技术资料

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

The Sleeping Beauty (SB) transposon system is a useful tool for genetic applications,including gene therapy. We discovered a hyperactive variant of the SB100X transposase,called SB200X. This mutant,resulting from a specific amino acid replacement (Q124C),showed an ∼2-fold increase in transposition activity in various human and murine cells. Other amino acid replacements in position 124 also led to a hyperactive phenotype. Position 124 is located at the very edge of the linker region that connects the DNA-binding and catalytic domains of the transposase. Consistent with a role of the linker in an autoregulatory mechanism called overproduction inhibition (OPI) in the monophyletic group of mariner transposases,we show that the hyperactivity of Q124C manifests at high concentrations of the transposase,suggesting a partial resistance of SB200X to OPI. We demonstrate that the hyperactive phenotype of Q124C can be combined with features of other useful mutations in the SB transposase. Namely,Q124C improves the transposition efficiency of the previously described K248R variant,while maintaining or even slightly improving its safer genome-wide integration profile. The SB200X transposase could enhance the utility of SB transposon-mediated genome engineering in preclinical and clinical applications. View Publication

Rationale: Immune checkpoint inhibitors (ICIs) have demonstrated significant efficacy against head and neck squamous cell carcinoma (HNSCC),but their overall response rate (ORR) remains limited. Previous studies have highlighted the crucial role of sphingosine kinases (SPHKs) in the tumor microenvironment (TME); however,their function in immunotherapy remains unclear. Methods: We conducted comprehensive bioinformatics analysis,functional studies,and clinical validation,to investigate the role of SPHK1 in the immunology of HNSCC. Results: Functionally,SPHK1 significantly promoted tumor growth by inhibiting anti-tumor immunity in immune-competent HNSCC mouse models and tumor-T cell co-cultures. Mechanistic analysis revealed that SPHK1 regulated matrix metalloproteinase-1 (MMP1) expression via the MAPK1 pathway,which subsequently influenced tumor programmed cell death ligand 1 (PD-L1) expression. Furthermore,SPHK1 and MMP1 could predict the efficacy of programmed cell death 1 monoclonal antibody (PD-1 mAb) immunotherapy in HNSCC and were independent risk factors for survival in patients with HNSCC. Conclusion: Our study reveals a novel role for SPHK1 in mediating immune evasion in HNSCC through the regulation of the MMP1-PD-L1 axis. We identified SPHK1 and MMP1 as predictive biomarkers for the therapeutic response to PD-1 mAb and provided new therapeutic targets for patients with HNSCC. View Publication

It is advantageous to culture the ex vivo retina and other tissues at the air-liquid interface to allow for more efficient gas exchange. However,gene delivery to these cultures can be challenging. Electroporation is a fast and robust method of gene delivery,but typically requires submergence in liquid buffer for electrical current flow. We have developed a submergence-free electroporation technique that incorporates an agarose hydrogel disk between the positive electrode and retina. Inner retinal neurons and Müller glia are transfected with increased propensity toward Müller glia transfection after extended time in culture. We also observed an increase in BrdU incorporation in Müller glia following electrical stimulation,and variation in detection of transfected cells from expression vectors with different promoters. This method advances our ability to use ex vivo retinal tissue for genetic studies and should be adaptable for other tissues cultured at an air-liquid interface. Subject areas: Genetic engineering,Methodology in biological sciences,Bioelectrical engineering View Publication

In clinical chimeric antigen receptor (CAR) T cell therapy,one of the strongest correlates of favorable patient responses is lower levels of differentiation in T cells from the peripheral blood mononuclear cell (PBMC) starting material or the CAR T cell product. T cells from older patients are inherently more differentiated,but we hypothesised that specific activation protocols could be used to limit CAR T cell differentiation during manufacturing,particularly in older patients. We used PBMCs from young (20–30 years old) and older (60+ years old) healthy donors to generate CAR T cells using two activation protocols: soluble anti‐(α) CD3 monoclonal antibody (mAb) vs immune complexes of αCD3 and αCD28 mAbs. Products were assessed for yield,function and differentiation,which was used as a measure of CAR T cell quality. T cells in PBMCs were assessed for CD28 expression and correlative analyses were performed. Older samples generated fewer,more differentiated CAR T cells than young samples,and the αCD3/CD28 mAb protocol exacerbated this,further reducing yield and quality. CD28 expression by T cells correlated with CAR T cell differentiation,but T cell differentiation in PBMC starting material was a stronger correlate of CAR T cell differentiation. Choice of activation protocol can substantially impact on the yield and quality of CAR T cells during manufacturing. This is a key consideration for older patients whose samples already generate a poorer yield and lower quality of CAR T cells. View Publication

Erythropoiesis is a crucial process in hematopoiesis,yet it remains highly susceptible to disruption by various diseases,which significantly contribute to the global challenges of anemia and blood shortages. Current treatments like erythropoietin (EPO) or glucocorticoids often fall short,especially for hereditary anemias such as Diamond-Blackfan anemia (DBA). To uncover new erythropoiesis-stimulating agents,we devised a screening system using primary human hematopoietic stem and progenitor cells (HSPCs). We discovered that BRAF inhibitors (BRAFi),commonly used to treat BRAF V600E melanoma,can unexpectedly and effectively promote progenitor cell proliferation by temporarily delaying erythroid differentiation. Notably,these inhibitors exhibited pronounced efficacy even under cytokine-restricted conditions and in patient samples of DBA. Mechanistically,although these BRAFi inhibit the MAPK cascade in BRAF V600E mutant cells,they paradoxically act as amplifiers in wild-type BRAF cells,potently enhancing the cascade. Furthermore,we found that while the oncogenic BRAF V600E mutation disrupts hematopoiesis and erythropoiesis through AP-1 hyperactivation,BRAFi minimally impact HSPC self-renewal and differentiation. In vivo studies have shown that BRAFi can enhance human hematopoiesis and erythropoiesis in severe immunodeficient mouse models and alleviate anemia in the Rpl11 haploinsufficiency DBA model,as well as other relevant anemia models. This discovery underscores the role of the MAPK pathway in hematopoiesis and positions BRAFi as a promising therapeutic option for improving hematopoietic reconstitution and treating anemias,including DBA. Subject terms: Drug screening,Molecular medicine View Publication

Apigenin (API),a traditionally sourced flavonoid,is recognized for its anti-neoplastic properties. Despite well-documented effects on tumorigenesis,the detailed therapeutic impact on breast cancer stem cells (BCSCs) and the associated molecular mechanisms are yet to be clarified. The objective of this study is to elucidate the therapeutic effects of API on BCSCs and to uncover its molecular mechanisms through network pharmacology and experimental validation. Interactions of API with candidate targets were examined through target screening,enrichment analysis,construction of protein-protein interaction networks,and molecular docking. MCF-7-derived BCSCs were utilized as a model system to investigate and substantiate the anti-BCSC effects of API and the underlying mechanism. Molecular docking studies have shown that API and TP53 exhibit favorable binding affinity. Compared with the negative control group,API effectively suppressed the expression of BCSC-related proteins such as ALDH1A1,NANOG,EpCAM,and MYC,downregulated p-PI3K and p-AKT,and upregulated p53. This study demonstrates that API can play an anti-BCSC role by regulating the PI3K/AKT/p53 pathway in BCSCs of MCF-7 cells,highlighting its potential as a therapeutic agent for targeting BCSCs. View Publication

Breast cancer is a heterogeneous disease comprising multiple molecularly distinct subtypes with varied prevalence,prognostics,and treatment strategies. Among them,triple-negative breast cancer,though the least prevalent,is the most aggressive subtype,with limited therapeutic options. Recent emergence of competing endogenous RNA (ceRNA) networks has highlighted how long noncoding RNAs (lncRNAs),microRNAs (miRs),and mRNA orchestrate a complex interplay meticulously modulating mRNA functionality. Focusing on TNBC,this study aimed to construct a ceRNA network using differentially expressed lncRNAs,miRs,and mRNAs. We queried the differentially expressed lncRNAs (DElncRNAs) between TNBC and luminal samples and found 389 upregulated and 386 downregulated lncRNAs,including novel transcripts in TNBC. DElncRNAs were further evaluated for their clinical,functional,and mechanistic relevance to TNBCs using the lnc2cancer 3.0 database,which presented LUCAT1 (lung cancer-associated transcript 1) as a putative node. Next,the ceRNA network (lncRNA–miRNA–mRNA) of LUCAT1 was established. Several miRNA–mRNA connections of LUCAT1 implicated in regulating stemness (LUCAT1-miR-375-Yap1,LUCAT1-miR181-5p-Wnt,LUCAT1-miR-199a-5p-ZEB1),apoptosis (LUCAT1-miR-181c-5p-Bcl2),drug efflux (LUCAT1-miR-200c-ABCB1,LRP1,MRP5,MDR1),and sheddase activities (LUCAT1-miR-493-5p-ADAM10) were identified,indicating an intricate regulatory mechanism of LUCAT1 in TNBC. Indeed,LUCAT1 silencing led to mitigated cell growth,migration,and stem-like features in TNBC. This work sheds light on the LUCAT1 ceRNA network in TNBC and implies its involvement in TNBC growth and progression. View Publication

The introduction of antibody–drug conjugates represents a significant advancement in targeted therapy of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Our study aims to investigate the role of the DNA damage response pathway and the impact of PARP1 inhibition,utilizing talazoparib,on the response of AML and ALL cells to Gemtuzumab ozogamicin (GO) and Inotuzumab ozogamicin (INO),respectively. AML and ALL cells were treated with GO,INO and γ-calicheamicin in order to induce severe DNA damage and activate the G2/M cell-cycle checkpoint in a dose- and time-dependent manner. The efficacy of PARP1 inhibitors and,in particular,talazoparib in enhancing INO or GO against ALL or AML cells was assessed through measurements of cell viability,cell death,cell cycle progression,DNA damage repair,accumulation of mitotic DNA damage and inhibition of clonogenic capacity. We observed that both ALL and AML cell lines activate the G2/M cell-cycle checkpoint in response to γ-calicheamicin-induced DNA damage,highlighting a shared cellular response mechanism. Talazoparib significantly enhanced the efficacy of INO against ALL cell lines,resulting in reduced cell viability,increased cell death,G2/M cell-cycle checkpoint override,accumulation of mitotic DNA damage and inhibition of clonogenic capacity. Strong synergism was observed in primary ALL cells treated with the combination. In contrast,AML cells exhibited a heterogeneous response to talazoparib in combination with GO. Our findings suggest a potential link between the differential responses of ALL and AML cells to the drug combinations and the ability of talazoparibto override G2/M cell-cycle arrest induced by antibody–drug conjugates. PARP1 emerges as a key player in the response of ALL cells to INO and represents a promising target for therapeutic intervention in this leukemia setting. Our study sheds light on the intricate interplay between the DNA damage response pathway,PARP1 inhibition,and response of γ-calicheamicin-induced DNA damages in AML and ALL. These findings underscore the importance of targeted therapeutic strategies and pave the way for future research aimed at optimizing leukemia treatment approaches. The online version contains supplementary material available at 10.1186/s12967-024-05838-9. View Publication