技术资料

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

Substantial progress has been made in the development of radiation countermeasures,resulting in the recent approval of several mitigators; however,there has yet to be an approved prophylactic radioprotectant. Research on countermeasure performance in mixed neutron and gamma radiation fields has also been scarce. Fibroblast-stimulating lipopeptide (FSL-1) is a novel synthetic agonist for toll-like receptor 2/6. In previous studies,the administration of FSL-1 before and after gamma radiation significantly improved survival outcomes for mice through the activation of the NF-κB pathway. In the current study,we tested FSL-1’s radioprotective abilities in a mixed radiation field that models one produced by a nuclear detonation in 11–14-week-old C57BL/6 male and female mice. We demonstrate that a single dose of 1.5 mg/kg of FSL-1 administered 12 h prior to 65% neutron 35% gamma mixed-field (MF) irradiation enhances survival,accelerates recovery of hematopoietic cell and stem cell populations,reduces inflammation,and protects innate immune function in mice. FSL-1’s ability to recover blood and protect immune functions is important in countering the high rate of incidence of sepsis caused by MF radiation’s damaging effects. These results demonstrate that FSL-1 is a promising prophylactic countermeasure where exposure to MF radiation is anticipated. View Publication

Accumulating data have shown that targeting breast cancer stem cells (CSCs) is an auspicious way for anticancer therapies. This study demonstrated that the antirheumatic drug auranofin is a potent CSC inhibitor with anti-CSC action on breast cancer. This research focused on investigating the effect of auranofin on breast cancer and CSCs and its cellular mechanism. Mammosphere formation,colony formation,levels of CD44 high /CD24 low,and aldehyde dehydrogenase 1 expression in the cells were evaluated after auranofin treatment. The anti-CSC properties of auranofin were further examined by gel shift assay and cytokine detection. Auranofin suppressed cell growth,colony formation,migration,and mammosphere formation and triggered apoptosis in breast cancer. Auranofin decreased the CD44 high /CD24 low - and aldehyde dehydrogenase-expressed subpopulations,as well as the Stat3-DNA interaction and phosphorylated Stat3 level. Auranofin also decreased the extracellular levels of interleukin-8 (IL-8) in the mammosphere media. Auranofin suppressed the Stat3/IL-8 signal and killed CSCs; therefore,it may be a potential target for CSCs. View Publication

Cerebral cavernous malformations (CCMs) are clusters of thin-walled enlarged blood vessels in the central nervous system that are prone to recurrent hemorrhage and can occur in both sporadic and familial forms. The familial form results from loss-of-function variants in the CCM1,CCM2,or CCM3 gene. Despite a better understanding of CCM pathogenesis in recent years,it is still unclear why CCM3 mutations often lead to a more aggressive phenotype than CCM1 or CCM2 variants. By combining high-throughput differentiation of blood vessel organoids from human induced pluripotent stem cells (hiPSCs) with a CCM1,CCM2,or CCM3 knockout,single-cell RNA sequencing,and high-content imaging,we uncovered both shared and distinct functions of the CCM proteins. While there was a significant overlap of differentially expressed genes in fibroblasts across all three knockout conditions,inactivation of CCM1,CCM2,or CCM3 also led to specific gene expression patterns in neuronal,mesenchymal,and endothelial cell populations,respectively. Taking advantage of the different fluorescent labels of the hiPSCs,we could also visualize the abnormal expansion of CCM1 and CCM3 knockout cells when differentiated together with wild-type cells into mosaic blood vessel organoids. In contrast,CCM2 knockout cells showed even reduced proliferation. These observations may help to explain the less severe clinical course in individuals with a pathogenic variant in CCM2 and to decode the molecular and cellular heterogeneity in CCM disease. Finally,the excellent scalability of blood vessel organoid differentiation in a 96-well format further supports their use in high-throughput drug discovery and other biomedical research studies. The online version contains supplementary material available at 10.1007/s10456-025-09985-5. View Publication

Anti-interleukin (IL)-4Rα monoclonal antibodies (mAb) improve lung function and decrease the number of exacerbations in patients with COPD type (T)2 inflammation. However,the involvement of early innate immune responses underlying these treatment effects is not well known. We sought to understand the effect and mechanisms of IL-4Rα mAb treatment on bronchial epithelial cells (BECs) from COPD patients under T2 inflammatory conditions with and without rhinoviral infection. Primary BECs from healthy and COPD patients were grown at an air–liquid interface and stimulated with IL-4 or IL-13 cytokines in the presence of IL-4Rα mAb. Cells were infected with human rhinovirus 1B and collected 24 h after infection. Antiviral mediators ( i.e.,interferons (IFNs) and pattern recognition receptors (PRRs)),as well as chemokine and alarmin expression,were measured by reverse transcriptase quantitative PCR and ELISA. Treatment with IL-4Rα mAb (100 nM) inhibited the eotaxin-3 (CCL26) gene after IL-4/IL-13 induction (p<0.05) in COPD BECs. However,no significant changes in rhinovirus-induced IFN-β,PRRs or thymic stromal lymphopoietin gene responses were observed with IL-4/IL-13 stimulation and IL-4Rα mAb treatment. A significant increase in mucin 5AC gene expression was observed with both IL-4 and IL-13 stimulation,but it was not reduced with IL-4Rα treatment in BECs. Inhibition of IL-4Rα reduced CCL26 levels without affecting antiviral immune responses in BECs from COPD patients. Inhibition of IL-4Rα reduced IL-4/IL-13 signalling without broadly suppressing the immune system,which might suggest that inhibition of the IL-4Rα pathways may prevent COPD exacerbations through reduction of eosinophil chemotaxis. View Publication

Drug resistance and relapse are still major challenges in acute myeloid leukemia (AML) because of the inability to effectively eradicate leukemia stem cells (LSCs). Senescence induction combined with immune killing may offer promising strategies for LSC eradication. However,whether and how drug-resistant LSCs retain stemness via senescence and immune regulation remains unknown. The immunoproteasome subunit PSMB10 expression levels were analyzed by single-cell RNA-seq data,along with the bioinformatics analysis of publicly available AML datasets,and quantified using RT-qPCR and flow cytometry (FCM) analysis on clinical samples from AML patients. The cellular senescence was evaluated by the assays of cell proliferation,cell cycle,senescence-associated β-galactosidase activity,and senescence-associated secretory phenotype factors. In vitro T-cell killing assay was played to determine immune escape reprogramming of AML cells. FCM was conducted to estimate intracellular drug concentration and cellular apoptosis rates. Human AML xenografts and PSMB10 knockout syngeneic mouse bone marrow transplantation models were utilized to investigate the function of PSMB10. Various techniques were employed for mechanism studies,including Lentivirus transduction or siRNA transfection,western blotting,co-immunoprecipitation assays,luciferase reporter assays,polysome profiling assays,quantitative proteomics,etc. PSMB10 mRNA was significantly upregulated in the surviving nonsenescent LSCs,exhibiting a 13-fold increase compared to senescent LSCs following chemotherapy. The specific high expression of PSMB10 in post-chemotherapy nonsenescent LSCs predicts a poor AML prognosis. The genetic inactivation of PSMB10 resulted in increased senescence and cytotoxic T lymphocyte (CTL) killing,as well as increased intracellular drug concentrations and drug-induced cellular senescence in different types of human AML cells,which also impeded human and murine leukemia initiation and stemness maintenance in vivo with a 19-fold decrease in the frequency of human LSCs and a 7.6-fold decrease of drug-resistant mouse LSCs,while normal hematopoietic cells remained unaffected. Mechanistically,the downregulation of PSMB10 boosted SLC22A16-mediated drug endocytosis and further induced chemotherapy drug-mediated senescence through the RPL6/RPS6-MDM2-P21 pathway in AML cells. Additionally,downregulating PSMB10 also impeded MHC-I protein degradation-induced escape of CTL killing. PSMB10 is a key candidate molecular target for eradicating drug-resistant LSCs via senescence and immune regulation. The online version contains supplementary material available at 10.1186/s13046-025-03420-9. View Publication

Testicular somatic cells play an important role in supporting spermatogenesis. Leydig cells (LCs) and peritubular myoid cells (PTMs) originate from a common progenitor population and show similar expression signatures in adulthood,making it difficult to distinguish and isolate the two in vitro. In this study,new surface markers for identifying adult LCs (ALCs) and PTMs were discovered by reanalyzing testicular single-cell dataset. Differential expressions of ITGA9 and NGFR were confirmed through immunofluorescence staining of human testes. A novel Fluorescence activated Cell Sorting (FACS) protocol is established for the isolation of ALCs and PTMs based on the two markers. Long-term culture of both cells were performed and their characteristics were characterized and explored. ITGA9+ /NGFR + cells were positive for markers of PTMs (SMA,CNN1) and negative for markers of ALCs (HSD3B,STAR),and were able to form tubular and spheroid structures in vitro. In contrast,ITGA9-/NGFR + cells were positive for ALC markers and negative for PTM markers,and showed a capacity of testosterone production in vitro. Also,both cells were negative for Sertoli cell marker SOX9. When the two cells were cultured,they can expand for more than 15 passages. Our study established a novel and efficient method for identifying and isolating human ALCs and PTMs,which provides a great potential for researches of the two cell types in human. The online version contains supplementary material available at 10.1186/s12958-025-01389-w. View Publication

Dynamic regulation of metabolic activities in astrocytes is critical to meeting the demands of other brain cells. During neuronal stress,lipids are transferred from neurons to astrocytes,where they are stored in lipid droplets (LDs). However,it is not clear whether and how neuron-derived lipids trigger metabolic adaptation in astrocytes. Here,we uncover an endolysosomal function that mediates neuron-astrocyte transcellular lipid signaling. We identify Tweety homolog 1 (TTYH1) as an astrocyte-enriched endolysosomal protein that facilitates autophagic flux and LD degradation. Astrocyte-specific deletion of mouse Ttyh1 and loss of its Drosophila ortholog lead to brain accumulation of neutral lipids. Computational and experimental evidence suggests that TTYH1 mediates endolysosomal clearance of ceramide 1-phosphate (C1P),a sphingolipid that dampens autophagic flux and LD breakdown in mouse and human astrocytes. Furthermore,neuronal C1P secretion induced by inflammatory cytokine interleukin-1β causes TTYH1-dependent autophagic flux and LD adaptations in astrocytes. These findings reveal a neuron-initiated signaling paradigm that culminates in the regulation of catabolic activities in astrocytes. Subject terms: Organelles,Glial biology,Lipid signalling View Publication

Trogocytosis is an underappreciated phenomenon that shapes the immune microenvironment surrounding many types of solid tumors. The consequences of membrane-bound proteins being deposited from a donor immune cell to a recipient cancer cell via trogocytosis are still unclear. Here,we report that human clear cell renal carcinoma tumors stably express the lymphoid markers CD45,CD56,CD14,and CD16. Flow cytometry performed on fresh kidney tumors revealed consistent CD45 expression on tumor cells,as well as varying levels of the other markers mentioned previously. These results were consistent with our immunofluorescent analysis,which also revealed colocalization of lymphoid markers with carbonic anhydrase 9,a standard kidney tumor marker. RNA analysis showed a significant upregulation of genes typically associated with immune cells by tumor cells. Finally,we show evidence of chromosomal DNA being transferred from immune cells to tumor cells through physical contact. This horizontal gene transfer has transcriptional consequences in the recipient tumor cell,resulting in a fusion phenotype that expresses both immune and cancer specific proteins. This work demonstrates a novel mechanism by which tumor cell protein expression is altered through the acquisition of surface membrane fragments and genomic DNA from infiltrating lymphocytes. These results alter the way in which we understand tumor-immune cell interactions and may reveal new insights into the mechanisms by which tumors develop. Additionally,further studies into trogocytosis and other mechanisms of contact-mediated cellular transfer will help push the field towards the next generation of immunotherapies and biomarkers for treating renal cell carcinoma and other cancers. View Publication

This study investigates the impact of minimal residual disease (MRD) on relapse in patients with acute myeloid leukemia (AML),focusing on its interaction with immune cells function. A total of 49 AML patients were enrolled in this prospective study and categorized into four groups: MRD − positive with relapse,MRD − positive without relapse,MRD − negative with relapse,and MRD − negative without relapse. Peripheral blood T lymphocyte subpopulations were analyzed using ten-color flow cytometry. CD4 + T cells were co-cultured with leukemia cell lines to assess the impact of CD4 + T cells on leukemia cell proliferation,apoptosis,and cytokine release. In MRD − positive patients,relapsed individuals exhibited significantly higher levels of CD4 + T cells,regulatory T (Treg) cells,and CD4 + CD45RA + naïve T cells compared to non-relapsed patients ( P < 0.0001,P = 0.0016,and P = 0.0066,respectively). Conversely,in MRD − negative patients,relapsed individuals showed a significantly lower percentage of Treg cells ( P = 0.0068). Furthermore,we observed that CD4 + T cells were associated with enhanced leukemia cell proliferation and reduced apoptosis,along with markedly increased IL-10 expression. The available data raise the possibility that CD4 + T cell-derived IL-10 participates in immune microenvironment regulation,a process that may have implications for MRD maintenance and disease recurrence in AML. View Publication