Cell Reports Medicine

We studied the effect of stimulating innate immune function in tumor-associated myeloid cells (TAMs) in medulloblastoma (MB) and diffuse midline glioma (DMG), using a polyoxazoline nanoparticle formulation of the TLR7/8 agonist resiquimod (ResiPOx). Children with MB and DMG need novel therapeutic strategies to improve outcomes and reduce recurrence. We investigated the effect of systemically administered ResiPOx on TAMs in MB and DMG using endogenous MB and DMG models in immune-competent mice and identified multiple mechanisms of anti-tumor effect. We packaged resiquimod into polyoxazoline micelles to generate ResiPOx. We studied ResiPOx efficacy as a single agent or paired with radiation therapy (RT). We determined ResiPOx pharmacokinetics (PK) using tritium-labeled resiquimod and mass spectroscopy imaging (MSI). We determined ResiPOx pharmacodynamics (PD) using flow cytometry immunohistochemistry, bulk and single-cell RNA-seq and immunoblotting. We then studied ResiPOx safety and PD in a non-human primate model using rhesus macaques. ResiPOx formulation improved the blood-brain barrier penetration and anti-tumor efficacy of resiquimod. ResiPOx treatment extended progression-free survival (PFS) in mice with MB and DMG. In both tumor types, ResiPOx expanded TAM populations and reprogrammed TAMs toward anti-tumoral states, characterized by activation of IFN{beta} and extrinsic apoptosis pathway signaling, antigen presentation, and T cell activation signatures. In rhesus macaques, systemic ResiPOx administration was well tolerated and induced brain transcriptional responses that resembled ResiPOx responses in DMG and MB mouse models, indicating common effects across species from mice to non-human primates, and highlighting potential for similar effects in patients. ResiPOx is a brain-penetrant immunomodulatory therapeutic that reshapes the immune-privileged brain tumor microenvironment. Systemic administration activates myeloid-driven anti-tumoral immunity mediated by microglial and macrophage TAMs, and improves survival in preclinical models of DMG and MB.

GLP-1 receptor agonists have reshaped obesity therapeutics, but their impact on neuropsychiatric outcomes remains poorly characterized. From 29 million patients in a large federated data platform across the USA, including 489,785 semaglutide treated patients, we conducted an observational study integrating longitudinal neuropsychiatric outcomes. From this population, we assembled a cohort of 63,215 patients with baseline neuropsychiatric conditions before treatment initiation and evaluated 24 incident neuropsychiatric outcomes. In propensity-matched comparator analyses, during the 2 year time-period from treatment initiation, semaglutide was associated with broadly lower neuropsychiatric event risk than metformin, SGLT2 inhibitors, and DPP-4 inhibitors. Within the semaglutide-treated cohort, higher attained dose during the first two years after the first prescription ("pre-landmark period") was associated with significantly lower incidence during the following two years ("post-landmark period") of diagnostic codes associated with substance-related disorders (P<0.001), mood disorders (P<0.001), anxiety- and stress-related disorders (P<0.001), CNS atrophies (P<0.001), neuromuscular disorders (P=0.013), eating/sleep/behavioral disorders (P=0.022), and personality/impulse-control disorders (P=0.028). Consistent with previous clinical trials, the post-landmark incidence of dementia or CNS degenerative diseases was similar between the high-dose and low-dose semaglutide cohorts (P=0.15). For most neuropsychiatric diagnoses, post-landmark incidence was strongly associated with the maximum attained semaglutide dose during the pre-landmark period, but incident cognitive symptoms and speech/language symptoms were more closely linked to the pre-landmark weight-loss magnitude (p<0.001 and p<0.003, respectively). Bulk and single-cell transcriptomic analyses demonstrated GLP1R expression in CNS tissues (hypothalamus, caudate, putamen, nucleus accumbens, cerebellum) and peripheral nerves. Age-associated heterogeneity in GLP1R expression was evident in several of these compartments including the caudate nucleus, suggesting dynamic changes in the availability of the neurobiological substrate for semaglutide response. Together, these data support a model in which semaglutide confers a sustained, dose-dependent, weight loss-independent benefit across multiple neuropsychiatric conditions via direct CNS target engagement. This observational study motivates prospective clinical studies and mechanistic analyses to clarify the impact of GLP-1 receptor agonists on human neuropsychiatric pathways and disease processes.

The intestinal microbiome influences immune recovery and long-term outcomes after allogeneic hematopoietic stem cell transplantation (allo-SCT). While reduced bacterial diversity and depletion of immunomodulatory microbial metabolites during peri-engraftment have been linked to acute graft-versus-host disease (aGvHD) and mortality, it remains unclear whether microbiome recovery after engraftment and immune reconstitution is better reflected by bacterial diversity or by microbial metabolic output. We aimed to define microbiome recovery in the late post-transplant period and test whether a metabolite-based biomarker improves the prediction of clinical outcomes, including overall survival (OS) and chronic (c) GvHD. In this two-center longitudinal observational study, serial stool samples were collected from pre-transplant baseline to day +100 after allo-SCT in a discovery cohort (n = 20, Technical University Munich University Hospital (TUM)) and an independent validation cohort (n = 100, University Hospital Regensburg (UKR)). Gut microbiome composition was assessed by 16S rRNA gene amplicon sequencing, with metagenomic profiling in selected patients, and stool metabolites were quantified using targeted mass spectrometry. Patients were classified as RECOVERY or NO RECOVERY based on changes in bacterial richness between baseline and the post-transplant period. To capture microbial metabolic output, the previously established Immune-Modulatory Metabolite Risk Index (IMM-RI), comprising butyric, propionic, and isovaleric acids, desaminotyrosine and indole-3-carboxaldehyde, was adapted to the late post-transplant period (IMM-RI post-TX). Bacterial alpha diversity frequently improved by day +100; however, this did not consistently indicate restoration of baseline community structure and was not paralleled by recovery of stool metabolite profiles. Accordingly, RECOVERY status showed a limited association with survival or transplant-related mortality (TRM). In contrast, IMM-RI post-TX low-risk identified patients with preserved butyrate-associated biosynthetic capacity and was significantly associated with improved OS in both cohorts (UKR: HR 0.2052, 95% CI 0.07703 - 0.5466, p < 0.0001). In the validation cohort, IMM-RI post-TX low-risk was significantly associated with reduced relapse-related mortality. Interestingly, stool butyric-, propionic and valeric acid concentrations were increased in cGvHD of the skin, indicating context-dependent metabolite effects. These findings suggest that metabolite profiling outperforms bacterial diversity for predicting outcomes after allo-SCT and support microbial metabolites as promising biomarkers for risk stratification and actionable candidates for precision microbiome interventions after allo-SCT.

For mRNA-based cancer gene therapy, we engineered a membrane-bound fusion protein combining interferon-{gamma} (IFN{gamma}) with the Fas intracellular domain (FasICD) to couple local IFN{gamma} signaling with Fas-driven apoptotic tumor cell death. IFN{gamma}-FasICD was robustly expressed on the plasma membrane after mRNA transfection. In murine cancer cell lines, IFN{gamma}-FasICD mRNA reduced viability within 24 h, resulting in [~]50% cell death in MC38 cells and [~]75% in B16OVA cells, exceeding the cytotoxicity of the FasICD-deleted control (IFN{gamma}-Fas{Delta}). Mechanistically, IFN{gamma}-FasICD induced predominantly apoptotic rather than necrotic cell death. IFN{gamma}-FasICD also activated IFN{gamma} receptor signaling in both cancer and the immune cells, inducing IFN{gamma}-responsive genes in IFN{gamma}R-high B16OVA cells and triggering STAT1 phosphorylation in co-cultured splenocytes. For in vivo delivery, IFN{gamma}-FasICD mRNA was formulated in lipid nanoparticles (LNPs), enabling strong intratumoral expression that peaked at [~]3 h and persisted for more than 48 h. Repeated intratumoral injections of LNP-formulated IFN{gamma}-FasICD mRNA suppressed the growth of established B16OVA and MC38 tumors and improved survival, with [~]40% and [~]20% of mice surviving beyond 30 days, respectively. IFN{gamma}-FasICD treatment remodeled the tumor microenvironment by increasing tumor-infiltrating CD45+ cells and CD8+ T cells, while further reducing FOXP3+ regulatory T cells. Moreover, NK/NKT cells and cDC1/cDC2 populations were increased, and their activation was enhanced. In tumor-draining lymph nodes, IFN{gamma}-FasICD mRNA promoted dendritic cell migration and increased priming and differentiation of CD8+ T cells toward effector and memory phenotypes, accompanied by enhanced functional activation of IFN{gamma}-producing CD8+ T cells and highly cytotoxic NK cells in peripheral blood. Overall, our findings provide a mechanistic foundation for cytokine-death receptor fusion proteins as an in vivo antitumor strategy that can reprogram tumor cells into localized sources of both apoptotic signals and immune-activating cues.

BackgroundCoronavirus outbreaks remain a persistent threat to global health, and vaccines based primarily on spike-specific immune responses are susceptible to antigenic variation. T-cell immunity directed against conserved internal viral proteins may provide a complementary and more variant-tolerant strategy for next-generation coronavirus vaccines. MethodsWe combined machine learning-guided antigen prioritization with ex vivo functional immunological validation to identify conserved non-spike T-cell targets across betacoronaviruses. Candidate sequences were screened for immunogenicity using primary human peripheral blood mononuclear cells from healthy donors using intracellular cytokine staining and activation-induced marker assays. Top-ranked conserved regions were incorporated into multiepitope mRNA constructs, and their intracellular expression and HLA class I presentation were confirmed by immunopeptidomics. Immunogenicity was further evaluated ex vivo and in vivo using mRNA immunization of mice and T-cell FluoroSpot assays. FindingsAcross a panel of 97 peptides derived from 19 viral proteins, evolutionary conservation across distinct betacoronavirus taxa was strongly associated with functional T-cell immunogenicity in human donors. Highly conserved peptides elicited significantly stronger and more frequent CD4 and CD8 T-cell responses than taxon-restricted peptides. Multiepitope mRNA constructs encoding conserved regions were efficiently expressed and presented on HLA class I molecules and induced T-cell responses in human PBMCs. In mice, mRNA immunization with conserved multiepitope constructs generated robust interferon-{gamma}- and interleukin-2-producing T-cell responses that exceeded those induced by unconserved control constructs. InterpretationThese results link evolutionary conservation to functional cellular immunogenicity and demonstrate the feasibility of multiepitope mRNA delivery for inducing conserved coronavirus-directed T-cell responses. Although protective efficacy remains to be established, conservation-guided antigen selection represents a scalable strategy for developing T-cell-focused vaccines with broad lineage coverage, supporting pandemic preparedness beyond spike-centered immunity. FundingThe research was supported by CEPI, NEC, University of Oslo and Oslo university hospital. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPrior coronavirus vaccine development has focused predominantly on spike protein-directed neutralizing antibodies. While highly effective against matched strains, spike-centered immunity is vulnerable to antigenic drift and lineage-specific escape. Multiple observational and experimental studies have shown that T-cell responses, particularly against internal viral proteins, are more conserved and correlate with reduced disease severity and cross-variant recognition. Epitope prediction algorithms and immunoinformatics approaches have been widely used to nominate candidate T-cell targets; however, systematic functional validation of conserved non-spike antigens across betacoronaviruses in primary human immune systems, combined with antigen presentation data and in vivo vaccine testing, has remained limited. Searches of PubMed and bioRxiv up to December 2025 using terms including "coronavirus T-cell vaccine," "conserved coronavirus epitopes," "betacoronavirus cross-reactive T cells," and "mRNA T-cell vaccine" identified studies demonstrating cross-reactive T-cell immunity and computational epitope selection, but few integrated machine-learning-guided antigen prioritization with ex vivo human functional screening, immunopeptidomics, and in vivo mRNA immunization in a unified workflow. Added value of this studyThis study provides an integrated experimental and computational framework for identifying and validating conserved non-spike T-cell antigens across betacoronaviruses. We functionally screened a panel of candidate peptides derived from multiple viral proteins and demonstrated that evolutionary conservation across species is strongly associated with T-cell immunogenicity. We further demonstrate that multiepitope mRNA constructs encoding these top-ranked conserved regions can be intracellularly expressed, presented on HLA class I molecules to induce polyfunctional T-cell responses in primary human PBMCs. Finally, in vivo mRNA immunization in mice induces robust interferon-{gamma} and interleukin-2 T-cell responses exceeding those induced by unconserved control constructs. Together, these findings link evolutionary conservation to functional cellular immunogenicity and extend beyond in silico prediction by demonstrating antigen processing, presentation, and immunogenicity across human and murine systems. Implications of all the available evidenceCollectively, the available evidence indicates that T-cell immunity directed toward conserved internal coronavirus proteins represents a complementary and potentially more variant-tolerant axis of vaccine design than spike-only strategies. Our findings suggest that evolutionary conservation can serve as a practical selection principle for prioritizing T-cell antigens with broad lineage coverage and that multiepitope mRNA delivery is a feasible platform for inducing such responses. While direct protection and heterologous challenge studies will be required to establish clinical efficacy, the integration of computational prioritization with functional validation supports a scalable approach to pandemic preparedness that may be applicable to other rapidly evolving viral families.

Semaglutide is often optimized for weight loss, but whether longer-term cardiovascular benefit tracks achieved weight loss or therapeutic exposure levels remains unclear. Using a federated deidentified U.S. electronic health record network of 29 million patients, including 505,874 semaglutide-treated individuals, we leveraged multimodal AI technologies to analyze 47,199 patients with baseline cardiovascular disease. We quantified dose escalation and weight change during the 0-2-year period after semaglutide initiation (landmark period) and assessed cardiovascular outcomes during the 2-4-year period (post-landmark). In propensity-matched comparisons during the landmark period, semaglutide was associated with lower cardiovascular events than metformin, DPP-4 and SGLT2 inhibitors. Higher maximum semaglutide dose was associated with greater weight loss during the landmark period (3.15% additional weight loss per 1 mg increase; r=0.97, P<0.001), and lower post-landmark risk of all-cause mortality (RR 0.42, p<0.001), composite cardiovascular events (death, myocardial infarction, or stroke; RR 0.51, p<0.001), cerebrovascular disease (RR 0.50, p<0.001), heart failure (RR 0.55, p<0.001), and valvular/rheumatic heart disease (RR 0.71, p=0.025). In contrast, greater achieved weight loss during the landmark period did not show a consistent monotonic association with lower post-landmark cardiovascular risk (All-cause mortality p-value=0.14, composite cardiovascular endpoint p-value=0.55). Integrating insights from a single cell GLP1R expression atlas was used to infer how semaglutide pharmacology may tie into heart-specific signaling, beyond what is reflected by body-weight reduction alone. The strongest prevalence-weighted GLP1R signal was observed in the pancreas, followed by the heart, where GLP1R engagement potential (GEP) was considerable across cardiomyocyte, cardiac endothelial, and rarer immune cell populations. Together, semaglutide cardiovascular benefit appears organized more by maximum dose attained than by achieved weight-loss magnitude, setting the stage for beyond-obesity trial designs that integrate whole-body spatial intelligence.

GLP-1 receptor agonists induce substantial weight loss, but the extent to which lean tissue and physical function are preserved in routine care remains poorly understood. Using an EHR-linked body-composition digital phenotyping pipeline with LLM-based extraction, we performed a large-scale longitudinal analysis of 670,422 first-episode GLP-1RA users, including 456,742 treated with semaglutide and 213,680 treated with tirzepatide. Among these, 7,965 individuals with paired pre- and post-initiation body-composition measurements were analyzed over 12 months. Tirzepatide was associated with greater relative lean body mass (LBM) loss than semaglutide at each measured time point, with excess LBM losses of 1.1%, 1.5%, 1.3% and 2% at 3, 6, 9 and 12 months, respectively. A Depletive GLP-1 metabotype, defined as >20% total body weight (TBW) loss with >5% LBM loss, was significantly more frequent with tirzepatide than semaglutide during the first year of therapy (10.3% versus 6.7%, p<0.001). By contrast, a Prime GLP-1 metabotype, defined as >10% TBW loss with <5% LBM loss, was numerically more frequent with semaglutide than tirzepatide, but not significantly so (12.3% versus 11.8%, p=0.66). Higher drug dose and longer exposure were associated with progressively greater LBM decline in both treatment groups (both p<0.001). Among 3,746 examined EHR phenotypes, baseline musculoskeletal pain emerged as the most significant correlate of greater LBM loss (BH-adjusted q<0.001): cervicalgia (semaglutide, -4.1 percentage points; tirzepatide, -14.3 percentage points) and knee pain (semaglutide, -4.8 percentage points; tirzepatide, -13.4 percentage points), consistent with mobility-limited patients being more vulnerable to lean-tissue depletion during incretin therapy. Analysis of EHR notes for on-treatment functional features showed reduced exercise tolerance was the strongest correlate of greater LBM loss, increasing by 7.2 and 11.1 percentage points in semaglutide- and tirzepatide-treated patients, respectively. An independent analysis of all available Single-cell RNA-seq data from human musculature showed broader GIPR+ cellular distribution than GLP1R+ cells across immune, stromal, vascular, and contractile compartments, providing plausible biological context for the greater LBM loss observed in routine care with tirzepatide (dual GLP1R-GIPR agonist) relative to semaglutide (GLP1R-specific agonist). In this observational study, greater weight-loss efficacy did not necessarily translate into more favorable body-composition outcomes, underscoring the need for clinical decision-making and trial designs that maximize each patient's likelihood of achieving a Prime GLP-1 metabotype.

Mucinous Ovarian Carcinoma (MOC) is a rare ovarian cancer histological subtype with distinct pathology, genomics and clinical outcomes compared to other epithelial ovarian cancers. Accordingly, there is little evidence to guide clinical care, particularly in the use of systemic therapies, and the field has lacked informative and diverse pre-clinical models. We developed MOC-specific methods for generating tumour organoids with a success rate of 70% for long-term cultured lines (n=19). Organoid lines were developed from localised, advanced and recurrent tumours, including from biopsy tissue, and represent diverse genomic features not previously captured by existing cell lines. The organoid lines were highly similar to the tumours of origin for genomic and immunohistochemical markers. Screening using a panel of 11 chemotherapy agents highlighted resistance to standard-of-care agents such as carboplatin. Gastrointestinal cancer chemotherapy agents and their combination regimens lacked activity. Paclitaxel was often highly potent at low doses but failed to kill all cells. However, less frequently used drugs such as gemcitabine, topotecan and doxorubicin inhibited many of the lines more effectively than paclitaxel. Available, but non-standard of care, chemotherapy agents should be considered for clinical management of MOC. This is the largest (by [~]10 fold) cohort of fully characterised patient-derived MOC organoid lines described and the first with extensive drug screening data affording an opportunity for drug discovery and screening for personalised treatment.

BackgroundFluoropyrimidines, specifically 5-fluorouracil (5-FU), remain the cornerstone of colorectal cancer (CRC) therapy. However, intrinsic and acquired resistance, alongside dose-limiting systemic toxicities, often result in treatment failure and disease relapse. There is a pressing clinical need for next-generation fluoropyrimidines that can retain the antitumor activity in 5-FU-refractory CRC models while maintaining a favorable safety profile. MethodsWe evaluated the antitumor efficacy of CF10, a novel polymeric fluoropyrimidine designed for the sustained delivery of FdUMP, against equimolar 5-FU. We utilized a diverse panel of six patient-derived CRC organoid (PDO) models to assess 3D growth inhibition under both normoxic ([~]20% O2) and physioxic (5% O2) conditions. Mechanisms of action were investigated via {gamma}H2AX signaling (DNA damage), Annexin V/PI flow cytometry (death kinetics), and ALDEFLUOR assays (stem-like populations). Functional suppression of metastasis-associated phenotypes was evaluated using 3D Matrigel invasion assays. Finally, the therapeutic index and overall survival were validated in vivo using two independent patient-cell-derived xenograft (PCDX) models (TX-CC-199 and TX-CC-201). ResultsCF10 demonstrated significantly greater suppression of organoid growth compared to equimolar 5-FU across all patient-derived lines, regardless of morphological heterogeneity or oxygen tension. In 3D invasion assays, CF10 achieved superior anti-invasive activity even at a 10-fold lower molar dose than 5-FU. This functional advantage was mirrored by a marked depletion of the ALDH-high stem-like subpopulation, which was largely recalcitrant to 5-FU. Mechanistically, CF10 induced intensified replication stress, DNA damage and repair signaling ({gamma}H2AX, Top1cc/pRPA32, FANCD2), and pushed the CRC to irreversible/terminal, PI-positive death states. In vivo, CF10 treatment resulted in profound tumor growth inhibition and a robust survival advantage in two patient cell-derived xenograft (PCDX) models (Log-rank P<0.01) without inducing systemic weight loss or noticeable toxicity. ConclusionsBy integrating 3D patient-derived modeling with in vivo validation, we demonstrate that CF10 effectively overcomes the biological and pharmacological limitations of 5-FU. CF10 targets the aggressive, invasive, and stem-like subpopulations of CRC that drive clinical relapses. These findings provide a compelling translational rationale for the clinical development of CF10 as a superior alternative to standard fluoropyrimidines in both treatment-naive and refractory CRC. Significance StatementDespite the foundational role of 5-fluorouracil (5-FU) in colorectal cancer (CRC) therapy, resistance and systemic toxicity remain major barriers to curative outcomes. This study identifies CF10, a novel polymeric fluoropyrimidine, as a superior alternative that overcomes 5-FU resistance in biologically diverse patient-derived organoids and xenograft models. Crucially, CF10 demonstrates a unique capacity to suppress the invasive, aldehyde dehydrogenase (ALDH)-high stem-like subpopulations that likely survive standard chemotherapy (5-FU) by maintaining efficacy under physiological oxygen levels and providing a significant survival advantage in vivo with improved tolerability. CF10 represents a promising translational candidate for the treatment of both treatment-naive and refractory CRC.

Semaglutide has shown benefit in metabolic dysfunction-associated steatohepatitis (MASH), but real-world evidence across longitudinal liver phenotypes remains limited, particularly regarding how liver remodeling relates to weight loss and dose exposure. Using a de-identified federated electronic health record network spanning more than 29 million patients in the United States, including 489,785 semaglutide-treated adults, we analyzed 6,734 patients with baseline liver disease burden. We find that higher attained pre-landmark (0-2 years) semaglutide dose was associated with lower post-landmark (2-4 years) risk of steatohepatitis, alcoholic liver disease, and all-cause mortality, whereas greater pre-landmark weight loss was associated with lower post-landmark risk of steatohepatitis, steatotic liver disease, and hepatorenal syndrome, indicating distinct dose- and weight-linked patterns of long-term liver benefits. These associations were notable because semaglutide prescribing was generally lower during the post-landmark period, raising the possibility of durable benefit beyond peak exposure. Towards better understanding mechanistic bases for liver protection, we performed a complementary longitudinal study of 326 adults with paired noninvasive liver elastography measurements before and after treatment initiation. Median liver stiffness decreased from 4.85 [3.02 - 7.20] to 3.9 [2.6 - 5.8] kPa after semaglutide initiation (median change = -0.38 kPa; p<0.001), with 194 of 326 patients (59.5%) showing lower follow-up stiffness. A clinically meaningful reduction of at least 20% was observed in 133 of 326 patients (40.8%), and 69 of 326 (21.2%) shifted to a lower fibrosis stage by prespecified elastography thresholds. Larger improvements were also seen in patients with higher baseline stiffness (p<0.001); notably 80% of patients with cirrhosis-range baseline stiffness ([&ge;]12.5 kPa) achieved [&ge;]20% improvement versus 29.5% with minimal baseline disease (p <0.001). The proportion achieving at least 20% stiffness improvement was similar across weight-loss strata, including patients with no weight loss or weight gain and those with at least 10% weight loss (38.0% in each group), and liver stiffness change showed negligible correlation with changes in weight, BMI, HBA1c, alanine aminotransferase, or aspartate aminotransferase. To provide biological context, single cell RNA analyses demonstrated sparse overall hepatic GLP1R expression (0.0239%), with enrichment in non-parenchymal niches including cholangiocytes, intrahepatic cholangiocytes, liver sinusoidal endothelial cells, and hepatic stellate cells implicated in fibrogenesis and vascular remodeling. Together, this real-world evidence suggests diverse liver benefits for semaglutide beyond weight-loss with intricate dose response relationships.

Mechanistic understanding and biomarkers of gonadotropin-releasing hormone antagonist treatment effect in paedophilic disorder are absent but may enhance outcomes and reduce sexual-offending risk. 52 help-seeking self-referred Swedish men with paedophilic disorder enrolled in a double-blinded, placebo-controlled, randomized clinical trial. Participants underwent task-based fMRI before, and two weeks after, subcutaneous injection of 120mg of degarelix or equal volume of placebo. fMRI blood-oxygen-level-dependent activation was compared between child and adult (child>adult) stimuli in task-derived regions of interest. Primary outcome was within region-of-interest child>adult activation change, whereas secondary outcomes correlated region-of-interest child>adult activation change to change in clinical measurements of risk, paedophilic interest, sexual preoccupation, hyper- and hyposexuality. 19 degarelix and 22 placebo participants had sufficient fMRI data quality. Reductions in paedophilic interest were strongly correlated with increased child>adult cerebellar (vermis) region-of-interest activation following degarelix (r=-0.740, p<0.001) but not placebo (r=0.183, p=0.41; between-group correlation coefficient z=3.347, p<0.001). Treatment did not significantly change child>adult region-of-interest activity. Post hoc analysis indicated that baseline autism symptoms correlated with degarelix-induced changes in paedophilic interest (r=0.717, p<0.001; between-group correlation coefficient z=2.958, p=0.003) and cerebellar activation (r=-0.581, p=0.01; between-group correlation coefficient z=-1.930, p=0.05). Increased child>adult cerebellar activation was associated with degarelix-induced reductions of paedophilic interest, suggesting cerebellar activity as mechanistically important to, and a prospective biomarker of, degarelix treatment effect. Additionally, autism symptoms may inform treatment prediction. Together, these findings have mechanistic and clinical implications for degarelix treatment of paedophilic disorder. EU clinical trials register identifier: 2014-000647-32 https://www.clinicaltrialsregister.eu/ctr-search/trial/2014-000647-32/SE, registered on 05/06/2014.

High-grade serous ovarian carcinoma (HGSC) is an aggressive malignancy for which bulk transcriptomic subtypes are used to stratify tumors, interpret biology, and guide biomarker development. The four TCGA-derived subtypes, mesenchymal (C1.MES), immunoreactive (C2.IMM), proliferative (C5.PRO), and differentiated (C4.DIF), are consistently observed across cohorts. However, despite their prominence, these subtypes have not translated into therapeutic utility, and their biological basis remains unresolved. Here, we show that HGSC transcriptomic subtypes are largely determined by tumor cellular composition rather than intrinsic malignant transcriptional programs. By integrating controlled single-cell-derived pseudobulk simulations with deconvolution-based analysis of 1,834 primary HGSC tumors across RNA-seq and microarray cohorts, we demonstrate that subtype probabilities align along a composition-driven axis of stromal and immune variation. Cellular composition alone predicted subtype labels with high accuracy (ROC-AUC = 0.81-0.95) and explained a substantial fraction of subtype-associated transcriptomic variation, with the mesenchymal (C1.MES) subtype representing the most robust and reproducible example of composition-driven signal. Although a secondary, composition-independent expression signal is detectable, it does not define the dominant structure of subtype classification. These findings redefine HGSC transcriptomic subtypes as features of the tumor ecosystem rather than discrete malignant states. This reinterpretation has immediate implications for studies that use subtype labels to infer tumor-intrinsic biology and provides a generalizable framework for separating composition-driven and intrinsic signals in bulk tumor data. Significance StatementHGSC transcriptomic subtypes lack consistent clinical utility and remain biologically ambiguous. We show subtype assignments are largely driven by tumor cellular composition, and less so by distinct intrinsic tumor states.

Our understanding of human mucosal T cell clonotype distribution in health and disease has centered on immunodominant antigens. We performed single cell T cell receptor (TCR) and RNA sequencing as an untargeted approach to define distributions of T cell clonal groups in health and ulcerative colitis (UC) across 333,088 T cells in colon and peripheral blood. Healthy donor-specific TCR repertoires had limited blood-colon clonal sharing, which was highest in cytotoxic T effector memory (Tem) populations and lowest in regulatory T cells (Tregs), reflecting tissue-based compartmentalization. Within healthy colon, TCR repertoires showed high T cell clonal sharing independent of anatomic distance, associated with high intra-clonal phenotypic diversity. Colon cytotoxic and Th17 populations showed high dispersion across sites, while Tregs were compartmentalized. Clonal lineages dispersed across blood and colon upregulated trafficking markers, suggesting active movement between tissues, while those dispersed across colon sites upregulated residency markers, suggesting intra-colon repertoire sharing is mediated by long-term, slow moving clonal groups. In UC, Tregs were expanded across inflamed sites, and increased CD8 Tem clonal groups showed increased dispersion regardless of inflammation. These findings reveal principles of T cell clonal organization in the human colon during health and disease, identifying opposing patterns of clonal dispersion among Treg and Th17 clonal groups, high phenotypic diversity within dispersed clonal groups, and elevated cross-colon dispersion of CD8 Tem clonotypes in UC.

We previously developed synthetic Notch (synNotch)-chimeric antigen receptor (CAR)-T cells to improve the safety and efficacy of CAR-T therapy for glioblastoma. In this system, an anti-EphA2/IL13R2-dual-CAR is expressed only upon recognition of tumor- or brain-specific "priming" antigens, EGFRvIII (termed E-SYNC cells) or brevican (B-SYNC), respectively, with E-SYNC currently under phase I clinical evaluation (NCT06186401). However, tracking and profiling these engineered cells in vivo remain challenging, limiting our understanding of their activity and therapeutic potential. To address this gap, we developed a single-cell RNA-sequencing (scRNA-seq) workflow with custom spike-in probes for synNotch-CAR transcripts, enabling simultaneous detection of engineered cells and transcriptomic profiling. In vitro, integration of multiple probes using machine-learning-assisted classifiers detected 78.2% of E-SYNC cells and 60.0% of B-SYNC cells with 98.0% specificity. In a xenograft model, synNotch-positive cells were detected across the spleen, lung, and brain, with the highest frequency and most robust priming and activation observed in the brain. Single-cell transcriptomic analyses revealed tissue-specific differentiation programs, including cytotoxicity, proliferation, metabolic activity, and acquisition of tissue-resident memory phenotypes, shaped by both environmental cues and synNotch-mediated antigen recognition. In summary, this spike-in probe-enhanced scRNA-seq workflow enables robust detection and high-resolution characterization of synNotch-CAR-T cell dynamics and provides a broadly applicable platform for monitoring engineered immune cells in diverse clinical contexts. One Sentence SummaryOur spike-in probe-enhanced single-cell RNA-sequencing method enables analysis of tissue-dependent activation and transcriptional states of synNotch-CAR-T cells, providing a robust and scalable platform for in vivo tracking and transcriptomic profiling of engineered cell therapies.

KIF18A inhibition has emerged as a therapeutic strategy for chromosomally unstable cancers, but clinical development is limited by the absence of a deployable predictive biomarker. Here we identify strong, diffuse p16INK4a expression, a well-established surrogate marker of Rb-pathway inactivation, as a predictive biomarker of response to KIF18A inhibition, and show that Rb-pathway inactivation marks a biologically distinct subset of cancers sensitive to this therapeutic approach. In sensitive models, low Rb activity is associated with robust spindle assembly checkpoint signaling and prolonged mitotic arrest following KIF18A inhibition. Weakening the spindle assembly checkpoint in this context is sufficient to confer resistance. Across three independent pan-cancer sensitivity datasets generated with distinct KIF18A inhibitors, Rb-pathway altered models were significantly more sensitive than histology-matched Rb-intact comparators, with the strongest association observed in cancers harboring direct RB1 loss or inactivating mutation. Guided by this mechanism, we retrospectively analyzed p16INK4a expression by immunohistochemistry (IHC) in pre-treatment tumor biopsies from 79 heavily pre-treated high-grade serous ovarian cancer patients across three dose-escalation or expansion cohorts and treated with two different KIF18A inhibitors (sovilnesib and VLS-1488) sharing a common mechanism of action. p16INK4a-high tumors showed substantially higher objective response rates than their p16INK4a-low counterparts (36.0% versus 2.2%; P = 0.0002) and markedly longer progression-free survival (median 24.3 versus 7.9 weeks; hazard ratio, 0.16; P < 0.0001). These findings establish p16INK4a as a mechanistically-based, clinically implementable biomarker of clinical response to KIF18A inhibition that is poised to support pan-cancer development of KIF18A inhibitors guided by Rb-pathway inactivation.

Antibody-drug conjugates (ADCs) require high and homogeneous target expression for optimal efficacy, yet the spatial, temporal, and cellular heterogeneity of clinically approved ADC targets in high-grade serous ovarian cancer (HGSC) remains incompletely defined. We analyzed bulk RNA-sequencing, single-cell RNA-sequencing, and whole-genome sequencing data from 867 samples across 304 patients enrolled in the real-world DECIDER cohort to systematically evaluate 11 approved ADC targets. FOLR1, TACSTD2, and ERBB2 emerged as highly expressed candidates. Inter-patient variability exceeded intra-patient heterogeneity, which further decreased following neoadjuvant chemotherapy. Target expression was highly concordant across anatomical sites and largely stable from diagnosis to relapse. Single-cell RNA-sequencing results revealed that TACSTD2 and FOLR1 showed the most frequent cancer cell-restricted expression. In rare cases of gene amplification, ERBB2 and F3 emerged as potential targets alongside TACSTD2 and FOLR1. Overall, 80% of patients displayed homogeneous expression of at least one actionable target, with frequent co-expression of TACSTD2 and FOLR1. These findings indicate that ADC target expression in HGSC is broadly stable across space and time and support the prioritization and strategic integration of TACSTD2- and FOLR1-directed ADCs in this disease.

Elevated plasma trimethylamine N-oxide (TMAO) is an independent predictor of major adverse cardiovascular events and ischemic stroke. While inhibition of microbial TMA production has been explored, concerns regarding off-target effects and limited efficacy in complex microbial ecosystems have hindered clinical translation. Here, we report a microbiome-based therapeutic strategy based on the direct enzymatic degradation of intestinal TMA by Paracoccus aminovorans BM109. Through targeted screening, we identified BM109 as a commensal strain harboring a comprehensive set of enzymes capable of metabolizing TMA and TMAO into non-toxic end products under both aerobic and anaerobic conditions. In a chronic high-choline diet murine model, oral administration of BM109 resulted in a 38% reduction in systemic TMAO levels. In a rat model of transient middle cerebral artery occlusion (tMCAO), short-term pre-treatment reduced cerebral infarct size by 58% and significantly improved neurological outcomes. These effects were accompanied by favorable safety observations, including the absence of hemolytic activity and intestinal tissue damage. Collectively, our findings establish BM109 as a promising live biotherapeutic product that targets the gut microbiome-host metabolic axis. By reducing the systemic TMAO burden, BM109 represents a potential strategy for modulating cardiometabolic and cerebrovascular risk.

Neonatal sepsis caused by Klebsiella pneumoniae is a major cause of under-five mortality in sub-Saharan Africa, and the rapid increase of infections caused by bacteria resistant to most or all available antimicrobials severely limits treatment options. An effective, maternally-administered vaccine could make a substantial reduction in neonatal sepsis and associated negative outcomes, as well as reduce the overall need for antimicrobials, a key driver of antimicrobial resistance. This study explored the potential for a maternally administered protein-based vaccine to provide neonatal protection via antibodies transferred transplacentally and through breastfeeding. A case-control study of mother and baby dyads was designed with 20 neonates developing K. pneumoniae sepsis and 80 uninfected control neonates to analyse breastmilk IgA, cord blood IgG and maternal serum IgA and IgG antibodies on a protein microarray with 161 selected K. pneumoniae proteins representing 152 unique genes. This analysis identified a set of proteins eliciting antibody responses, some associated with lack of K. pneumoniae sepsis, that indicate the presence of potentially protective antibodies. This is an essential first step in exploring surface protein accessibility, despite the large capsule. We highlight fimbrial structures, conjugative pili, and small lipoproteins associated with large outer membrane complexes as potential protein vaccine targets.

1.The ABACUS study was a single arm, phase II trial evaluating neoadjuvant atezolizumab in operable urothelial carcinoma (UC). Initial bulk transcriptomic and immunohistochemistry analyses suggested links between immune activation, tissue remodeling, and resistance pathways such as transforming growth factor {beta} (TGF{beta}) that were associated with clinical outcome. To further characterize spatial and phenotypic changes at high resolution, artificial intelligence-assisted digital image analysis of hematoxylin and eosin sections and spatial transcriptomics (10x Genomics Visium) were performed on paired tissue samples. In baseline samples, cells residing in lymphoid aggregates and tertiary lymphoid structures (LAs/TLSs) were more abundant in stable disease than in relapse and exhibited gene expression programs associated with improved survival in UC. Most spatial features reflected shared pharmacodynamic changes between stable disease and relapse; however, carcinoma-endothelial adjacency was reduced significantly following treatment and differed between groups, accompanied by distinct transcriptional programs. Together, these findings indicate that atezolizumab induces localized immune and stromal remodeling within the tumor microenvironment, while non-response despite immune expansion is associated with persistent spatial immune exclusion and carcinoma-endothelial adjacency. Spatial and phenotypic biomarkers identified here may inform rational combination strategies for immune checkpoint inhibitor-refractory urothelial carcinoma.

Hypoxic-ischemic encephalopathy (HIE) is a neonatal brain injury in which a definitive diagnosis within the first 6 hours of life is essential for initiating therapeutic hypothermia and improving neurological outcomes. However, early clinical evaluation and currently available biomarkers lack quantitative specificity during this narrow therapeutic window. We are providing insights into how rapid disturbances in systemic metabolites that regulate cerebral energy metabolism, neurotransmitter cycling, redox balance, and membrane integrity generate a definitive biochemical signature of HIE immediately after birth. We performed quantitative blood-based 1H NMR metabolomics on collected blood samples within [~]1 hour of birth from 81 neonates (HIE, n = 42; non-HIE, n = 39), under optimized handling conditions to preserve metabolic integrity. Metabolite analysis revealed a distinct HIE-associated profile characterized by elevated lactate, alanine, succinate, glutamate, taurine, glycine, choline, and pyroglutamate, alongside significant depletion of glucose and glutamine compared with non-HIE controls (p < 0.05). These coordinated metabolic shifts reflect impaired mitochondrial respiration, enhanced anaerobic glycolysis, excitotoxic amino acid accumulation, altered membrane phospholipid turnover, and oxidative stress. Multivariate analysis demonstrated clear separation between groups (PLS-DA accuracy = 0.83, R{superscript 2}Y = 0.46, Q{superscript 2} = 0.82), with glutamine, lactate, glutamate, and pyroglutamate as key discriminators. Pathway enrichment highlighted perturbations in glycolysis, the glucose-alanine cycle, glutamate-glutamine metabolism, Warburg effect like metabolic reprogramming, and redox homeostasis. Integration into supervised machine-learning models (Random Forest, XGBoost, SVM, KNN) achieved strong diagnostic performance (AUC = 0.97 {+/-} 0.03; sensitivity {approx} 87%). Collectively, this minimally invasive NMR-to-machine-learning framework enables early, mechanistically grounded risk stratification of neonatal HIE within the therapeutic window.