scale (HDRS). As a measure of treatment response, delta HDRS was calculated. We found a significant moderation effect of sex on the relationship between NLR and Delta HDRS: a negative relation was found in females and a positive one in males. NLR was found to negatively affect hippocampal volumes in females. Both left and right hippocampal volume positively associated with Delta HDRS. Finally, left hippocampal volume mediated the effect of NLR on Delta HDRS in females. Sex moderated the relation between inflammation and treatment response in line with previous reports linking inflammation to hampered antidepressant effect in females. Further, this effect is partially mediated by hippocampal volume, suggesting that antidepressant response may be hampered by the detrimental effect of inflammation on the brain.

Bipolar disorder (BD) and major depressive disorder (MDD) are severe psychiatric illnesses that share among their environmental risk factors the exposure to adverse childhood experiences (ACE). Exposure to ACE has been associated with long-term changes in brain structure and the immune response. In the lasts decades, brain abnormalities including alterations of white matter (WM) microstructure and higher levels of peripheral immune/inflammatory markers have been reported in BD and MDD and an association between inflammation and WM microstructure has been shown. However, differences in these measures have been reported by comparing the two diagnostic groups. The aim of the present study was to investigate the interplay between ACE, inflammation, and WM in BD and MDD. We hypothesize that inflammation will mediate the association between ACE and WM and that this will be different in the two groups. A sample of 200 patients (100 BD, 100 MDD) underwent 3T MRI scan and ACE assessment through Childhood Trauma Questionnaire. A subgroup of 130 patients (75 MDD and 55 BD) underwent blood sampling for the assessment of immune/inflammatory markers. We observed that ACE associated with higher peripheral levels of IL-2, IL-17, bFGF, IFN-γ, TNF-α, CCL3, CCL4, CCL5, and PDGFBB only in the BD group. Further, higher levels of CCL3 and IL-2 associated with lower FA in BD. ACE were found to differently affect WM microstructure in the two diagnostic groups and to be negatively associated with FA and AD in BD patients. Mediation analyses showed a significant indirect effect of ACE on WM microstructure mediated by IL-2. Our findings suggest that inflammation may mediating the detrimental effect of early experiences on brain structure and different mechanism underlying brain alterations in BD and MDD.

Despite the increasing availability of antidepressant drugs, a high rate of patients with major depression (MDD) does not respond to pharmacological treatments. Brain-derived neurotrophic factor (BDNF)-tyrosine receptor kinase B (TrkB) signaling is thought to influence antidepressant efficacy and hippocampal volumes, robust predictors of treatment resistance. We therefore hypothesized the possible role of BDNF and neurotrophic receptor tyrosine kinase 2 (NTRK2)-related polymorphisms in affecting both hippocampal volumes and treatment resistance in MDD. A total of 121 MDD inpatients underwent 3T structural MRI scanning and blood sampling to obtain genotype information. General linear models and binary logistic regressions were employed to test the effect of genetic variations related to BDNF and NTRK2 on bilateral hippocampal volumes and treatment resistance, respectively. Finally, the possible mediating role of hippocampal volumes on the relationship between genetic markers and treatment response was investigated. A significant association between one NTRK2 polymorphism with hippocampal volumes and antidepressant response was found, with significant indirect effects. Our results highlight a possible mechanistic explanation of antidepressant action, possibly contributing to the understanding of MDD pathophysiology.

Due to the overlapping depressive symptomatology with major depressive disorder (MDD), 60% of patients with bipolar disorder (BD) are initially misdiagnosed, calling for the definition of reliable biomarkers that can support the diagnostic process. Here, we optimized a machine learning pipeline for the differentiation between depressed BD and MDD patients based on multimodal structural neuroimaging features. Diffusion tensor imaging (DTI) and T1-weighted magnetic resonance imaging (MRI) data were acquired for 282 depressed BD (n = 180) and MDD (n = 102) patients. Images were preprocessed to obtain axial (AD), radial (RD), mean (MD) diffusivity, fractional anisotropy (FA), and voxel-based morphometry (VBM) maps. Each feature was entered separately into a 5-fold nested cross-validated predictive pipeline differentiating between BD and MDD patients, comprising: confound regression for nuisance variables removal, feature standardization, principal component analysis for feature reduction, and an elastic-net penalized regression. The DTI-based models reached accuracies ranging from 75% to 78%, whereas the VBM model reached 61% of accuracy. All the models were significantly different from a null model distribution at a 5000-permutation test. A 5000 bootstrap procedure revealed that widespread differences drove the classification, with BD patients associated to overall higher values of AD and FA, and grey matter volumes. Our results suggest that structural neuroimaging, in particular white matter microstructure and grey matter volumes, may be able to differentiate between MDD and BD patients with good predictive accuracy, being significantly higher than chance-level.