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Aplastic anemia (AA) is a life-threatening bone marrow (BM) failure syndrome characterized by pancytopenia. Recent studies revealed that dysfunctional endothelial progenitor cells (EPCs),critical components of the BM microenvironment,are involved in hematopoietic-dysfunction-related diseases,including AA. However,the mechanism underlying EPC damage in AA remains unknown. Here we find that transforming growth factor-β (TGF-β) signaling is hyperactive in dysfunctional AA EPCs with impaired hematopoietic support and immune regulatory ability,and TGF-β inhibition promotes hematopoiesis and immune rebalance by repairing dysfunctional EPCs. Through impaired EPC and AA murine models,we validated that TGF-β inhibition restores EPC dysfunction to improve hematopoiesis and immune status in vitro and in vivo. RNA sequencing and real-time quantitative polymerase chain reaction provided further validation. These results indicate that dysfunctional BM EPCs with hyperactive TGF-β signaling are involved in AA. TGF-β inhibition promotes multilineage hematopoiesis recovery and immune balance by repairing dysfunctional EPCs,providing a potential therapeutic strategy for AA. Subject terms: Experimental models of disease,Translational research View Publication

Cancer stem cells (CSCs) are responsible for colorectal cancer (CRC) chemoresistance,recurrence,and metastasis. Therefore,identifying molecular stemness targets that are involved in tumor growth is crucial for effective treatment. Here,we performed an extensive in vitro and in vivo molecular and functional characterization,revealing the pivotal role of the lysine methyltransferase SET and MYND Domain Containing 3 (SMYD3) in colorectal cancer stem cell (CRC-SC) biology. Specifically,we showed that SMYD3 interacts with and methylates c-MYC at K158 and K163,thereby modulating its transcriptional activity,which is implicated in stemness and colorectal malignancy. Our in vitro data suggest that SMYD3 pharmacological inhibition or its stable genetic ablation affects the clonogenic and self-renewal potential of patient-derived CRC-SCs and organoids by altering their molecular signature. Moreover,we found that SMYD3 stable knock-out or pharmacological inhibition drastically reduces CRC tumorigenicity in vivo and CRC-SC metastatic potential. Overall,our findings identify SMYD3 as a promising therapeutic target acting directly on c-MYC,with potential implications for countering CRC-SC proliferation and metastatic dissemination. Subject terms: Gastrointestinal cancer,Cancer stem cells View Publication

CD38,an ecto-enzyme involved in NAD + catabolism,is highly expressed in exhausted CD8 + T cells and has emerged as an attractive target to improve response to immune checkpoint blockade (ICB) by blunting T cell exhaustion. However,the precise role(s) and regulation of CD38 in exhausted T cells and the efficacy of CD38-directed therapeutic strategies in human cancer remain incompletely defined. Here,we show that CD38 + CD8 + T cells are induced by chronic TCR activation and type I interferon stimulation and confirm their association with ICB resistance in human melanoma. Disrupting CD38 restores cellular NAD + pools and improves T cell bioenergetics and effector functions. Targeting CD38 restores ICB sensitivity in a cohort of patient-derived organotypic tumor spheroids from explanted melanoma specimens. These results support further preclinical and clinical evaluation of CD38-directed therapies in melanoma and underscore the importance of NAD + as a vital metabolite to enhance those therapies. View Publication

Maturation of human pluripotent stem (hPS) cell-derived cardiomyocytes is critical for their use as a model system. Here we mimic human heart maturation pathways in the setting of hPS cell-derived cardiac organoids (hCOs). Specifically,transient activation of 5′ AMP-activated protein kinase and estrogen-related receptor enhanced cardiomyocyte maturation,inducing expression of mature sarcomeric and oxidative phosphorylation proteins,and increasing metabolic capacity. hCOs generated using the directed maturation protocol (DM-hCOs) recapitulate cardiac drug responses and,when derived from calsequestrin 2 ( CASQ2 ) and ryanodine receptor 2 ( RYR2 ) mutant hPS cells exhibit a pro-arrhythmia phenotype. These DM-hCOs also comprise multiple cell types,which we characterize and benchmark to the human heart. Modeling of cardiomyopathy caused by a desmoplakin ( DSP ) mutation resulted in fibrosis and cardiac dysfunction and led to identifying the bromodomain and extra-terminal inhibitor INCB054329 as a drug mitigating the desmoplakin-related functional defect. These findings establish DM-hCOs as a versatile platform for applications in cardiac biology,disease and drug screening. Subject terms: Tissue engineering,Differentiation,Cardiomyopathies View Publication

Natural killer (NK) cell-based therapies represent a promising approach for acute myeloid leukemia (AML) relapse,yet their efficacy is hindered by immunosuppressive factors such as transforming growth factor beta (TGF-β) in the tumor microenvironment. This study investigated the effects of TGF-β on NK cell cytotoxicity and migration using 2D and 3D co-culture models that mimic the leukemic microenvironment. TGF-β production was evaluated in AML-derived leukemic cell lines and mesenchymal stromal cells (hTERT-MSCs) using ELISA. Bulk RNA sequencing (RNA-seq) was performed to analyze global gene expression changes in TGF-β-treated primary human NK cells. NK cell cytotoxicity and migration were assessed in 2D monolayer and 3D spheroid co-cultures containing hTERT-MSCs and leukemic cells using flow cytometry and confocal microscopy. Both leukemic cells and MSCs produced TGF-β,with increased levels observed in MSCs after co-culture with primary AML blasts. RNA sequencing revealed that TGF-β altered key gene pathways associated with NK cell cytotoxicity,adhesion,and migration,supporting its immunosuppressive role. In functional assays,TGF-β exposure significantly reduced NK cell-mediated cytotoxicity in a time-dependent manner and impaired NK cell infiltration into 3D spheroids,particularly in models incorporating MSCs. Additionally,MSCs themselves provided a protective environment for leukemic cells,further reducing NK cell effectiveness in 2D co-cultures. TGF-β suppresses both NK cell cytotoxicity and migration,limiting their ability to eliminate leukemic cells and infiltrate the bone marrow niche (BMN). These findings provide novel insights into TGF-β–mediated immune evasion mechanisms and provide important insights for the future design of NK-based immunotherapies and clinical trials. View Publication

Genetic variations in the Trpm8 gene that encodes the cold receptor TRPM8 have been linked to protection against polygenic migraine,a disabling condition primarily affecting women. Noteworthy,TRPM8 has been recently found in brain areas related to emotional processing,suggesting an unrecognized role in migraine comorbidities. Here,we use mouse behavioural models to investigate the role of Trpm8 in migraine-related phenotypes. Subsequently,we test the efficacy of rapamycin,a clinically relevant TRPM8 agonist,in these behavioural traits and in human induced pluripotent stem cell (iPSC)-derived sensory neurons. We report that Trpm8 null mice exhibited impulsive and depressive-like behaviours,while also showing frequent pain-like facial expressions detected by an artificial intelligence algorithm. In a nitroglycerin-induced migraine model,Trpm8 knockout mice of both sexes developed anxiety and mechanical hypersensitivity,whereas wild-type females also displayed depressive-like phenotype and hypernociception. Notably,rapamycin alleviated pain-related behaviour through both TRPM8-dependent and independent mechanisms but lacked antidepressant activity,consistent with a peripheral action. The macrolide ionotropically activated TRPM8 signalling in human sensory neurons,emerging as a new candidate for intervention. Together,our findings underscore the potential of TRPM8 for migraine relief and its involvement in affective comorbidities,emphasizing the importance of addressing emotional symptoms to improve clinical outcomes for migraine sufferers,especially in females. The online version contains supplementary material available at 10.1186/s10194-025-02082-4. View Publication

Severe cutaneous adverse drug reactions (SCARs) are life-threatening diseases,which are associated with human leukocyte antigen ( HLA ) risk variants. However,the low positive predictive values of HLA variants suggest additional factors influence disease susceptibility. Using dapsone hypersensitivity syndrome (DHS) as a paradigm for SCARs,we show that the DHS patients harbor a sex-related global reduction in blood NK cells,contributing to the higher incidence of reactions in females. Single-cell RNA sequencing revealed a decrease in the immunoregulatory CD56 low XCL1/2 low NK cell subset and an expansion of CD56 high XCL1/2 high NK cell subsets with an effector phenotype in DHS patients compared to dapsone-tolerant individuals. Functionally,interleukin-15 superagonist-induced activation of NK cells exacerbated SCARs-like symptoms in a murine model. Mechanistically,TSC22 domain family member 3 (TSC22D3) deficiency enhanced NK cell effector function,shifting the immune response from CD4+ T cell to CD8+ T cell function. These results demonstrate that TSC22D3-regulated NK cells play a critical role in predisposing to drug hypersensitivity reactions,bridging innate and adaptive immune dysregulation in SCARs pathogenesis. Our study highlights the importance of NK cell heterogeneity and TSC22D3 in immune-mediated hypersensitivity disorders,offering potential therapeutic targets for SCARs and related conditions. Subject terms: Innate immunity,Innate immunity View Publication

Circulating tumor cells (CTCs) hold significant promise for cancer diagnosis,prognosis,and treatment monitoring. We previously developed a technique for a single‐cell filtering device known as the microcavity array (MCA),specifically designed for the efficient recovery of CTCs from whole blood samples. Efficient enrichment and release of cells from the MCA remains challenging because of cell adhesion that occurs on the MCA surface during the enrichment phase. This study investigated the effects of surface modification with 2‐methacryloyloxyethyl phosphorylcholine (MPC) on the recovery efficiency of cancer cell lines from MCA. Scanning electron microscope (SEM) demonstrated reduced cell‐substrate interactions,leading to improved recovery efficiency. Comparative analyses showed that the MCA method provided superior recovery efficiency and reduced processing time compared to traditional methods such as density gradient centrifugation (DGC),while maintaining cell viability and proliferative capacity. CTCs were successfully detected in patients with gastric cancer,and short‐term cultures were achieved even when fewer than 20 CTCs per milliliter of blood were isolated. These findings emphasize the importance of surface modification for enhancing CTC isolation and the need for optimized culture conditions. The optimized MCA method offers a promising approach for rapid CTC recovery and potential integration with automated systems. Practical application : The Microcavity array (MCA) is a device specifically designed for efficient recovery of CTCs from whole blood. However cell adhesion on the MCA surface can limit release efficiency. This study demonstrated that surface modification with MPC signigicantly reduces cell‐substrate adhesion,improving recovery efficiency while maintaining cell viability and proliferative capacity. Compared to traditional density gradient centrifugation,the MPC‐modified MCA offers shorter processing time and better performance. CTCs were successfully detected in gastric cancer,and short‐term cultures were achieved even when fewer than 20 CTCs per mL of blood were isolated. The method supports downstearm applications such as cancer cell characterization and treatment monitoring. With potential for integration into automated system,the optimized MCA provides a practical,scalable solution for clinical liquid biopsy and personalized oncology. View Publication

Histologic variant (HV) subtypes of bladder cancer are clinically aggressive tumors that are more resistant to standard therapy compared to conventional urothelial carcinoma (UC). Little is known about the transcriptional programs that account for their biological differences. Here we show using single cell analysis that HVs harbor a tumor cell state characterized by expression of MUC16 (CA125),MUC4,and KRT24 . This cell state is enriched in metastases,predicted to be highly resistant to chemotherapy,and linked with poor survival. We also find enriched expression of TM4SF1,a transmembrane protein,in HV tumor cells. Chimeric antigen receptor (CAR) T cells engineered against TM4SF1 protein demonstrated in vitro and in vivo activity against bladder cancer cell lines in a TM4SF1 expression-dependent manner,highlighting its potential as a therapeutic target. Subject terms: Bladder cancer,Tumour biomarkers,Targeted therapies View Publication

Targeted therapy interventions using tyrosine kinase inhibitors (TKIs) provide encouraging treatment responses in patients with ALK -rearranged lung adenocarcinomas,yet resistance occurs almost inevitably. In addition to tumor cell-intrinsic resistance mechanisms,accumulating evidence suggests that cancer-associated fibroblasts (CAFs) within the tumor microenvironment contribute to therapy resistance. This study aimed to investigate CAF-driven molecular networks that shape the therapeutic susceptibility of ALK -driven lung adenocarcinoma cells. Three-dimensional (3D) spheroid co-cultures comprising ALK -rearranged lung adenocarcinoma cells and CAFs were utilized to model the tumor microenvironment. Single-cell RNA sequencing was performed to uncover transcriptional differences between TKI-treated homotypic and heterotypic spheroids. Functional assays assessed the effects of CAF-conditioned medium and CAF-secreted factors on tumor cell survival,proliferation,lipid metabolism,and downstream AKT signaling. The therapeutic potential of targeting metabolic vulnerabilities was evaluated using pharmacological inhibition of lipid metabolism and by ferroptosis induction. CAFs significantly diminished the apoptotic response of lung tumor cells to ALK inhibitors while simultaneously enhancing their proliferative capacity. Single-cell RNA sequencing identified lipogenesis-associated genes as a key transcriptional difference between TKI-treated homotypic and heterotypic lung tumor spheroids. CAF-conditioned medium and the CAF-secreted factors HGF and NRG1 activated AKT signaling in 3D-cultured ALK-rearranged lung tumor cells,leading to increased de novo lipogenesis and suppression of lipid peroxidation. These metabolic adaptations were critical for promoting tumor cell survival and fostering therapy resistance. Notably,both dual inhibition of ALK and the lipid-regulatory factor SREBP-1,as well as co-treatment with ferroptosis inducers such as erastin or RSL3,effectively disrupted the CAF-driven metabolic-supportive niche and restored sensitivity of resistant lung tumor spheroids to ALK inhibition. This study highlights a critical role for CAFs in mediating resistance to ALK-TKIs by reprogramming lipid metabolism in ALK-rearranged lung cancer cells. It suggests that targeting these metabolic vulnerabilities,particularly through inhibition of lipid metabolism or induction of ferroptosis,could provide a novel therapeutic approach to overcome resistance and improve patient outcomes. The online version contains supplementary material available at 10.1186/s40170-025-00400-7. View Publication

CACNA1A encodes the pore-forming α 1A subunit of the Ca V 2.1 calcium channel,whose altered function is associated with various neurological disorders,including forms of ataxia,epilepsy,and migraine. In this study,we generated isogenic iPSC-derived neural cultures carrying CACNA1A loss-of-function mutations differently affecting Ca V 2.1 splice isoforms. Morphological,molecular,and functional analyses revealed an essential role of CACNA1A in neurodevelopmental processes. We found that different CACNA1A loss-of-function mutations produce distinct neurodevelopmental deficits. The F1491S mutation,which is located in a constitutive domain of the channel and therefore causes a complete loss-of-function,impaired neural induction at very early stages,as demonstrated by changes in single-cell transcriptomic signatures of neural progenitors,and by defective polarization of neurons. By contrast,cells carrying the Y1854X mutation,which selectively impacts the synaptically-expressed Ca V 2.1[EFa] isoform,behaved normally in terms of neural induction but showed altered neuronal network composition and lack of synchronized activity. Our findings reveal previously unrecognized roles of CACNA1A in the mechanisms underlying neural induction and neural network dynamics and highlight the differential contribution of the divergent variants Ca V 2.1[EFa] and Ca V 2.1[EFb] in the development of human neuronal cells. The online version contains supplementary material available at 10.1007/s00018-025-05740-7. View Publication

Cystic Fibrosis (CF) is a common genetic disease in the United States,resulting from mutations in the Cystic Fibrosis transmembrane conductance regulator (cftr) gene. CFTR modulators,particularly Elexacaftor/Tezacaftor/Ivacaftor (ETI),have significantly improved clinical outcomes for patients with CF. However,many CFTR mutations are not eligible for CFTR modulator therapy due to their rarity. In this study,we report that a patient carrying rare complex CFTR mutations,c.1680-877G>T and c.3067_3072delATAGTG,showed positive clinical outcomes after ETI treatment. We demonstrate that ETI was able to increase the expression of CFTR harboring c.3067_3072delATAGTG in a heterologous system. Importantly,patient-derived nasal epithelial cells in an air–liquid interface (ALI) culture showed improved CFTR function following ETI treatment. These findings supported the initiation of ETI with the patient. Retrospective studies have suggested that the patient has shown small but steady improvement over the past two years in several clinical metrics,including lung function,body mass index (BMI),and sweat chloride levels. Our studies suggest that ETI could be beneficial for patients carrying c.3067_3072delATAGTG. View Publication