Cell Reports Medicine

Severe preeclampsia (sPE) is a major cause of maternal and fetal morbidity worldwide, yet its placental molecular heterogeneity remains poorly defined by current clinical diagnosis. To resolve the molecular architecture of sPE, here we integrated DNA methylation and proteomic profiling from a multi-ethnical cohort of 444 placentas from the Hawaiian Biorepository (HiBR), including 169 sPE cases, matched preterm controls and full-term controls. To address cellular heterogeneity in bulk placental tissue, we developed HOMED (Hierarchically Optimized Methylation Deconvolution), a single-cell-guided hierarchical framework for inferring placental cell-type composition from DNA methylation data. HOMED-adjusted integrative analyses identified extensive subtype-specific alterations involving hypoxia, angiogenesis, immune activation, trophoblast differentiation and metabolic remodeling. Molecular stratification revealed two reproducible sPE subtypes with divergent placental aging trajectories. One subtype exhibited a pre-mature placental state marked by accelerated placental aging, whereas the other displayed slower accelerated placental aging but a substantially increased risk of small-for-gestational-age birth (P = 0.028). These subtypes were independently replicated across six external cohorts and further supported by proteomic signatures achieving a classification accuracy of 0.88. Integrative epigenomic and proteomic analyses linked the growth-restricted subtype to hypoxia-associated glycolytic remodeling, suggesting distinct pathogenic mechanisms underlying clinically diagnosed sPE. Together, our findings redefine severe preeclampsia as a biologically heterogeneous placental disorder composed of molecularly distinct subtypes with divergent aging trajectories and fetal growth outcomes, providing a framework for mechanism-based stratification and precision obstetric medicine.

Alpha-gal Syndrome (AGS) is a potentially life-threatening allergy caused by an IgE-mediated immune response to galactose--1,3-galactose (alpha-gal), a carbohydrate epitope present in most mammalian meats. Currently, strict avoidance of mammalian meat remains the primary management strategy for affected individuals, and alpha-gal-free beef is not commercially available. Here, we leverage cultivated meat as a biotechnology plat-form to address this unmet clinical need by engineering alpha-gal-free bovine muscle cells. Using CRISPR/Cas9 genome editing, we disrupted GGTA1, the gene encoding 1,3-galactosyltransferase, in immortalized bovine satellite cells (iBSCs). High-efficiency editing produced clonal GGTA1 knockout iBSCs harboring a homozygous frameshift mutation. Flow cytometry and immunofluorescence confirmed loss of the alpha-gal epitope, while bulk RNA-seq indicated minimal disruption of global gene expression and preserved myogenic differentiation capacity. Importantly, lysates from GGTA1 knockout iBSCs elicited substantially reduced basophil activation in assays using plasma from a patient with AGS, indicating reduced basophil activation consistent with reduced allergenic potential. Together, these findings establish a proof of concept for engineering AGS-compatible cultivated meat and demonstrate the potential of cultivated meat technologies to address human health challenges. HIGHLIGHTS{circ} CRISPR/Cas9-mediated disruption of GGTA1 eliminated alpha-gal from bovine satellite cells {circ}GGTA1 knockout cells retained myogenic identity and differentiation capacity {circ}GGTA1 knockout reduced basophil activation in an alpha-gal syndrome immune assay {circ}Genome-edited bovine cells provide a proof of concept for AGS-compatible cultivated meat

To better understand how pregnancy impacts humoral immunity, we conducted an in-depth longitudinal analysis of the kinetics and characteristics of vaccine responses in a prospective cohort of pregnant and non-pregnant women. Humoral immune responses observed among pregnant participants who received the mRNA-delivered SARS-CoV-2 vaccination, including their effector functions, were in some cases marginally lower than those among non-pregnant controls, while prior infection was associated with some potentiation in humoral responses. Importantly, vaccine-induced antibodies were efficiently transferred across the placenta, providing the fetus with passive immunity and underscoring the dual benefit of maternal vaccination for both mother and neonate against COVID-19. Delayed induction of spike-specific IgG4 following the primary two-dose vaccination series was observed in vaccine recipients, independent of pregnancy status. In a subset (n=6) of pregnant women whose spike-specific serum IgG repertoires were extensively profiled at the clonotypic level over time as part of another study, we proteomically identified secreted IgG clonotypes that had class-switched to IgG4. Matching of these clonotypes detected as IgG4 to those defined as SARS-CoV-2 spike-specific revealed that, while a minority of total clonotypes, they were elicited early in the immunization series and tended to be more highly mutated, more prevalent, and more persistent than clonotypes in the serological repertoire that were not detected as IgG4. Consistent with the increase in secreted vaccine-specific IgG4 over time, but its poorer placental transfer, these clonotypes were detected at greater levels in maternal but not cord blood at the time of delivery as compared to 28 days post the second vaccine dose. These findings indicate some impact in the kinetics, characteristics, and functions of the humoral response that may be associated with pregnancy-related immune modulation. Conservation of the late class-switch recombination to IgG4 that has previously been associated with mRNA-based SARS-CoV-2 vaccines raises questions about how different immunological states and vaccine components influence short- and long-term characteristics of the humoral immune response.

Subfertility and recurrent pregnancy loss (RPL) affect a significant proportion of couples worldwide. Genetic causes can be seen in up to 30% of these individuals but require multiple genetic tests, which often impede a comprehensive work up. Newer genomic technologies, such as PacBio HiFi long read sequencing (LRS) can detect most subclasses of variations (such as structural rearrangement, monogenic disorders) through one single test. In this multicenter study, we enrolled couples with unexplained subfertility and/or RPL and performed HiFi LRS to determine the underlying genetic etiology. Participants were recruited using a standardized inclusion/ exclusion criteria to rule out other known causes of subfertility and/or RPL. 96 individuals were recruited across the 5 sites. Average age of participants was 36 years (range 30-46 years). Among the 84 individuals who completed sequencing, 4.8% were identified with a likely genetic diagnosis and variants of uncertain significance were identified in another 14.2% of individuals. One individual was identified with an ACMG secondary finding, and while multiple carriers for recessive genetic disorders were identified, none of the couples were identified to be at increased risk. This study highlights the utility of performing genomic sequencing in couples with unexplained subfertility and/or RPL, with 1 in 10 couples harboring a clinically significant variant. In addition, use of HiFi LRS allowed for characterization of different subclasses of genomic variations through a single test. Future studies, including exploring the cost effectiveness and resource utilization of LRS as first line test, will help in optimizing care for such couples. TWEETABLE STATEMENTA single long-read genome sequencing test can consolidate multiple genetic investigations and uncover clinically relevant causes in couples with unexplained subfertility and recurrent pregnancy loss. AT A GLANCEO_LIWhy was this study conducted? O_LIMany couples with subfertility and recurrent pregnancy loss remain undiagnosed after multiple conventional genetic tests C_LIO_LIExisting workflows require sequential testing and may miss complex genomic variants C_LI C_LIO_LIWhat are the key findings? O_LILong-read genome sequencing identified clinically relevant variants in [~]1 in 10 couples with unexplained subfertility or recurrent pregnancy loss C_LIO_LIA single assay enabled detection of multiple variant types, including structural and sequence variants C_LI C_LIO_LIWhat does this study add to what is already known? O_LIDemonstrates feasibility of a unified genomic testing approach in a real-world multicenter cohort C_LIO_LISupports a potential shift from fragmented testing toward a single comprehensive genomic workflow C_LI C_LI

mRNA-lipid nanoparticle (LNP) vaccines are detectable in human blood after vaccination, but platform-specific differences in systemic persistence and transcript integrity remain poorly defined. We analyzed serial blood samples from 73 participants receiving Moderna mRNA-1273 (three formulations), Pfizer/BioNTech BNT162b2, or an investigational receptor-binding domain (RBD) mRNA vaccine (three different doses). Using droplet digital polymerase chain reaction (ddPCR) assays, we quantified total and long-range linked ("intact") vaccine mRNA, and we measured vaccine-specific ionizable lipids by liquid chromatography-mass spectrometry (LC-MS). Across platforms, mRNA decay was fastest for mRNA-1273, intermediate for BNT162b2, and slowest for the RBD vaccine, with ionizable lipid decay following the same rank order. Notably, intact spike mRNA declined two-fold faster after mRNA-1273 than BNT162b2 vaccination. Kinetics modelling revealed platform-dependent coupling of mRNA and lipid kinetics: intact mRNA tracked closely with SM-102 for mRNA-1273, whereas ALC-0315 persisted longer than intact mRNA for BNT162b2. A ten-fragment linkage ddPCR panel spanning the spike transcript showed lower linkage toward 3'-proximal regions that mirrored the administered mRNA-1273 formulation. Together, these data establish a quantitative framework for benchmarking mRNA-LNP platform kinetics and transcript integrity in humans.

Dexamethasone is widely used to control cerebral edema and inflammation in glioblastoma, but its benefits are limited by systemic toxicities and adverse prognostic associations. We evaluated local administration of dexamethasone via convection-enhanced delivery (CED) to maximize intratumoral anti-inflammatory effects by increasing local corticosteroid exposure while minimizing systemic exposure. In two glioma mouse models, continuous intraparenchymal infusion of dexamethasone was well tolerated with no adverse effects. Pharmacokinetic analyses supported preferential intratumoral distribution and reduced systemic exposure with CED compared with systemic dosing. Single-nucleus RNA sequencing (snRNA-seq) and immunohistochemistry showed attenuation of glioma-associated inflammation with downregulation of reactive microglial/macrophage programs and reduced tumor-infiltrating myeloid cells with a morphology consistent with a less activated state. Experiments in human induced pluripotent stem cell (iPSC)-derived microglia confirmed that dexamethasone directly suppresses inflammatory gene expression, indicating a conserved mechanism across species. This inflammatory suppression was recapitulated in both immortalized microglial (HMC3) and macrophage (THP1) cell lines. These findings suggest that localized dexamethasone delivered by CED reprograms the glioma immune microenvironment and achieves control of inflammation without the systemic adverse effects associated with standard systemic dexamethasone therapy. This clinically translatable strategy may improve symptom management and provide a platform for integrating local immunomodulation with future glioblastoma therapies.

Polycystic ovary syndrome (PCOS) is linked to adverse pregnancy outcomes and increased cardiometabolic risk in offspring, yet the placental mechanisms underlying these risks remain poorly understood. Metformin is prescribed during PCOS pregnancies despite limited mechanistic justification. Using multi-modal molecular analyses of placentas from healthy controls and women with PCOS randomized to placebo or metformin (PregMet trial), restricted to uncomplicated pregnancies, we characterized direct PCOS associated placental alterations independent of confounding complications. PCOS placentas showed transcriptional downregulation across multiple cell types and shifts in cell type proportions. Specifically, syncytiotrophoblasts exhibited reduced expression activity of growth hormone receptor signaling and glycosaminoglycan biosynthesis. Endothelial cells displayed diminished receptor tyrosine kinase pathway activity, including VEGFC, despite increased cell proportion and hypervascularity. Intercellular communication networks were globally suppressed, including reductions in PDGF signaling from Hofbauer cells to fibroblasts. Notably, metformin did not reverse most PCOS-associated molecular alterations and induced transcriptional changes correlated to birth weight and childhood BMI. These findings indicate that PCOS-associated placental features are driven by cell type specific dysregulation of growth factor, angiogenic signaling pathways that are largely unresponsive to metformin. This underscores the need to develop mechanism based, placenta targeted therapeutic alternatives for future pregnancy management.

PurposeBispecific T cell engagers (BiTEs) have recently been approved as a locoregional immunotherapy for malignant ascites. Although ascites is recognised as a lipid-rich, immunosuppressive environment, the mechanisms by which ascites, particularly its lipid components, suppress antitumour immunity remain poorly understood. Here, we investigated the impact of ascites-associated lipids on T cell immunosuppression and assessed whether lipid modulation could enhance the efficacy of BiTE therapy. Experimental DesignTranscriptomic profiling was performed on T cells treated with acellular ascites fluid to identify gene expression signatures associated with ascites exposure. Functional assays were conducted to evaluate the effects of ascites-associated lipids on T cell activation and cytotoxicity. In parallel, T cells were cocultured with ovarian cancer cells and EpCAM-targeting BiTEs in the presence or absence of a lipid-removal agent to assess how lipid depletion affected BiTE efficacy. ResultsT cells exposed to acellular ascites fluid exhibited an enriched transcriptomic signature associated with cholesterol efflux and incomplete fatty acid oxidation, which are metabolic features often found in exhausted T cells. These alterations converged on a metabolically imbalanced state linked to impaired plasma membrane signalling. Lipid removal from ascites selectively rescued CD137 expression but not CD25, and restored BiTE-mediated cytotoxicity, suggesting a differential impact of lipid metabolism on TCR complex-dependent versus cytokine-driven activation pathways. ConclusionsThese findings identified lipid as a driver for T cell dysfunction in ovarian cancer ascites. Removal of ascites lipids restored T cell activation and augmented BiTE-mediated cytotoxicity, supporting a combination approach to potentiate BiTE therapy in malignant ascites. Translational RelevanceMalignant ascites represents a lipid-rich, immunosuppressive tumour microenvironment that is increasingly targeted by emerging T cell-based therapies. Although EpCAM-targeting bispecific T cell engagers (BiTEs) have recently been approved for malignant ascites and multiple similar BiTEs are in clinical development, the mechanisms by which ascites impairs T cell function and potentially limits therapeutic efficacy remain poorly understood. Using patient-derived ascites throughout, this study demonstrated that lipid metabolic reprogramming, rather than immune checkpoint upregulation, was a driver of T cell dysfunction. Importantly, we demonstrated that lipid removal from ascites rescued T cell function and restored BiTE efficacy, identifying a targetable metabolic barrier to immunotherapy. While EpCAM was used as a proof-of-concept target, we anticipate the metabolic insights and therapeutic strategies identified here will be equally applicable to other BiTE and CAR-T platforms, supporting a new combination approach for the treatment of malignant ascites.

Patient-derived organoids from breast cancer brain metastases enable real-time drug sensitivity testing integrated with genomic profiling. Drug response varied by subtype and molecular alterations. PI3K inhibitors showed activity regardless of PIK3CA mutation status. Pronounced tumor heterogeneity highlighted the urgent need for effective therapies personalized for each patient. Functional assays and molecular matching can help tailor therapy for patients who need the most effective next treatment quickly and warrant further translational evaluation to address this unmet need.

Head and neck squamous cell carcinoma (HNSCC) remains associated with substantial morbidity and a 5-year overall survival rate of approximately 60%, reflecting persistent radio- and chemo-resistance and the lack of effective precision medicine strategies. Patient-Derived Tumor Organoids (PDTO) constitute promising functional models that may predict individual treatment response. In this study, we generated PDTO from surgically resected HNSCC of the oral cavity, oropharynx, larynx, and hypopharynx. A total of 20 long-term PDTO lines were established, maintaining growth over seven passages and successfully cryopreserved, capturing the molecular and clinical diversity of the patient cohort. These PDTO faithfully recapitulated histological features, major tumor marker expression, and the genomic and transcriptomic landscapes of their tumors of origin, with stability over time. Functional assays revealed heterogeneous responses to cisplatin and X-rays. Importantly, in vitro sensitivity of PDTO was associated with clinical outcome of patients at 24 months. Cisplatin response of PDTO predicted prognosis with 66.7% sensitivity and 100% specificity, while X-ray response showed 91.7% sensitivity and 75% specificity. Notably, all patients whose PDTO were classified as resistant to both cisplatin and X-rays experienced relapse and/or death within 24 months. Collectively, the successful long-term expansion and cryopreservation of HNSCC PDTO establish a stable and scalable preclinical resource that captures the molecular and clinical heterogeneity of the disease. This biobank provides a valuable platform for mechanistic studies and for the evaluation of innovative therapeutic strategies. This cohort represents one of the largest clinically annotated HNSCC PDTO collections to date, demonstrating a robust association between PDTO response to cisplatin and X-rays and patient prognosis. These findings support the predictive potential of PDTO-based functional assays and argue for their integration into standardized, rapid, and miniaturized precision oncology workflows for HNSCC.

BackgroundPrimary and secondary resistance to immune checkpoint blockade (ICB) remains a critical challenge in advanced melanoma. Oncolytic Viruses (OV) selectively lyse tumor cells while generating systemic anti-tumor immune responses with minimal side effects. Yet their clinical use is limited to refractory melanoma patients and are only given in combination with second-line ICB regimens. ICB can both help and hinder OV efficacy depending on the source of checkpoint interactions across the tumor-immune microenvironment (TiME). However, functional checkpoint interactions are typically inferred from gene or protein expression and rarely contextualized within myeloid- and antigen presenting cell-associated immune niches during OV therapy, despite these populations dominating melanoma TiMEs and serving as key regulators of anti-viral immunity. MethodsAn integrated multi-omics framework combining Nanostring GeoMx digital spatial profiling (DSP), COMET sequential immunofluorescence (seqIF) and functional oncology mapping (FuncOmap) was applied to melanoma patient tissues collected pre- and post-neoadjuvant Talimogene Laherparepvec (T-VEC) to characterize immune remodeling and directly quantify checkpoint interaction dynamics associated with pathologic responses to OV therapy. ResultsT-VEC induced broad lymphocyte- and myeloid-associated immune transcriptional activation across melanoma TiMEs; however, pathologic responses could not be defined by bulk transcriptomics or cellular deconvolution alone. COMET seqIF analysis identified that HSV-associated M1/APC-like tumor-associated macrophages (TAMs) were enriched in complete pathologic response (CR) tissues and were a major source of PD-1/PD-L1 interaction niches. While partial (PR) and non-pathologic response (NR) tissues retained melanoma-centered PD-1/PD-L1 interaction niches and were enriched for B cell and M2-like TAM populations. FuncOmap analysis indicated that post-T-VEC PD-1/PD-L1 interaction states were consistently elevated in tumor bed, but not in lymph node tissues, across all pathologic response groups. Suggesting that immune checkpoint interactions may benefit T-VEC therapeutic responses depending on their spatial and immune context relative to OV infection. ConclusionsThese findings highlight the importance of integrated transcriptomic and functional proteomic analyses for resolving the spatial distribution and functional status of immune niches during OV therapy. Resolving PD-1/PD-L1 interaction states to specific M1/APC-like TAM and B cell niches may define mechanisms of responses and resistance to OV therapy.

BRAF-targeted therapy (BRAFi/MEKi) and immune checkpoint blockade (anti-PD-1/anti-CTLA-4) have transformed the treatment of BRAF-mutant metastatic melanoma. While most patients who respond to targeted therapy eventually progress, a subset derives durable benefit, and biomarkers to identify this subset would inform optimal treatment selection. In this study, we analyzed pre-treatment tumor samples from a clinically annotated cohort of 155 patients with BRAF-mutant metastatic melanoma treated with first-line BRAFi/MEKi and followed for up to five years. We stratified patients into durable responders (PFS [&ge;] 24 months) and rapid progressors (PFS < 6 months with progression) and found that a global metric of tumor genomic heterogeneity, rather than individual gene alterations, distinguished these groups. Combining genomic heterogeneity with baseline tumor burden (e.g., lactate dehydrogenase (LDH) or radiographic lesion dimensions), we developed a parsimonious model that predicted durable responders with high precision and specificity. Notably, the analogous population of patients treated instead with immunotherapy were not durable responders, suggesting that the selected predictors of durable responders are targeted therapy specific. Spatial profiling of a subset of pre-treatment biopsies (n = 47) demonstrated that high intratumoral, but not peritumoral, CD8+ T-cell infiltration correlated with prolonged survival on BRAF-targeted therapy and served as an independent predictive factor when considered with genomic heterogeneity and features of clinical tumor burden. Together, these findings highlight the distinct baseline intrinsic and extrinsic features underlying durable response to BRAF-targeted therapy and support their potential implication in guiding treatment selection for patients with BRAF-mutant metastatic melanoma. One-Sentence SummaryIntegrated clinical, tumor genomic, and immune microenvironmental features predict durable responses to BRAF-targeted therapy.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is rapidly increasing worldwide, yet effective targeted therapies remain limited. To better understand the molecular mechanisms underlying MASLD, we performed an integrated proteogenomic analysis of human liver tissue. Using mass spectrometry, we quantified 2,744 proteins in 504 liver biopsies from the Quebec Obesity Biobank and examined changes across disease stages. To investigate causality, we integrated liver proteomics with RNA sequencing and genome-wide genotyping to map thousands of protein quantitative trait loci (pQTLs) and expression quantitative trait loci (eQTLs). These molecular data were combined with summary statistics from a meta-analysis of genome-wide association studies including 16,532 MASLD cases and 1,240,188 controls. Mendelian randomization and genetic colocalization analyses revealed that most proteins differentially expressed across MASLD stages were not causally implicated in disease risk, whereas several genetically predicted liver proteins showed evidence of causal effects. Among these, higher hepatic levels of the MTARC1 protein were causally associated with MASLD and hepatic fat accumulation. Phenome-wide analyses suggested that MTARC1 inhibition may reduce the risk of cirrhosis, hepatocellular carcinoma, and cholelithiasis while improving lipid profiles. Notably, the causal MTARC1 variant influenced liver protein levels but not gene expression. Genetic analyses also identified ERLIN1 and HSD17B13 as potential therapeutic targets. In contrast, eQTLs and pQTLs at other loci such as GCKR showed opposite effects on MASLD risk. These findings highlight the importance of integrating tissue proteomics with human genetics to distinguish biomarkers from causal drivers and to identify promising therapeutic targets for MASLD.

Background & AimsClarithromycin (CLA) resistance severely compromises the efficacy of triple therapy (TT) against Helicobacter pylori (H. pylori). Bismuth-based regimens exhibit greater efficacy against CLA-resistant H. pylori than against strains resistant to other antibiotics, suggesting a resistance-specific vulnerability rather than broad antimicrobial activity. The mechanistic basis for this selectivity, however, remains unknown. We hypothesized that high-level CLA resistance confers a metabolically targetable vulnerability that can be exploited by bismuth, and that a quantitative MIC of CLA threshold could identify this responsive subset. MethodsWe conducted a real-world retrospective analysis of 4,610 pediatric patients with H. pylori infection treated between 2019 and 2024, among whom 1,844 (40%) had complete follow-up data for eradication assessment. In parallel, we prospectively enrolled 51 patients with culture-positive isolates--the largest liquid checkerboard panel reported to date--to evaluate bismuth-CLA interactions and track treatment outcomes. Mechanistic validation included transcriptomic profiling and functional assays of iron and ATP metabolism, with iron chelation and supplementation experiments. ResultsIn the retrospective real-world cohort (n = 4,610; 1,844 with follow-up), bismuth quadruple therapy (BQT) achieved superior eradication specifically in CLA-resistant infections (93.1% vs 68.8% with TT; p = 0.017). In vitro, bismuth-CLA synergy was exclusive to resistant strains and intensified with increasing MIC of CLA. Mechanistically, bismuth triggered coordinated depletion of intracellular iron and ATP--a phenotype mimicked by iron chelation and reversed by iron supplementation. A baseline MIC of CLA [&ge;]16 g/mL robustly predicted this synergy (AUC = 0.991) and was prospectively validated in an independent patient subset: bismuth cured 96% of high-level resistant patients (MIC [&ge;] 16 g/mL) versus 0% with triple therapy (p < 0.001). ConclusionHigh-level CLA resistance defines an iron-dependent metabolic vulnerability in H. pylori that is selectively targeted by bismuth. The MIC threshold of [&ge;] 16 g/mL provides the first clinically actionable biomarker for resistance-guided therapy, transforming a marker of treatment failure into a positive predictor of bismuth response. These findings establish the mechanistic and clinical foundation for MIC-stratified eradication strategies and inform future randomized trials aimed at precision management of antibiotic-resistant H. pylori infection. Graphical abstractO_ST_ABSLeftC_ST_ABSHigh-level clarithromycin (CLA) resistance defines a distinct physiological phenotype in Helicobacter pylori, in which an elevated MIC of CLA ([&ge;] 16 {micro}g/mL) predicts poor eradication with triple therapy (TT) but favorable response to bismuth-containing quadruple therapy (BQT). MiddleMechanistically, CLA resistance is associated with upregulation of the ferric uptake regulator Fur, leading to reprogrammed iron homeostasis and an increased metabolic burden. Colloidal bismuth subcitrate (CBS) disrupts Fur-dependent iron regulation, exacerbates iron-restricted metabolic stress, and compromises cellular integrity, thereby selectively sensitizing CLA-resistant bacteria to antibiotic killing. RightTranslational implication of reframing antibiotic resistance as a therapeutic vulnerability--bismuth-based regimens function as a "key" that unlocks resistance-associated metabolic liabilities, delays resistance evolution, and improves treatment outcomes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/26351907v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@28fb58org.highwire.dtl.DTLVardef@8d5190org.highwire.dtl.DTLVardef@1e5fc9dorg.highwire.dtl.DTLVardef@2bc102_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Monocyte-derived DC therapies programmed for robust IL-12p70 production have been associated with favorable outcomes in cancer clinical trials. However, clinical responses remain inconsistent even under standardized protocols, and the cellular basis for this variability is unknown. We leveraged single-cell multiomics to characterize two widely used DC platforms, the high-IL-12p70-producing alpha-Type-1-polarized DC (DC1) and the IL-12p70-deficient DC induced in the presence of PGE2 (PGE2-DC), at baseline and following rhCD40L activation. DC generated from 7 healthy participants representing the spectrum of rhCD40L-induced IL-12p70 production were profiled by transcriptome analysis with concurrent 42-plex surface proteomics, multiplex ELISA, and ELISpot quantification of IL-12p70-producing cells. While DC1 and PGE2-DC distinctly responded to rhCD40L, DC1 alone unexpectedly comprised 3 transcriptionally and phenotypically distinct subpopulations in resting and stimulated states. Only a limited fraction of DC1 coexpressed IL12A and IL12B (IL-12p70 producers), which was confirmed by ELISpot at the protein level. The distribution of DC1 subclusters varied markedly between individuals and correlated with bulk cytokine and chemokine secretion profiles. Heterogeneity within DC1 preparations may underlie inconsistent clinical trial outcomes, and identification of associated surface proteins provides a prospective strategy for subcluster enrichment to enhance DC release criteria and patient stratification for optimized therapeutic efficacy.

High grade serous ovarian cancer patients initially respond to platinum-based chemotherapy, but usually relapse within two years and ultimately develop therapy resistance. Management of response and effective clinical decisions are currently based on unspecific biomarkers and limited imaging techniques, illustrating the clear clinical need for reliable predictors of response. In this work, we evaluated the performance of patient-derived organoids generated from ascitic fluid and functionally tested in parallel to the patients clinical course, in the prediction of treatment response, and guiding clinical decision-making in a patient-specific manner. Ascites derived organoids reliably recapitulated the histological and molecular features of a paradigmatic HGSOC patient with an apparent dissociated response, and demonstrated chemoresistance months before laparoscopy confirmed persistent inoperable disease with poor pathological response. Drug screening identified alternative therapeutic options, while multi-omics provided additional insights into the tumor-specific biological features, to assist in the personalized clinical management in ovarian cancer.

Leptomeningeal disease (LMD) is a rapidly fatal complication of systemic cancer for which sensitive diagnostic tools and informative biomarkers remain limited. Here, we introduce CSF-Seq, a method for whole-transcriptome sequencing of cell-free RNA (cfRNA) from human cerebrospinal fluid (CSF), designed to enable molecular profiling of LMD and other central nervous system (CNS) conditions. Using a prospectively collected CSF biobank, we analyzed 125 samples spanning multiple pathologies, including breast and lung LMD, glioblastoma, traumatic brain injury, and non-cancer neurological controls. Through optimized RNA extraction, library preparation, and deep sequencing, CSF-Seq generated robust and reproducible transcriptome-wide profiles despite the low abundance and fragmentation of cfRNA in CSF. CSF transcriptomes exhibited disease-specific expression, separating LMD from non-cancer controls and from non-LMD cancers, independent of CSF collection modality. Tumor-associated epithelial transcripts, including CEACAM6 and MUC1, were consistently enriched in LMD samples, whereas immune and CNS-associated transcripts were broadly detected across disease states, consistent with contributions from both tumor and non-tumor sources. Cross-site processing of matched samples demonstrated high concordance, indicating preservation of sample-specific transcriptional signatures across independent workflows. Importantly, we identified a collection method- independent LMD gene expression signature that was significantly associated with overall survival, supporting its potential prognostic relevance. Together, these findings establish CSF-Seq as a technically robust and clinically informative platform for transcriptomic biomarker discovery in CNS metastatic disease, offering a minimally invasive approach for disease characterization, risk stratification, and longitudinal monitoring in patients with LMD.