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AbstractAimsP-glycoprotein (P-gp), an efflux transporter with diverse compound effects, is a vital part of cardiac function. To determine if the selective substrate tracer [18F]MC225 also functions in cardiac P-gp, micro-engineered heart tissues (µ-EHTs) utilizing human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes were used. This model offers advantages in potentially reducing animal experiments and allowing direct evaluation on human cells. However, its adoption in nuclear medicine remains very limited. This study aims to evaluate [18F]MC225 as a measurement method for cardiac P-gp function using a heart-on-chip model.Methods and resultsµ-EHTs were treated with the P-gp inhibitor Tariquidar (200 nM for 30 min) or the P-gp inducer Doxorubicin (1 µM for 24 h) and incubated with [18F]MC225 (1 MBq/mL for 30 min). First, we identified and confirmed the expression of P-gp in the µ-EHTs using immunofluorescent staining, which showed an increase of P-gp expression after Doxorubicin treatment. According to γ-counter measurements, Tariquidar-treated tissues exhibited a higher uptake (117.5 ± 33.67%, n = 24) (P = 0.035) than the control, compared to Doxorubicin-treated tissues which exhibited a lower uptake (63.97 ± 21.89%, n = 20) (P < 0.001) compared to its controls. Autoradiography visualized radioactive distribution in each µ-EHT and confirmed the γ-counter measurements.Conclusion[18F]MC225 effectively evaluates and measures cardiac P-gp function in µ-EHTs on the heart-on-chip platform. This research sets the stage for future studies using P-gp function to evaluate the efficacy and safety of novel cardiovascular drugs using µ-EHTs. Graphical Abstract Graphical Abstract

BackgroundLong-term persistence of spike-specific memory B cells (MBC) upon mRNA SARS-CoV-2 vaccination remains poorly explored in people living with HIV (PLWH). We previously observed that the primary two-dose immunization elicited a B cell response quantitatively similar but phenotypically different compared to healthy participants (HC), with higher prevalence of CD27-IgD- double-negative cells and lower proportion of CD27+ Ig-switched MBC.MethodsThe PatoVac_COV longitudinal prospective single-centre study included 74 PLWH vaccinated with mRNA SARS-CoV-2 vaccines. Building upon our previous findings, blood samples were collected before and after the third and fourth doses, and two years after the first dose. PBMC were analysed for the RBD-specific MBC response by multidimensional flow cytometry, while spike-specific IgG were tested in plasma samples by ELISA and by surrogate virus neutralization assay. A supervised decision tree model was used to identify clinical and demographic predictors of RBD-specific MBC persistence.ResultsThe third vaccine dose robustly recalled RBD-specific MBC, driving their differentiation into Ig-switched resting MBC, a subset undetectable before boosting and dominant also in HC. The fourth vaccine dose minimally impacted on the MBC response. IgG+ resting MBC, recognizing both wild type and Omicron BA.2 strains, were still present two years after vaccination. Clinical and demographic factors associated to the persistence of IgG+ resting RBD-specific MBC were time since initiation of current ART, BMI, CD8+ cells count, granulocytes count and blood creatinine levels.ConclusionsThese findings highlight the impact of boosting, particularly the third dose, in enhancing and shaping the RBD-specific MBC in PLWH. Clinical and demographic variables associated with long-term MBC persistence may serve as potential biomarkers for predicting durable vaccine responses in PLWH.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-07414-1.

Pediatric brain tumors (PBTs) are the leading cause of cancer-related mortality in children. Despite advances in next-generation sequencing (NGS) deepening our understanding of the molecular features of PBT, the lack of preclinical models that capture their complexity and diversity remains a significant barrier to developing less toxic and more effective treatments. We have established 20 ex-vivo patient-derived tumoroid (PDTs) cultures from fresh surgical material of a wide range of PBTs, including low- and high-grade gliomas, medulloblastomas, and even rarer tumor entities such as a CNS tumor with BCOR alteration. Immunofluorescence, NGS analysis, and DNA methylation profiling revealed that the PDTs faithfully mirrored the cellular nature, the genetic and epigenetic landscape of their matched primary tumors. Our study shows the feasibility of generating PDTs, even from rarer entities, that recapitulate the genetic and epigenetic features of primary tumors, highlighting their potential as models for tumor biology studies and precision medicine.

Vertebrate radial glia progenitors (RGPs) balance self-renewal and differentiation through asymmetric cell division (ACD), which involves unequal centrosome inheritance. How centrosome asymmetry directs cell fate remains poorly understood. Here, we identify Pericentriolar material 1 (Pcm1) as a key player in this process. In zebrafish embryonic RGPs, Pcm1 is asymmetrically associated with Cep83, a mother centrosome marker. Using in vivo time-lapse imaging and nanoscale-resolution expansion microscopy, we detect Pcm1 on Notch ligand-containing endosomes, where it interacts–either directly or indirectly–with Par-3 and dynein. Loss of pcm1 disrupts endosome dynamics, increasing neuronal differentiation at the expense of RGP self-renewal. Mechanistically, Pcm1 facilitates the transition from Rab5b to Rab11a and promotes the assembly of Par-3 and dynein macromolecular complexes on recycling endosomes. Furthermore, we find conserved PARD3-PCM1-CEP83-RAB11 associations in human cortical brain organoids. Our findings uncover that Pcm1 links centrosome asymmetry to polarized endosome trafficking, thereby regulating RGP fate decisions. Pcm1 bridges centrosome asymmetry and polarized endosome trafficking to regulate radial glia progenitor fate decisions, balancing self-renewal and differentiation in zebrafish and human cortical organoids.

Myelodysplastic syndrome (MDS) originates from hematopoietic stem cell (HSC) clones with acquired gene mutations. However, the molecular characteristics of MDS stem cells remain poorly understood. Here, we show that the chromatin accessibility profiles of MDS stem cells more accurately reflect disease status than those of progenitor cells and reveal the process of stem cell alterations during disease progression. Characterization of differentially accessible regions (DARs) shows that MDS stem cells acquire progenitor-like chromatin accessibility during disease progression, leading to disruption of the normal stem-progenitor hierarchy. Profiling of transcription factor-binding motifs at DARs further uncovers precocious activation of myeloid transcriptional networks in MDS stem cells, with a concurrent loss of HSC-associated regulatory programs. In particular, increased chromatin accessibility at CEBP target sites represents the myeloid reprogramming status of MDS stem cells. Newly developed “progenitor scores” based on chromatin accessibility stratify disease status and correlate well with prognosis. These findings indicate that chromatin landscapes of MDS stem cells define their cell-autonomous behavior and contribute to disease progression. This study reveals that MDS stem cells progressively acquire progenitor-like chromatin features during disease progression, and that a scoring system based on chromatin accessibility in MDS stem and progenitor cells correlates strongly with disease progression.

Mechanical force controls bone development and homeostasis, with mechanosensitive osteocytes playing a key role. Osteocytes form interconnected dendrites to respond to mechanical stimuli and regulate bone formation and resorption. However, the molecular mechanisms underlying osteocyte maturation and dendrite formation remain elusive. Here we show that Piezo1 loss in late osteoblasts and osteocytes reduces bone mass and disrupts osteocyte differentiation with reduced dendrite number, length and network formation. Decreased YAP activity and unexpected Wnt/β-catenin signaling activation cause these deficiencies, largely restored by intermittent YAP activation. We further identify YAP targets CCN1/2 as crucial secreted factors in the Piezo1-YAP mechanotransduction. CCN1/2 overexpression in the liver rescues dendritic and bone defects in the Piezo1 mutant mice by activating Src, promoting actin polymerization, and activating YAP in a positive feedforward loop. Our findings highlight the pivotal roles of Piezo1 and YAP-CCN1/2-Src signaling, offering potential therapeutic targets for improving osteocyte dendritic networks. Mechanical force controls bone development and homeostasis. Here, the authors show that Piezo1-driven YAP–CCN1/2 signalling is essential for osteocyte maturation and dendrite formation.

Endometrial cancer (EC) is one of the most common gynecologic malignancies amongst women worldwide. Its incidence and mortality rates have been increasing in the last decade. In the present work, we built a patient EC-derived organoid (PDOs) platform that faithfully recapitulated tumor phenotype, genomic alterations, and expression profiles of matched-primary cancer tissues. Interestingly, we found that the response of EC-derived PDOs to both standard therapy and a wide range of targeted drugs accordingly to their specific druggable genetic alterations was congruent with that of the originating patients. We also isolated and genomically characterized matched-PDO stromal cells, specifically cancer-associated fibroblasts (CAFs). Unlike PDOs matched CAFs were poorly responsive and underwent to pro-inflammatory senescence upon treatment with standard therapy. Collectively, our findings established a EC-PDOs preclinical platform which allows assessing the therapeutic response of tumor and surrounding tumor microenvironment cellular landscape.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy; however, their use is frequently associated with immune-related adverse events (irAEs). In this study, anti-PD-L1 therapy exacerbates muscle wasting in tumor-bearing male mice despite its anti-tumor efficacy, accompanied by an accumulation of CD8+ T cells in muscle. Single-cell RNA sequencing identifies these cells as tissue-resident memory-like CD49a+ CD8+ T cells. While CD8+ T cell depletion prevents muscle wasting, it compromises the anti-tumor efficacy of anti-PD-L1. To resolve this paradox, we identify cathepsin L (CTSL) as a dual-target capable of suppressing both tumor progression and CD8+ T cell-mediated muscle wasting, through integrative transcriptomic analysis. Pharmacological inhibition of CTSL not only mitigates anti-PD-L1-induced muscle wasting but also further suppresses tumor growth, potentially via downregulation of BNIP3. Here, we show that CTSL is a dual-action target to uncouple anti-tumor efficacy from muscle-specific irAEs, offering a strategy to improve clinical outcomes of ICIs. Immune checkpoint inhibitors (ICIs) are associated with side effects such as muscle wasting. Here, the authors discover that targeting cathepsin L not only suppresses ICI-induced CD8 + T cell-mediated muscle wasting but also enhances ICI anti-tumor efficacy.

PurposeCastration and enzalutamide induce BCL-2 to drive therapy resistance in prostate cancer (PCa). We conducted a phase Ib trial to test that metastatic castration-resistant PCa (mCRPC) can be effectively targeted by combining enzalutamide with the BCL-2 inhibitor venetoclax.Experimental designThis phase Ib single-arm trial of enzalutamide (160 mg/d) with venetoclax in patients with progressive mCRPC assessed dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Three dose levels (DL) of venetoclax (DL1 at 400 mg/d; DL2 at 600 mg/d; and DL3 at 800 mg/d) were evaluated using a 3 + 3 design. We also analyzed enzalutamide and venetoclax pharmacokinetics and conducted pharmacodynamic studies in peripheral blood mononuclear cells (PBMCs) to determine the impact of venetoclax on BCL-2 expression.ResultsA total of 10 patients were enrolled across 3 DL and no DLT was observed. Mean duration on treatment was 29 weeks (range: 8–140 weeks). Treatment-related adverse events (TRAEs) were mostly grade 1–2, and Grade 3 TRAEs included fatigue (10%) and thrombocytopenia (10%). 1/10 (10%) attained PSA50 response and 4/10 (40%) had stable disease. Estimated median overall survival (OS) was 19 months (95% CI 5–28 months) and median time to next systemic therapy (TNST) was 5 months (95% CI 1–35 months). Pharmacokinetic results revealed sub-optimal plasma levels of venetoclax. Pharmacodynamic studies demonstrated that venetoclax enhanced BCL-2β generation and promoted BCL-2 degradation.ConclusionsEnzalutamide with venetoclax has an acceptable toxicity profile in patients with mCRPC. Despite sub-optimal venetoclax levels, the treatment elicited pharmacodynamic and clinical response in a subset of patients.Clinical trial IDNCT03751436.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00280-025-04840-2.

BackgroundAt present, healthcare facilities often face blood shortages because of the low supply of donated blood relative to the high demand. Therefore, efforts to develop red blood cell (RBC) production methods have gained traction. In this work, Lin−CD45−CD133+ cells were isolated from human umbilical cord blood (UCB) and subsequently differentiated into erythrocytes in vitro in serum-free culture medium.MethodsLin−CD45−CD133+ cells were prepared from mononuclear cells (MNCs) using magnetic-activated cell sorting (MACS). The characteristics of Lin−CD45−CD133+ cells were confirmed using flow cytometry analysis, colony-forming unit (CFU) assays, morphological analysis, immunocytochemistry (ICC) analysis, and real-time fluorescent quantitative polymerase chain reaction (RT–PCR). Erythrocytes were differentiated in serum-free medium supplemented with stem cell factor (SCF), interleukin-3 (IL-3), erythropoietin (EPO), and FK506 for 13 days, after which autoplasma derived from UCB was added at a concentration of 5% beginning on day 14. Erythroid differentiation and maturation were examined using electron microscopy and flow cytometric analysis.ResultsLin−CD45−CD133+ cells were successfully obtained from UCB. These cells were slightly smaller than normal RBCs and had a high nucleus-to-cytoplasm ratio. Oct-4 and Nanog were expressed at both the mRNA and protein levels in Lin−CD45−CD133+ cells. Most of the colonies were burst-forming unit-erythroid (BFU-E). After 7 days of in vitro culture, the Lin−CD45−CD133+ cells were negative for CD133 expression and positive for CD45 expression. The percentage of CD71+ cells gradually increased, peaked on day 10, and then started decreasing on day 13. The percentage of CD235a+ cells increased gradually after day 7 and peaked on day 13. CD240 expression was detected on day 18, with the highest level detected on day 20. The number of erythroid cells increased persistently during differentiation, and their morphology was consistent with that of normal erythrocytes.ConclusionAn ex vivo culture system was developed that can generate human erythrocytes from Lin−CD45−CD133+ cells isolated from human UCB.

The clinical outcome of SARS-CoV-2 infection spans from asymptomatic viral elimination to lethal COVID-19 pneumonia, which is due to type I interferon (IFN) deficiency in at least 15–20% of cases. We report two unrelated male patients with critical COVID-19 who are heterozygous for rare deleterious variants in RB1CC1, encoding the autophagy-related FIP200 protein. Airway epithelial cells genetically deprived of FIP200 or cell lines expressing the RB1CC1/FIP200 patient variants exhibit elevated SARS-CoV-2 replication and impaired autophagic flux. The antiviral function of FIP200 is independent of canonical autophagy and type I IFN, but involves the selective autophagy receptor NDP52. We identify a non-canonical function of FIP200 in a novel lysosomal degradation pathway, in which SARS-CoV-2 virions are targeted to single-membrane compartments for degradation of viral RNA in LC3B-positive acidified vesicles. This pathway is impaired in FIP200-deficient cells and in cells expressing FIP200 patient haplotypes. Collectively, we describe a cell-autonomous anti-SARS-CoV-2 restriction pathway, dependent on FIP200 and NDP52, and independent of canonical autophagy and type I IFN, which can underlie critical COVID-19 pneumonia. The variability in clinical outcomes of SARS-CoV-2 infection is partly due to deficiencies in production or response to type I interferons (IFN). Here, the authors describe a FIP200-dependent lysosomal degradation pathway, independent of canonical autophagy and type I IFN, that restricts SARS-CoV-2 replication, offering insights into critical COVID-19 pneumonia mechanisms.

ABSTRACTStem cell niches are complex multi‐signalling networks comprised of molecular cues and physical interactions, orchestrated by niche‐resident cells and the extracellular factors they produce. The bone niche specifically houses haematopoietic stem cells (HSCs), a critical cell type responsible for producing all blood and immune cells throughout life. Currently, how niches facilitate an ideal environment with simultaneously coordinating both intrinsic and extrinsic cellular signals is unknown. Studies presented here identify the existence of unique extracellular vesicle (EV)‐defined niches within the haematopoietic system of human individuals. Bridging studies using proteomic signatures, nanoparticle characterization at single‐vesicle resolution and machine learning‐based techniques reveal that EVs can be grouped by blood, bone marrow and trabeculae within a human individual. Stem cell assays demonstrate that these niche‐defined EVs impart functional effects on stem cells/progenitors based on location within the haematopoietic system. Finally, using single‐cell transcriptomic analyses, results identify for the first time how niche‐sourced EVs differentially affect the most primitive human HSCs and progenitors. This study highlights the significance of nanoparticles on human immunity and blood production and provides evidence for a new role for EVs, namely the demarcation of distinct nano‐niches within biological systems.