技术资料

Monoclonal antibody-based immune checkpoint inhibitors,which have brought breakthrough effects in cancer treatments,are expected to assist in the treatment of viral diseases. However,antibody therapies may cause immune-related side effects,such as inflammation and pneumonia,due to cytokine storms. Small-molecule PD-1/PD-L1 inhibitors are an alternative to monoclonal antibody-based therapeutics. We have identified a novel small-molecule PD-1/PD-L1 inhibitor having a functional group (disulfide group),namely compound 2 (molecular weight: 456.6),from our library of sulfur-containing protein–protein interaction inhibitor compounds. Compound 2 selectively bound to PD-L1 over PD-1,with the dissociation rate constant (K D ) of 77.60 ± 4.44 nM (obtained by affinity analysis) and showed promising T cell activation recovery. A molecular docking simulation study between 2 and PD-L1 suggested that 2 binds to PD-L1 in a binding mode different from those of other small-molecule PD-L1/PD-1 inhibitors. Notably,oral administration of 2 to mice pre-infected with influenza A virus (A/NWS/33,H1N1 subtype) caused a significant increase in the neutralizing antibody titers,as well as recovery from influenza-induced pneumonia. Overall,2 provides insight for the development of therapeutic drugs against early viral infections,with both virus titer-reducing and antibody titer-boosting effects. Moreover,2 is widely used as a rubber peptizing agent in the production process of tires and other rubber products. Our findings may provide useful information for investigating its influence on living organisms. The online version contains supplementary material available at 10.1038/s41598-025-17982-3. Subject terms: Drug discovery and development,Pharmacology,Screening,Structure-based drug design View Publication

Immune checkpoint blockers (ICBs) have demonstrated substantial efficacy across various malignancies,yet the benefits of ICBs are limited to a subset of patients. Therefore,it is essential to identify novel therapeutic targets. By integrating multi-omics data from cohorts of patients with melanoma treated with ICBs,a positive correlation is observed between tumor CD137L expression and the efficacy of PD-1 blockade. Functionally,CD137L induction in cancer cells significantly enhances anti-tumor immunity by promoting CD8 + T cell survival,both in vivo and in vitro. Mechanistically,helicase-like transcription factor (HLTF) is identified as a pivotal transcriptional regulator of CD137L,controlling its expression through phosphorylation of serine at position 398. Therapeutically,the AMPK agonist AICAR (acadesine) as an inducer of CD137L,exhibiting synergistic effects with PD-1 or CTLA-4 blockade. In summary,our findings elucidate a mechanism controlling CD137L expression and highlight a promising combination therapy to enhance the efficacy of ICBs in melanoma. One Sentence Summary: Inducing co-stimulatory immune checkpoint CD137L expression in melanoma cells enhances T cell-mediated anti-tumor immunity. Subject terms: Tumour immunology,Cancer immunotherapy View Publication

Hsp90 is a molecular chaperone that is often overexpressed across multiple cancer types and has a potential value as a prognostic marker as well as a therapeutic target. Given the high interest in Hsp90 therapies,positron emission tomography or PET imaging of Hsp90 can be a valuable tool for patient selection. The limitations of the previously developed Hsp90 tracers prompted us to evaluate the recently developed brain-permeable [ 11 C]HSP990 PET probe to advance the development of Hsp90-targeted therapeutics. Given the brain accumulation of [ 11 C]HSP990 probe,application for glioblastoma imaging of this tracer is of particular interest. In vitro [ 11 C]HSP990 binding was assessed in breast cancer and glioma cell lines including patient-derived cells using Hsp90 inhibitors and RNA interference knockdown of Hsp90 isoforms. Saturation binding studies were conducted on these cells and tumour tissue homogenates,and autoradiography was performed on tissue sections. Ex vivo biodistribution and in vivo dynamic µPET/CT studies were performed in healthy mice and tumour-bearing mice,including immunocompromised subcutaneous human U87 and MDA-MB-231models and immunocompetent intracranial murine NS/CT-2A models at baseline and following a pre-treatment with Hsp90 inhibitors. High Hsp90-specific tracer uptake was observed in breast cancer and glioma cells,with Hsp90β inhibition resulting in the most substantial reduction in uptake. In vivo uptake was high in U87 tumours but low in MDA-MB-231,presumably due to the differences in Hsp90 expression in tumour tissue versus cultured cells. Differences in maximum binding capacity or B max across cell and tissue types support this hypothesis,especially given that the affinity measured as dissociation constant K d remained similar across all tissue types. Despite high NS/CT-2A tumour uptake in vitro,no contrast between the healthy brain tissue and the NS/CT-2A glioma was observed in vivo due to the high uptake by the healthy brain. [ 11 C]HSP990 is a promising tracer for identifying Hsp90-overexpressing tumours and may hold potential for patient stratification,prognosis,and therapy monitoring of novel Hsp90 therapeutics. High healthy brain uptake of this tracer precluded the differentiation of the tumour in the intracranial NS/CT-2A tumour model,therefore [ 11 C]HSP990 might not be a suitable tracer for the glioblastoma imaging. Tracer with a longer half-life might be needed to compare the washout of the tracer from the brain and the tumour tissue over several hours to identify a suitable imaging window. The online version contains supplementary material available at 10.1186/s41181-025-00386-z. View Publication

Antibiotic resistance is a threat to human health,yet recent work highlights how loss of resistance may drive pathogenesis in some bacteria. In two recent studies,we found that β-lactam antibiotics and nutrient stresses faced during infection selected for genetic inactivation of the Pseudomonas aeruginosa antibiotic efflux pump mexEFoprN . Unexpectedly,efflux pump mutations increased P. aeruginosa virulence during infection; however,neither the prevalence of mexEFoprN inactivating mutations in real human infections,nor the mechanisms driving increased virulence of efflux pump mutants are known. We hypothesized that human infection would select for virulence enhancing mutations. Using genome sequencing of clinical isolates,we show that mexEFoprN efflux pump inactivating mutations are enriched in P. aeruginosa isolates from cystic fibrosis infections relative to isolates from acute respiratory infections. Combining RNA-seq,metabolomics,genetic approaches,and infection models we show that efflux pump mutants have elevated quorum sensing driven expression of elastase and rhamnolipids which increase P. aeruginosa virulence during acute and chronic infections. Restoration of the efflux pump in a representative respiratory isolate and the notorious cystic fibrosis Liverpool epidemic strain reduced their virulence. These findings suggest that mutations inactivating antibiotic resistance mechanisms could lead to greater patient mortality and morbidity. Subject terms: Antimicrobial resistance,Pathogens,Bacteriology,Molecular evolution View Publication

Suspension bath bioprinting,whereby bioinks are extruded into a yield stress bath with rapid recovery from shearing,has enabled the printing of low viscosity bioinks into constructs with high geometric complexity. Previous studies have often relied upon external stabilisation of the suspension bath (e.g. collagen) in order to culture soft materials without loss of printed structure. Here,we report a systematic investigation of suspension bath properties that support the printing,fusion,and culture of spheroid-based bioinks without added stabilisation. Specifically,agarose fluid gels of varied polymer concentrations and dilutions were produced and characterised morphologically and rheologically. Juvenile bovine chondrocytes or mesenchymal stromal cells (MSCs) were formed into spheroids of ∼150 µ m in diameter and investigated within agarose suspension baths either for their fusion in hanging drop cultures or as jammed bioinks. MSC spheroids were also printed when mixed with hydrogel microparticles to demonstrate additional versatility to the approach. Suspension baths of lower polymer concentrations and increased dilution enabled faster spheroid fusion; however,the most heavily diluted suspension bath was unable to maintain print fidelity. Other formulations supported the printing,fusion,and culture of spheroid-based inks,either as simple lines or more complex patterns. These findings help to inform the design of suspension baths for bioprinting and culture. View Publication

Tissue remodeling is a key feature of allergic rhinitis (AR),but its underlying molecular mechanisms remain unclear. Lysyl oxidase-like 2 (LOXL2),a regulator of tissue remodeling,has not been studied in AR. Proteomic analysis was performed on nasal mucosal tissues from 8 AR patients and 8 healthy controls (HCs) to identify differentially expressed proteins (DEPs). The top three upregulated DEPs and their association with tissue remodeling markers were validated by immunofluorescence,Western blot,and RT-qPCR in an independent cohort of 30 AR patients and 30 HCs. In vitro,human nasal epithelial cells (HNECs) were treated with IL-4,and the effects of candidate protein inhibitors on remodeling were assessed. An AR mouse model was used to evaluate the impact of these inhibitors on nasal inflammation and remodeling. Proteomic analysis revealed a disease-specific protein expression profile in the nasal mucosa of AR patients,with the top three upregulated proteins being LOXL2,TGF-β1,and TIRAP. Tissue validation showed that LOXL2 was significantly upregulated in the nasal mucosa of AR patients compared to HCs and was significantly correlated with EMT markers (TGF-β1,α-SMA,and E-cadherin). In vitro,IL-4 stimulation significantly upregulated LOXL2,TGF-β1,and α-SMA,while downregulating E-cadherin in a dose-dependent manner in human nasal epithelial cells. These effects were reversed by inhibition of LOXL2. Further investigations demonstrated that LOXL2 promotes tissue remodeling through activation of the TGF-β1/Smad signaling pathway. In the AR mouse model,LOXL2 inhibitors significantly reduced nasal mucosal inflammation and tissue remodeling. Our proteomic analysis suggests that LOXL2 may be involved in the pathological remodeling processes of AR,potentially through modulation of the TGF-β1/Smad signaling pathway. These findings provide preliminary evidence that LOXL2 could serve as a candidate biomarker and a possible therapeutic target in AR,warranting further investigation. View Publication

Despite recent approval of monoclonal antibodies that reduce amyloid (Aβ) accumulation,the development of disease-modifying strategies targeting the underlying mechanisms of Alzheimer's disease (AD) is urgently needed. We demonstrate that mitochondrial complex I (mtCI) represents a druggable target,where its weak inhibition activates neuroprotective signalling,benefiting AD mouse models with Aβ and p-Tau pathologies. Rational design and structure‒activity relationship studies yielded mtCI inhibitors profiled in a drug discovery funnel designed to address safety,selectivity,and efficacy. The lead compound C458 is highly protective against Aβ toxicity,has favourable pharmacokinetics,and minimal off-target effects. C458 exhibited excellent brain penetrance,activating neuroprotective pathways with a single dose. Preclinical studies in APP/PS1 mice were conducted using functional tests,metabolic assessment,in vivo 31 P-NMR spectroscopy,blood cytokine panels,ex vivo electrophysiology,and Western blotting. Chronic oral administration improved long-term potentiation,reduced oxidative stress and inflammation,and enhanced mitochondrial biogenesis,antioxidant signalling,and cellular energetics. Efficacy against Aβ and p-Tau was confirmed in human organoids. These studies provide further evidence that the restoration of mitochondrial function in response to mild energetic stress represents a promising disease-modifying strategy for AD. This research was supported by grants from NIH AG 5549-06,NS1 07265,AG 062135,UG3/UH3 NS 113776,and ADDF 291204 (all to ET); U19 AG069701 (to TK); the Alzheimer’s Association Research Fellowship grant 23AARF-1027342 (to TKON). View Publication

Cholesterol is an essential plasma membrane component,and altered cholesterol metabolism has been linked to cholesterol accumulation in the airways of COPD and cystic fibrosis patients. However,its role in airway epithelial differentiation is not well understood. Tandem mass spectrometry-based proteomic analysis of differentiating primary human bronchial epithelial cells (phBECs) revealed an overall inhibition of the cholesterol biosynthesis pathway. We hypothesized that excess cholesterol impairs the differentiation of phBECs into a fully functional bronchial epithelium. PhBECs were differentiated in the presence of 80 µM cholesterol for 21 days,the main airway cell type populations monitored using qRT-PCR and immunofluorescent stainings,and epithelial barrier integrity was analyzed via transepithelial electrical resistance measurements. Chronic cholesterol exposure led to a significant increase in CC10 + secretory cells at the expense of ciliated cells. Pathway enrichment analysis suggested the tumor protein p53 as a master regulator of genes during normal differentiation of phBECs. Chronic cholesterol exposure drastically impaired the nuclear translocation of p53. Our findings suggest that this inhibition underlies the cholesterol-induced expansion of CC10 + secretory cell populations at the expense of ciliated cells. In conclusion,we identify cholesterol as an important regulator of normal bronchial epithelial cell differentiation through inhibition of p53 nuclear translocation. View Publication

Atezolizumab plus bevacizumab and lenvatinib are standard treatments for unresectable hepatocellular carcinoma; however,tumor markers such as alpha-fetoprotein and des-gamma-carboxy prothrombin have a limited ability to reflect treatment responses. Circulating tumor cells are non-invasive biomarkers associated with cancer stemness and treatment resistance. We assessed circulating tumor cell subsets expressing cancer stem cell markers (CD90,epithelial cell adhesion molecule,CD133,vimentin) using multiparametric flow cytometry at early and maximal response phases in patients receiving atezolizumab plus bevacizumab or lenvatinib. Early decreases in CD90-positive circulating tumor cells after therapy initiation were associated with tumor shrinkage and longer progression-free survival in both groups,as well as prolonged overall survival in the atezolizumab plus bevacizumab group. At maximal response,changes in CD90-positive circulating tumor cells reflected tumor burden more accurately than alpha-fetoprotein or des-gamma-carboxy prothrombin. These findings highlight the potential of CD90-positive circulating tumor cells to become dynamic biomarkers in systemic therapy for unresectable hepatocellular carcinoma. View Publication

Cancer research has long focused on mutations in normal body cells,but this approach has not produced major breakthroughs for most cancers. Our study explores a different concept that some aggressive cancers may actually arise from early reproductive cells called primordial germ cells,which normally develop into eggs and sperm. We created a new experimental model showing how a virus can transform human primordial germ cell-like cells into virus-positive Merkel cell carcinoma,a rare but deadly skin cancer. This model shows that cancers can emerge through changes in developmental states rather than relying solely on genetic mutations. By linking cancer development to early germ cells,our findings suggest a unifying explanation for both germ cell cancers and body cancers. This new perspective may guide more effective approaches to study,diagnose,and treat cancer by focusing on early human development rather than only DNA mutations and later developmental stages. View Publication

With the approval of the antibody-drug conjugate enfortumab vedotin (EV),NECTIN4 has emerged as a bona fide therapeutic target in urothelial carcinoma (UC). Here,we report the development of a NECTIN4-directed chimeric antigen receptor (CAR) T cell,which exhibits reactivity across cells expressing a range of endogenous NECTIN4,with enhanced activity in high expressors. We demonstrate that the PPARγ pathway,critical for luminal differentiation,transcriptionally controls NECTIN4,and that the PPARγ agonist rosiglitazone primes and augments NECTIN4 expression,thereby increasing sensitivity to NECTIN4-CAR T cell-mediated killing. NECTIN4-CAR T cells have potent anti-tumor activity even against EV resistant cells,which largely retain NECTIN4 expression,including in a post-EV biopsy cohort. Our results elucidate a therapeutically actionable mechanism that UC cells use to control NECTIN4 expression and suggest therapeutic approaches that leverage PPARγ agonists for rational combinations with NECTIN4-targeting agents in UC,as well as future potential treatment options for EV-refractory patients. Subject terms: Bladder cancer,Cancer immunotherapy,Cancer therapeutic resistance,Oncology,Bladder cancer View Publication

In bone marrow,cell numbers are balanced between production and loss. After chemotherapy,blood cell counts decrease initially but later recover as hematopoietic progenitor cells expand,although the mechanisms underlying this recovery are still unclear. We investigated the influence of red blood cells (RBCs) on hematopoietic stem cell (HSC) function during bone marrow recovery. Following chemotherapy,RBC concentrations in bone marrow peaked on day 5 posttreatment,coinciding with the recovery of hematopoiesis. Coculture of HSCs with RBCs resulted in a significant increase in hematopoiesis. Direct contact between RBCs and HSCs was essential for enhancement of hematopoiesis,and HSCs precultured with RBCs resulted in greater numbers of donor‐derived mature hematopoietic cells after transplantation. RNA‐sequencing analysis showed that Hes1 was the most significantly upregulated transcription factor in RBC coculture,and the response to RBC‐induced hematopoiesis of Hes1‐deficient HSCs was reduced. These findings imply a role of RBCs and Hes1 in the enhancement of hematopoietic recovery following bone marrow stress. View Publication