技术资料

During lung cancer metastasis,tumor cells undergo epithelial-to-mesenchymal transition (EMT),enabling them to intravasate through the vascular barrier and enter the circulation before colonizing secondary sites. Here,a human in vitro microphysiological model of EMT-driven lung cancer intravasation-on-a-chip was developed and coupled with machine learning (ML)-assisted automatic identification and quantification of intravasation events. A robust EMT-inducing cocktail (EMT-IC) was formulated by augmenting macrophage-conditioned medium with transforming growth factor-β1. When introduced into microvascular networks (MVNs) in microfluidic devices,EMT-IC did not affect MVN stability and physiologically relevant barrier functions. To model lung cancer intravasation on-a-chip,EMT-IC was supplemented into co-cultures of lung tumor micromasses and MVNs. Wihin 24 h of exposure,EMT-IC facilitated the insertion of membrane protrusions of migratory A549 cells into microvascular structures,followed by successful intravasation. EMT-IC reduced key basement membrane and vascular junction proteins - laminin and VE-Cadherin - rendering vessel walls more permissive to intravasating cells. ML-assisted vessel segmentation combined with co-localization analysis to detect intravasation events confirmed that EMT induction significantly increased the number of intravasation events. Introducing metastatic (NCI-H1975) and non-metastatic (BEAS-2B) cell lines demonstrated that both,baseline intravasation potential and responsiveness to EMT-IC,are reflected in the metastatic predisposition of lung cancer cell lines,highlighting the model's universal applicability and cell-specific sensitivity. The reproducible detection of intravasation events in the established model provides a physiologically relevant platform to study processes of cancer metastasis with high spatio-temporal resolution and short timeframe. This approach holds promise for improved drug development and informed personalized patient treatment plans. View Publication

Chronic myeloid leukemia (CML) remains a therapeutic challenge,particularly in patients who develop resistance to standard tyrosine kinase inhibitors (TKIs) such as imatinib. Here,we present the first demonstration of the potent anti-leukemic activity of the histone deacetylase (HDAC) inhibitor martinostat in both TKI-sensitive and TKI-resistant CML. Structural and biochemical analyses confirmed the efficient and selective binding of martinostat to HDAC isoenzyme ligand-binding pockets,resulting in histone and tubulin hyperacetylation in both imatinib-sensitive and resistant CML cells,outperforming vorinostat,a clinically used HDAC inhibitor (HDACi). It selectively impaired CML cell proliferation and viability and induced apoptosis across various CML models,including resistant cell models and patient blasts,with minimal toxicity to healthy cells and low developmental toxicity in zebrafish. In addition to its single-agent efficacy,martinostat demonstrated enhanced anticancer effects when combined with imatinib,both in vitro and in vivo,significantly reducing tumor growth in resistant CML xenograft models. Mechanistically,mRNA-seq data showed that martinostat disrupted key survival signaling pathways and amplified apoptotic responses,contributing to its anticancer activity. These findings highlight the potential of martinostat as a selective,low-toxicity HDACi that,combined with TKIs,could provide an effective strategy to overcome drug resistance in CML and improve therapeutic outcomes. The online version contains supplementary material available at 10.1186/s13148-025-01921-0. View Publication

Secondary and tertiary lymphoid structures are a critical target of suppression in many autoimmune disorders,protein replacement therapies,and in transplantation. Although antigen-specific regulatory T cells (Tregs),such as chimeric antigen receptor (CAR) Tregs,generally persist longer and localize to target tissues more effectively than polyclonal Tregs in animal models,their numbers still progressively decline over time. A potential approach to maximize Treg activity in vivo is the expression of chemokine receptors such as CXCR5,which would enable localization of a greater number of engineered cells at sites of antigen presentation. Indeed,CXCR5 expression on follicular T helper cells and follicular Tregs enables migration toward lymph nodes,B cell zones,and tertiary lymphoid structures that appear in chronically inflamed non-lymphoid tissues. In this study,we generated human and murine CXCR5 co-expressing engineered receptor Tregs and tested them in preclinical mouse models of allo-immunity and hemophilia A,respectively. Additionally,we engineered a murine CXCR5 co-expressing clotting factor VIII (FVIII) specific T cell receptor fusion construct epsilon (FVIII TRuCe CXCR5) Treg to suppress anti-drug antibody development in a model of FVIII protein replacement therapy for hemophilia A. In vitro,anti-HLA-A2 CXCR5+ CAR-Tregs showed enhanced migratory and antigen-specific suppressive capacities compared to untransduced Tregs. When injected into an NSG mouse model of HLA-A2+ pancreatic islet transplantation,anti-HLA-A2 CXCR5+ CAR-Tregs maintained a good safety profile allowing for long-term graft survival in contrast to anti-HLA-A2 CXCR5+ conventional CAR-T (Tconv) cells that eliminated the graft. Similarly,FVIII TRuCe CXCR5 Treg demonstrated increased in vivo persistence and suppressive capacity in a murine model of hemophilia A. Collectively,our findings indicate that CXCR5 co-expression is safe and enhances in vivo localization and persistence in target tissues. This strategy can potentially promote targeted tolerance without the risk of off-target effects in multiple disease models. View Publication

CRISPR/Cas9 genome editing has emerged as a promising treatment for genetic diseases like β-thalassemia. Editing γ-globin promoters to disrupt ZBTB7A/LRF or BCL11A binding sites has shown potential for reactivating fetal hemoglobin and treating sickle cell disease. However,its application to β 0 -thalassemia/HbE disease remains unclear. This study utilized CRISPR/Cas9 to disrupt these sites in mobilized CD34 + hematopoietic stem /progenitor cells from healthy donors and β 0 -thalassemia/HbE patients. The editing efficiency for the BCL11A site (75–92%) was higher than for the ZBTB7A/LRF site (57–60%). Both disruptions similarly increased fetal hemoglobin production in healthy donors ( BCL11A 26.2 ± 1.4%,ZBTB7A/LRF 27.9 ± 1.5%) and β 0 -thalassemia/HbE cells ( BCL11A 62.7 ± 0.9%,ZBTB7A/LRF 64.0 ± 1.6%). Off-target effects were absent in BCL11A -edited cells but observed at low frequencies in ZBTB7A/LRF -edited cells. Neither disruption significantly affected erythroid differentiation. These findings highlight the comparable contributions of ZBTB7A/LRF and BCL11A binding sites to γ-globin reactivation. CRISPR/Cas9 editing of either site may offer a potential therapeutic strategy for β 0 -thalassemia/HbE disease. View Publication

MED12 is a key regulator of transcription and chromatin architecture,essential for normal hematopoiesis. While its dysregulation has been implicated in hematological malignancies,the mechanisms driving its upregulation in acute myeloid leukemia (AML) remain poorly understood. We investigated MED12 expression across AML subgroups by integrating chromatin accessibility profiling,histone modification landscapes,and DNA methylation (DNAm) patterns. Functional assays using DNMT inhibition were performed to dissect the underlying regulatory mechanisms. MED12 shows subtype-specific upregulation in AML compared to hematopoietic stem and progenitor cells,independent of somatic mutations. Chromatin accessibility profiling reveals that the MED12 locus is epigenetically primed in AML blasts,with increased DNase hypersensitivity at regulatory elements. Histone modification analysis demonstrates strong H3K4me3 and H3K27ac enrichment around the transcription start site (TSS),consistent with promoter activation,while upstream and intragenic regions exhibit enhancer-associated marks (H3K4me1,H3K27ac). Notably,hypermethylation within TSS-proximal regulatory regions (TPRRs)—including promoter-overlapping and adjacent CpG islands—correlates with ectopic MED12 overexpression,challenging the canonical view of DNAm as strictly repressive. Functional studies show that DNMT inhibition via 5-azacytidine reduces MED12 expression despite promoter demethylation in cells with hypermethylated TPRRs,suggesting a noncanonical role for DNA methylation in maintaining active transcription. Furthermore,MED12 expression positively correlates with DNMT3A and DNMT3B expression,implicating these methyltransferases in sustaining its epigenetic activation. This study identifies a novel regulatory axis in which aberrant DNA methylation,rather than genetic mutation,drives MED12 upregulation in AML. Our findings suggest that TPRR hypermethylation may function noncanonically to support transcriptional activation,likely in cooperation with enhancer elements. These results underscore the importance of epigenetic mechanisms in AML and highlight enhancer-linked methylation as a potential contributor to oncogene dysregulation. Future studies should further explore the role of noncanonical methylation-mediated gene activation in AML pathogenesis and therapeutic targeting. The online version contains supplementary material available at 10.1186/s13072-025-00610-9. View Publication

This work focused on generating a three-dimensional (3D) in vitro dynamic model to study chronic lymphocytic leukemia (CLL) cell dissemination,homing,and mechanisms of therapy resistance. We used a gelatin-based,hard porous biomaterial as a support matrix to develop 3D tissue-like models of the human lymph node and bone marrow,which were matured inside bioreactors under dynamic perfusion of medium. Comparing static and dynamic cultures of these 3D constructs revealed that perfusion promoted a tissue-like internal organization of cells,characterized by the expression of specific functional markers and deposition of an intricate extracellular matrix protein network. Recirculation of CLL cells within the dynamic system led to changes in leukemic cell behavior and in the expression of key markers involved in tumor progression. These findings suggest that the model is well suited for investigating the pathophysiological mechanisms of CLL and potentially other hematological malignancies. View Publication

Chronic myeloid leukemia (CML) is a leading example of a malignancy where a molecular targeted therapy revolutionized treatment but has rarely led to cures. Overcoming tyrosine kinase inhibitor (TKI) drug resistance remains a challenge in the treatment of CML. We have recently identified miR-185 as a predictive biomarker where reduced expression in CD34 + treatment-naïve CML cells was associated with TKI resistance. We have also identified PAK6 as a target gene of miR-185 that was upregulated in CD34 + TKI-nonresponder cells. However,its role in regulating TKI resistance remains largely unknown. In this study,we specifically targeted PAK6 in imatinib (IM)-resistant cells and CD34 + stem/progenitor cells from IM-nonresponders using a lentiviral-mediated PAK6 knockdown strategy. Interestingly,the genetic and pharmacological suppression of PAK6 significantly reduced proliferation and increased apoptosis in TKI-resistant cells. Cell survivability was further diminished when IM was combined with PAK6 knockdown. Importantly,PAK6 inhibition in TKI-resistant cells induced cell cycle arrest in the G2-M phase and cellular senescence,accompanied by increased levels of DNA damage-associated senescence markers. Mechanically,we identified a PAK6-mediated MDM2-p21 axis that regulates cell cycle arrest and senescence. Thus,PAK6 plays a critical role in determining alternative cell fates in leukemic cells,and targeting PAK6 may offer a therapeutic strategy to selectively eradicate TKI-resistant cells. View Publication

This study focuses on improving the identification of chicken primordial germ cells (PGCs),which are vital for genetic transmission and biotechnological applications. Traditional markers like SSEA1 and CVH have limitations—SSEA1 lacks specificity,and CVH is intracellular. A monoclonal antibody was generated by injecting chicken PGCs into mice,producing one that specifically binds to PGCs and decreases with cell differentiation. Mass spectrometry identified its target as the MYH9 protein. The resulting αMYH9 antibody effectively labels PGCs at various developmental stages,offering a valuable tool for isolating viable PGCs and advancing avian genetics,agriculture,and biotechnology. View Publication

Colorectal cancer (CRC) is a prevalent malignant tumor globally,ranking third in incidence and second in mortality. Metastasis is the main cause of death in patients with CRC. Solanum nigrum L. (SNL),a traditional Chinese medicinal herb endowed with detoxification,blood circulation enhancement,and anti-swelling properties,has been widely used in folk prescriptions for cancer treatment in China. Solamargine (SM) is the major steroidal alkaloid glycoside purified from SNL. However,its role and mechanism against metastatic CRC are not yet clear. The purpose of this study was to evaluate the inhibitory effect of SM on human hepatic metastatic CRC and investigate its underlying mechanism. CCK-8 assay,colony-formation assay,transwell assay,flow cytometry,tumoursphere formation assay,reverse-transcription quantitative PCR (RT-qPCR),Western blotting,transcriptomic sequencing and ferroptosis analysis were performed to reveal the efficacy and the underlying mechanism of SM in CRC cell lines. In vivo,allograft model,patient-derived xenograft (PDX) model,and liver metastatic model were performed to verify the effect of SM on the growth and metastasis of CRC. SM was found to suppress hepatic metastasis in CRC by effectively targeting key cellular processes,including proliferation,survival,and stemness. RNA sequencing showed that SM could induce ferroptosis,which was confirmed by elevated lipid reactive oxygen species (ROS) and downregulated glutathione peroxidase 4 (GPX4) and glutathione synthetase (GSS) in CRC cells and xenografts. Induction of ferroptosis by SM was regulated by nuclear factor erythroid 2-related factor 2 (Nrf2). Furthermore,downregulation of β-catenin was found to be fundamental for the SM-enabled cancer stem cells (CSCs) elimination and metastasis blockage in CRC. Our results indicated that SM is a promising therapeutic drug to inhibit hepatic metastasis in CRC by inducing ferroptosis and impeding CSCs. The online version contains supplementary material available at 10.1186/s13020-025-01171-5. View Publication

Cell therapy is promising for heart failure treatment,with growing interest in cardiovascular progenitor cells (CPCs) from pluripotent stem cells. A major challenge is managing the immune response,due to their allogeneic source. Regulatory T cells (Treg) offer an alternative to pharmacological immunosuppression by inducing immune tolerance. This study assesses whether Treg therapy can mitigate the xeno-immune response,improving cardiac outcomes in a mouse model of human CPC intramyocardial transplantation. CPCs stimulated immune responses in allogeneic and xenogeneic settings,causing proliferation in T cell subsets. Tregs showed immunosuppressive effects on T lymphocyte populations when co-cultured with CPCs. Post infarction,CPCs were transplanted intramyocardially into an immune-competent mouse model 3 weeks after myocardial infarction. Human or murine Tregs were intravenously administered on transplantation day and three days later. Control groups received CPCs without Tregs or saline (PBS). CPCs with Tregs improved LV systolic function in three weeks,linked to reduced myocardial fibrosis and enhanced angiogenesis. This was accompanied by decreased splenocyte NK cell populations and pro-inflammatory cytokine levels in cardiac tissue. Treg therapy with CPC transplantation enhances cardiac functional and structural outcomes in mice. Though it does not directly avert graft rejection,it primarily affects NKG2D+ cytotoxic cells,indicating systemic immune modulation and remote heart repair benefits. View Publication

The growth factor and small molecule protocol are the two primary approaches for generating human induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs). We compared the efficacy of the growth factor and small molecule protocols across fifteen different human iPSC lines. Morphological assessment,relative quantification of gene expression,protein expression and proteomic studies were carried out. HLCs derived from the growth factor protocol displayed mature hepatocyte morphological features including a raised,polygonal shape with well-defined refractile borders,granular cytoplasm with lipid droplets and/or vacuoles with multiple spherical nuclei or a large centrally located nucleus; significantly elevated hepatocyte gene and protein expression including AFP,HNF4A,ALBUMIN,and proteomic and metabolic features that are more aligned with a mature phenotype. HLCs derived from the small molecule protocol showed a dedifferentiated,proliferative phenotype that is more akin to liver tumor-derived cell lines. These experimental results suggest that HLCs derived from growth factors are better suited for studies of metabolism,biotransformation,and viral infection. View Publication

In search for broad-spectrum antivirals,we discover a small molecule inhibitor,RMC-113,that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrate selective inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Lipidomics analysis reveals alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and links its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We identify PIP4K2C’s roles in SARS-CoV-2 entry,RNA replication,and assembly/egress,validating it as a druggable antiviral target. Integrating proteomics,single-cell transcriptomics,and functional assays,reveals that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced autophagic flux impairment. Promoting viral protein degradation by reversing autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C,an understudied kinase,and propose dual PIP4K2C and PIKfyve inhibition as a candidate strategy to combat emerging viruses. Subject terms: SARS-CoV-2,Target identification,Autophagy View Publication