技术资料

Micropapillary adenocarcinoma (MPC) is an aggressive histological subtype of lung adenocarcinoma (LUAD). MPC is composed of small clusters of cancer cells exhibiting inverted polarity. However,the mechanism underlying its formation is poorly understood. Here we show that hypoxia is involved in MPC formation. Hypoxia induced the formation of MPC-like structures (MLSs) in a three-dimensional culture system using A549 human LUAD cells,and HIF-1α was indispensable for MLS formation. RNA sequencing analysis demonstrated that A549 cells forming MLSs exhibited a gene expression signature similar to that of lung MPC. Moreover,MLS formation enhanced the resistance of A549 cells to natural killer cell cytotoxicity. Our findings suggest that hypoxia drives lung MPC formation through HIF-1α and that immune escape from natural killer cells might underlie the aggressiveness of MPC. View Publication

The lack of a robust system to reproducibly propagate HRV-C,a family of viruses refractory to cultivation in standard cell lines,has substantially hindered our understanding of this common respiratory pathogen. We sought to develop an organoid-based system to reproducibly propagate HRV-C,and characterize virus-host interaction using respiratory organoids. We demonstrate that airway organoids sustain serial virus passage with the aid of CYT387-mediated immunosuppression,whereas nasal organoids that more closely simulate the upper airway achieve this without any intervention. Nasal organoids are more susceptible to HRV-C than airway organoids. Intriguingly,upon HRV-C infection,we observe an innate immune response that is stronger in airway organoids than in nasal organoids,which is reproduced in a Poly(I:C) stimulation assay. Treatment with α-CDHR3 and antivirals significantly reduces HRV-C viral growth in airway and nasal organoids. Additionally,an organoid-based immunofluorescence assay is established to titrate HRV-C infectious particles. Collectively,we develop an organoid-based system to reproducibly propagate the poorly cultivable HRV-C,followed by a comprehensive characterization of HRV-C infection and innate immunity in physiologically active respiratory organoids. The organoid-based HRV-C infection model can be extended for developing antiviral strategies. More importantly,our study has opened an avenue for propagating and studying other uncultivable human and animal viruses. Subject terms: Virus-host interactions,Viral pathogenesis,Respiratory tract diseases View Publication

Although acute myeloid leukemia (AML) affects hematopoietic stem cell (HSC)-supportive microenvironment,it is largely unknown whether leukemia-modified bone marrow (BM) microenvironment can be remodeled to support normal hematopoiesis after complete remission (CR). As a key element of BM microenvironment,endothelial progenitor cells (EPCs) provide a feasible way to investigate BM microenvironment remodeling. Here,we find reduced and dysfunctional BM EPCs in AML patients,characterized by impaired angiogenesis and high ROS levels,could be partially remodeled after CR and improved by N-acetyl-L-cysteine (NAC). Importantly,HSC-supporting ability of BM EPCs is partially recovered,whereas leukemia-supporting ability is decreased in CR patients. Mechanistically,the transcriptome characteristics of leukemia-modified BM EPCs return to near-normal after CR. In a classic AML mouse and chemotherapy model,BM vasculature and normal hematopoiesis are reversed after CR. In summary,we provide further insights into how leukemia-modified BM microenvironment can be remodeled to support normal hematopoiesis after CR,which can be further improved by NAC. Subject terms: Translational research,Acute myeloid leukaemia View Publication

Metaplastic breast cancer (MpBC) is a highly chemoresistant subtype of breast cancer with no standardized therapy options. A clinical study in anthracycline-refractory MpBC patients suggested that nitric oxide synthase (NOS) inhibitor NG-monomethyl-l-arginine (L-NMMA) may augment anti-tumor efficacy of taxane. We report that NOS blockade potentiated response of human MpBC cell lines and tumors to phosphoinositide 3-kinase (PI3K) inhibitor alpelisib and taxane. Mechanistically,NOS blockade leads to a decrease in the S-nitrosylation of c-Jun NH 2 -terminal kinase (JNK)/c-Jun complex to repress its transcriptional output,leading to enhanced tumor differentiation and associated chemosensitivity. As a result,combined NOS and PI3K inhibition with taxane targets MpBC stem cells and improves survival in patient-derived xenograft models relative to single-/dual-agent therapy. Similarly,biopsies from MpBC tumors that responded to L-NMMA+taxane therapy showed a post-treatment reversal of epithelial-to-mesenchymal transition and decreased stemness. Our findings suggest that combined inhibition of iNOS and PI3K is a unique strategy to decrease chemoresistance and improve clinical outcomes in MpBC. Subject terms: Breast cancer,Cell signalling,Cancer therapy View Publication

Irradiation with X-rays has been widely utilized in the clinical treatment of solid tumors and certain hematopoietic malignancies. However,this method fails to completely distinguish between malignant and normal cells. Prolonged or repeated exposure to radiation,whether due to occupational hazards or therapeutical interventions,can cause damage to normal tissues,particularly impacting the hematopoietic system. Therefore,it is important to investigate the effects of total body irradiation on the hematopoietic system of mice and to compare the inhibitory effects of various doses of irradiation on this system. In this study,we primarily employed flow cytometry to analyze mature lineage cells in the peripheral blood,as well as immature hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and spleen. Additionally,we evaluated the multilineage differentiation capacity of HSPCs through colony-forming cell assays. Our results indicated that peripheral B and T cells demonstrated increased sensitivity to irradiation,with significant cell death observed 1-day post-irradiation. Common lymphoid progenitor cells exhibited greater radiotolerance compared to other progenitor cell types,enabling them to maintain a certain population even at elevated doses. Moreover,notable differences were observed between intramedullary and extramedullary hematopoietic stem cells and common lymphoid progenitor cells regarding the extent of damage and recovery rate following irradiation. The multilineage differentiation capacity of HSPCs was also compromised during radiation exposure. In conclusion,different types of mature blood cells,along with immature HSPCs,exhibited varying degrees of sensitivity and tolerance to irradiation,resulting in distinct alterations in cell percentages and numbers. View Publication

The hippocampus is associated with mood disorders,and the activation of quiescent neurogenesis has been linked to anxiolytic effects. Near-infrared (NIR) light has shown potential to improve learning and memory in human and animal models. Despite the vast amount of information regarding the effect of visible light,there is a significant gap in our understanding regarding the response of neural stem cells (NSCs) to NIR stimulation,particularly in anxiety-like behavior. The present study aimed to develop a new optical manipulation approach to stimulate hippocampal neurogenesis and understand the mechanisms underlying its anxiolytic effects. We used 940 nm NIR (40 Hz) light exposure to stimulate hippocampal stem cells in C57BL/6 mice. The enhanced proliferation and astrocyte differentiation of NIR-treated NSCs were assessed using 5-ethynyl-2’-deoxyuridine (EdU) incorporation and immunofluorescence assays. Additionally,we evaluated calcium activity of NIR light-treated astrocytes using GCaMP6f recording through fluorescence fiber photometry. The effects of NIR illumination of the hippocampus on anxiety-like behaviors were evaluated using elevated plus maze and open-field test. NIR light effectively promoted NSC proliferation and astrocyte differentiation via the OPN4 photoreceptor. Furthermore,NIR stimulation significantly enhanced neurogenesis and calcium-dependent astrocytic activity. Moreover,activating hippocampal astrocytes with 40-Hz NIR light substantially improved anxiety-like behaviors in mice. We found that flickering NIR (940 nm/40Hz) light illumination improved neurogenesis in the hippocampus with anxiolytic effects. This innovative approach holds promise as a novel preventive treatment for depression. The online version contains supplementary material available at 10.1186/s13287-024-04114-3. View Publication

Mild therapeutic hypothermia showed potential neuroprotective properties during and after cerebral hypoxia or ischemia in experimental animal studies. However,in clinical trials,where hypothermia is mainly applied after reperfusion,results were divergent and neurophysiological effects unclear. In our current study,we employed human-derived neuronal networks to investigate how treatment with hypothermia during hypoxia influences neuronal functionality and whether it improves post-hypoxic recovery. We differentiated neuronal networks from human induced pluripotent stem cells on micro-electrode arrays (MEAs). We studied the effect of hypothermia (34°C)–as well hyperthermia (39°C) ‐ on neuronal functionality during and after hypoxia using MEAs. We also studied the effects on the number of synaptic puncta and cell viability by immunocytochemistry. In comparison to neuronal networks under normothermia,we found that hypothermia during hypoxia improved functional neuronal network recovery,expressed as enhanced neuronal network activity. This was associated with prevention of synaptic loss during and after the hypoxic phase. Furthermore,hypothermia improved cell viability after the hypoxic phase. Instead,hyperthermia during hypoxia had detrimental effects,with an irreversible loss of neuronal network function,loss of synaptic puncta and decreased cell viability. Our results show potential neuroprotective properties of hypothermia occurring during hypoxia,indicating that administering hypothermia to bridge the time to reperfusion may be beneficial in clinical settings. View Publication

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by the accumulation of pathological proteins and synaptic dysfunction. This study aims to investigate the molecular and functional differences between human induced pluripotent stem cells (hiPSCs) derived from patients with sporadic AD (sAD) and age-matched controls (healthy subjects,HS),focusing on their neuronal differentiation and synaptic properties in order to better understand the cellular and molecular mechanisms underlying AD pathology. Skin fibroblasts from sAD patients ( n = 5) and HS subjects ( n = 5) were reprogrammed into hiPSCs using non-integrating Sendai virus vectors. Through karyotyping,we assessed pluripotency markers (OCT4,SOX2,TRA-1–60) and genomic integrity. Neuronal differentiation was evaluated by immunostaining for MAP2 and NEUN. Electrophysiological properties were measured using whole-cell patch-clamp,while protein expression of Aβ,phosphorylated tau,Synapsin-1,Synaptophysin,PSD95,and GluA1 was quantified by western blot. We then focused on PAK1-LIMK1-Cofilin signaling,which plays a key role in regulating synaptic structure and function,both of which are disrupted in neurodegenerative diseases such as AD. sAD and HS hiPSCs displayed similar stemness features and genomic stability. However,they differed in neuronal differentiation and function. sAD-derived neurons (sAD-hNs) displayed increased levels of AD-related proteins,including Aβ and phosphorylated tau. Electrophysiological analyses revealed that while both sAD- and HS-hNs generated action potentials,sAD-hNs exhibited decreased spontaneous synaptic activity. Significant reductions in the expression of synaptic proteins such as Synapsin-1,Synaptophysin,PSD95,and GluA1 were found in sAD-hNs,which are also characterized by reduced neurite length,indicating impaired differentiation. Notably,sAD-hNs demonstrated a marked reduction in LIMK1 phosphorylation,which could be the underlying cause for the changes in cytoskeletal dynamics that we found,leading to the morphological and functional modifications observed in sAD-hNs. To further investigate the involvement of the LIMK1 pathway in the morphological and functional changes observed in sAD neurons,we conducted perturbation experiments using the specific LIMK1 inhibitor,BMS-5. Neurons obtained from healthy subjects treated with the inhibitor showed similar morphological changes to those observed in sAD neurons,confirming that LIMK1 activity is crucial for maintaining normal neuronal structure. Furthermore,administration of the inhibitor to sAD neurons did not exacerbate the morphological alterations,suggesting that LIMK1 activity is already compromised in these cells. Our findings demonstrate that although sAD- and HS-hiPSCs are similar in their stemness and genomic stability,sAD-hNs exhibit distinct functional and structural anomalies mirroring AD pathology. These anomalies include synaptic dysfunction,altered cytoskeletal organization,and accumulation of AD-related proteins. Our study underscores the usefulness of hiPSCs in modeling AD and provides insights into the disease's molecular underpinnings,thus highlighting potential therapeutic targets. The online version contains supplementary material available at 10.1186/s13195-024-01632-3. View Publication

Cancer-associated fibroblasts (CAFs) play pivotal roles in solid tumor initiation,growth,and immune evasion. However,the optimal biomimetic modeling conditions remain elusive. In this study,we investigated the effects of 2D and 3D culturing conditions on human primary CAFs integrated into a modular tumor microenvironment (TME). Using single-nucleus RNA sequencing (snRNAseq) and Proteomics’ Proximity Extension Assays,we characterized CAF transcriptomic profiles and cytokine levels. Remarkably,when cultured in 2D,CAFs exhibited a myofibroblast (myCAF) subtype,whereas in 3D tumor spheroid cultures,CAFs displayed a more inflammatory (iCAF) pathological state. By integrating single-cell gene expression data with functional interrogations of critical TME-related processes [natural killer (NK)-mediated tumor killing,monocyte migration,and macrophage differentiation],we were able to reconcile form with function. In 3D TME spheroid models,CAFs enhance cancer cell growth and immunologically shield cells from NK cell-mediated cytotoxicity,in striking contrast with their 2D TME counterparts. Notably,3D CAF-secreted proteins manifest a more immunosuppressive profile by enhancing monocyte transendothelial migration and differentiation into M2-like tumor-associated macrophages (TAMs). Our findings reveal a more immunosuppressive and clinically relevant desmoplastic TME model that can be employed in industrial drug discovery campaigns to expand the cellular target range of chemotherapeutics. View Publication

The challenge of expanding haematopoietic stem/progenitor cells (HSPCs) in vitro has limited their clinical application. Human hair follicle mesenchymal stem cells (hHFMSCs) can be reprogrammed to generate intermediate stem cells by transducing OCT4 (hHFMSCs OCT4 ) and pre-inducing with FLT3LG/SCF,and differentiated into erythrocytes. These intermediate cells exhibit gene expression patterns similar to pre-HSCs,making them promising for artificial haematopoiesis. However,further investigation is required to elucidate the in vitro proliferation ability and mechanism underlying the self-renewal of pre-HSCs derived from hHFMSCs. hHFMSCs OCT4 were pre-treated with FLT3LG and SCF cytokines,followed by characterization and isolation of the floating cell subsets for erythroid differentiation through stimulation with hematopoietic cytokines and nutritional factors. Cell adhesion was assessed through disassociation and adhesion assays. OCT4 expression levels were measured using immunofluorescence staining,RT-qPCR,and Western blotting. RNA sequencing and Gene Ontology (GO) enrichment analysis were then conducted to identify proliferation-related biological processes. Proliferative capacity was evaluated through CCK-8,colony formation assays,Ki67 index,and cell cycle analysis. Cytoskeleton was observed through Wright‒Giemsa,Coomassie brilliant blue,and phalloidin staining. Expression of adherens junction (AJ) core members was confirmed through RT‒qPCR,Western blotting,and immunofluorescence staining before and after ZO-1 knockdown. A regulatory network was constructed to determine relationships among cytoskeleton,proliferation,and the AJ pathway. Student’s t tests (GraphPad Prism 8.0.2) were used for group comparisons. The results were considered significant at P < 0.05. Pre-treatment of hHFMSCs OCT4 with FLT3LG and SCF leads to the emergence of floating cell subsets exhibiting small,globoid morphology,suspended above adherent cells,forming colonies,and displaying minimal expression of CD45. Excessive OCT4 expression weakens adhesion in floating hHFMSCs OCT4 . Floating cells moderately enhanced proliferation and undergo cytoskeleton remodelling,with increased contraction and aggregation of F-actin near the nucleus. The upregulation of ZO-1 could impact the expressions of F-actin,E-cadherin,and β-catenin genes,as well as the nuclear positioning of β-catenin,leading to variations in the cytoskeleton and cell cycle. Finally,a regulatory network revealed that the AJ pathway cored with ZO-1 critically bridges cytoskeletal remodelling and haematopoiesis-related proliferation in a β-catenin-dependent manner. ZO-1 improved the self-renewal of pre-HSCs from OCT4-overexpressing hHFMSCs by remodeling the cytoskeleton via the ZO-1-regulated AJ pathway,suggesting floating hHFMSCs OCT4 as the promising seed cells for artificial hematopoiesis. The online version contains supplementary material available at 10.1186/s13287-024-04080-w. View Publication

Infusion of T cells modified with a chimeric antigen receptor (CAR) targeting CD19 has achieved exceptional responses in patients with non-Hodgkin’s lymphoma (NHL),which led to the approval of CAR targeting CD19 (CART19) (Axi-cel and Liso-cel) as second line of treatment for adult patients with relapsed/refractory NHL. Unfortunately,60% of patients still relapse after CART19 due to either a loss of expression of the target antigen (CD19) in the tumor cell,observed in 27% of relapsed patients,a limited CAR-T persistence,and additional mechanisms,including the suppression of the tumor microenvironment. Clinic strategies to prevent target antigen loss include sequential treatment with CARs directed at CD20 or CD22,which have caused loss of the second antigen,suggesting targeting other antigens less prone to disappear. CD79b,expressed in NHL,is a target in patients treated with antibody-drug conjugates (ADC). However,the limited efficacy of ADC suggests that a CAR therapy targeting CD79b might improve results. We designed three new CARs against CD79b termed CAR for Lymphoma (CARLY)1,2 and 3. We compared their efficacy,phenotype,and inflammatory profiles with CART19 (ARI0001) and CARTBCMA (ARI0002h),which can treat NHL. We also analyzed the target antigen’s expression loss (CD79b,CD19,and B-cell maturation antigen(BCMA)). We found that CARLY2 and CARLY3 had high affinity and specificity towards CD79b on B cells. In vitro,all CAR-T cells had similar anti-NHL efficacy,which was retained in an NHL model of CD19 − relapse. In vivo,CARLY3 showed the highest efficacy. Analysis of the loss of the target antigen demonstrated that CARLY cells induced CD79b and CD19 downregulation on NHL cells with concomitant trogocytosis of these antigens to T cells,being most notorious in CARLY2,which had the highest affinity towards CD79b and CD19,and supporting the selection of CARLY3 to design a new treatment for patients with NHL. Finally,we created a CAR treatment based on dual targeting of CD79b and BCMA to avoid losing the target antigen. This treatment showed the highest efficacy and did not cause loss of the target antigen. Based on specificity,efficacy,and loss of the target antigen,CARLY3 represents a potential novel CAR treatment for NHL. View Publication

Inflammation in cystic fibrosis (CF) airways is difficult to treat with well-established regimens often including azithromycin (AZ) as an immunomodulatory drug. As AZ has been reported to require CF transmembrane conductance regulator (CFTR) to be able to reduce interleukin (IL)-8 and given the emergence of highly effective CFTR “triple” modulator therapy (elexacaftor/tezacaftor/ivacaftor; ETI),the aim of this study was to investigate the effect of AZ and ETI,singly and in combination,on ion channel activity and to assess the potential anti-inflammatory effects. Electrophysiological assessment of ETI and AZ was performed on three-dimensional cultures of primary CF human bronchial epithelial (HBE) cells using a Multi Trans-Epithelial Current Clamp. IL-8 from NuLi-1 (non-CF) and CuFi-1 (CF) cells treated with AZ was measured by ELISA. Inflammatory mediators from primary CF HBE cells exposed to tumour necrosis factor-α in the presence of AZ,ETI and their combination,were screened using the Proteome Profiler™ Human Cytokine Array Kit,with selected targets validated by ELISA. AZ did not alter CFTR chloride efflux,nor did it have any synergistic/antagonistic effect in combination with ETI. AZ reduced IL-8 in NuLi-1 but not CuFi-1 cells. The Proteome Profiler™ screen identified several disease-relevant cytokines that were modulated by treatment. Subsequent analysis by ELISA showed IL-8,IL-6,CXCL1 and granulocyte–macrophage colony-stimulating factor to be significantly reduced by treatment with ETI,but not by AZ. Incorporating ETI into the standard of CF care provides an opportunity to re-evaluate therapeutic regimens to reduce treatment burden and safely discontinue chronic treatments such as AZ,without loss of clinical benefit. Identification of redundant treatments in the era of CFTR modulation may improve medication adherence and overcome potential adverse effects associated with the chronic use AZ and other drugs. View Publication