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The maturation of brain microvascular endothelial cells leads to the formation of a tightly sealed monolayer,known as the blood–brain barrier (BBB). The BBB damage is associated with the pathogenesis of age-related neurodegenerative diseases including vascular cognitive impairment and Alzheimer’s disease. Growing knowledge in the field of epigenetics can enhance the understanding of molecular profile of the BBB and has great potential for the development of novel therapeutic strategies or targets to repair a disrupted BBB. Histone deacetylases (HDACs) inhibitors are epigenetic regulators that can induce acetylation of histones and induce open chromatin conformation,promoting gene expression by enhancing the binding of DNA with transcription factors. We investigated how HDAC inhibition influences the barrier integrity using immortalized human endothelial cells (HCMEC/D3) and the human induced pluripotent stem cell (iPSC)-derived brain vascular endothelial cells. The endothelial cells were treated with or without a novel compound named W2A-16. W2A-16 not only activates Wnt/?-catenin signaling but also functions as a class I HDAC inhibitor. We demonstrated that the administration with W2A-16 sustained barrier properties of the monolayer of endothelial cells,as evidenced by increased trans-endothelial electrical resistance (TEER). The BBB-related genes and protein expression were also increased compared with non-treated controls. Analysis of transcript profiles through RNA-sequencing in hCMEC/D3 cells indicated that W2A-16 potentially enhances BBB integrity by influencing genes associated with the regulation of the extracellular microenvironment. These findings collectively propose that the HDAC inhibition by W2A-16 plays a facilitating role in the formation of the BBB. Pharmacological approaches to inhibit HDAC may be a potential therapeutic strategy to boost and/or restore BBB integrity. View Publication

The ovary is one of the first organs to exhibit signs of aging,characterized by reduced tissue function,chronic inflammation,and fibrosis. Multinucleated giant cells (MNGCs),formed by macrophage fusion,typically occur in chronic immune pathologies,including infectious and non-infectious granulomas and the foreign body response,but are also observed in the aging ovary. The function and consequence of ovarian MNGCs remain unknown as their biological activity is highly context-dependent,and their large size has limited their isolation and analysis through technologies such as single-cell RNA sequencing. In this study,we define ovarian MNGCs through a deep analysis of their presence across age and species using advanced imaging technologies as well as their unique transcriptome using laser capture microdissection. MNGCs form complex interconnected networks that increase with age in both mouse and nonhuman primate ovaries. MNGCs are characterized by high Gpnmb expression,a putative marker of ovarian and non-ovarian MNGCs. Pathway analysis highlighted functions in apoptotic cell clearance,lipid metabolism,proteolysis,immune processes,and increased oxidative phosphorylation and antioxidant activity. Thus,MNGCs have signatures related to degradative processes,immune function,and high metabolic activity. These processes were enriched in MNGCs compared to primary ovarian macrophages,suggesting discrete functionality. MNGCs express CD4 and colocalize with T-cells,which were enriched in regions of MNGCs,indicative of a close interaction between these immune cell types. These findings implicate MNGCs in modulation of the ovarian immune landscape during aging given their high penetrance and unique molecular signature that supports degradative and immune functions. Ovarian multinucleated giant cells are a unique macrophage population that arise within the aging mammalian ovary. This study characterizes their transcriptome in mice,uncovering a potential role in degradation of cellular debris and immune signaling,suggesting a potential contribution to ovarian inflammation during aging. View Publication

High-efficiency gene editing in primary human cells is critical for advancing therapeutic development and functional genomics,yet conventional electroporation platforms often require high cell input and are poorly suited to parallelized experiments. Here we introduce a next-generation digital microfluidics (DMF) electroporation platform that enables high-throughput,low-input genome engineering using discrete droplets manipulated on a planar electrode array. The system supports 48 independently programmable reaction sites and integrates seamlessly with laboratory automation,allowing efficient delivery of CRISPR-Cas9 RNPs and mRNA cargo into as few as 3,000 primary human cells per condition. The platform was validated across diverse primary human cell types and cargo modalities,demonstrating efficient delivery of various cargo,with high rates of transfection,gene knockout via non-homologous end joining,and precise knock-in through homology-directed repair. To showcase its utility in functional genomics,we applied the platform to an arrayed CRISPR-Cas9 screen in chronically stimulated human CD4⁺ T cells,identifying novel regulators of exhaustion,including epigenetic and transcriptional modulators. These findings establish our DMF-based electroporation platform as a powerful tool for miniaturized genome engineering in rare or precious cell populations and provide a scalable framework for high-content genetic screening in primary human cells.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-13532-z. View Publication

Of all gynecologic cancers,epithelial-ovarian cancer (OCa) stands out with the highest mortality rates. Despite all efforts,90% of individuals who receive standard surgical and cytotoxic therapy experience disease recurrence. The precise mechanism by which leukemia inhibitory factor (LIF) and its receptor (LIFR) contribute to the progression of OCa remains unknown. Analysis of cancer databases revealed that elevated expression of LIF or LIFR was associated with poor progression-free survival of OCa patients and a predictor of poor response to chemotherapy. Using multiple primary and established OCa cell lines or tissues that represent five subtypes of epithelial-OCa,we demonstrated that LIF/LIFR autocrine signaling is active in OCa. Moreover,treatment with LIFR inhibitor,EC359 significantly reduced OCa cell viability and cell survival with an IC 50 ranging from 5-50 nM. Furthermore,EC359 diminished the stemness of OCa cells. Mechanistic studies using RNA-seq and rescue experiments unveiled that EC359 primarily induced ferroptosis by suppressing the glutathione antioxidant defense system. Using multiple in vitro,ex vivo and in vivo models including cell-based xenografts,patient-derived explants,organoids,and xenograft tumors,we demonstrated that EC359 dramatically reduced the growth and progression of OCa. Additionally,EC359 therapy considerably improved tumor immunogenicity by robust CD45 + leukocyte tumor infiltration and polarizing tumor-associated macrophages (TAMs) toward M1 phenotype while showing no impact on normal T-,B-,and other immune cells. Collectively,our findings indicate that the LIF/LIFR autocrine loop plays an essential role in OCa progression and that EC359 could be a promising therapeutic agent for OCa. Subject terms: Molecular medicine,Ovarian cancer View Publication

Liver cancer stem cells (LCSCs) are thought to drive the metastasis and recurrence,however,the heterogeneity of molecular markers of LCSCs has hindered the development of effective methods to isolate them. This study introduced an effective approach to isolate and culture LCSCs from human primary liver cancer (HPLC),leveraging mouse embryonic fibroblasts (MEFs) as feeder cells in conjunction with using defined medium. Isolated LCSCs were further characterized by multiple approaches. Transcriptome sequencing data analysis was conducted to identify highly expressed genes in LCSCs and classify different subtypes of liver cancers. Total sixteen cell strains were directly isolated from 24 tissues of three types of HPLC without sorting,seven of which could be maintained long-term culture as colony growth on MEFs,which is unique characteristics of stem cells. Even 10 of cloned cells formed the tumors in immunodeficient mice,indicating that those cloned cells were tumorgenic. The histologies and gene expression pattern of human xenografts were very similar to those of HPLC where these cloned cells were isolated. Moreover,putative markers of LCSCs were further verified to all express in cloned cells,confirming that these cells were LCSCs. These cloned LCSCs could be cryopreserved,and still maintained the feature of colony growth on MEFs after the recovery. Compared to suspension culture as conventional approach to culture LCSCs,our approach much better maintained stemness of LCSCs for a long time. To date,these cloned cells could be cultured on MEFs over 12 passages. Moreover,bioinformatics analysis of sequencing data revealed the gene expression profiles in LCSCs,and liver cancers were classified into two subtypes C1 and C2 based on genes associated with the prognosis of LCSCs. Patients of the C2 subtype,which is closely related to the extracellular matrix,were found to be sensitive to treatments such as Cisplatin,Axitinib,JAK1 inhibitors,WNT-c59,Sorafenib,and RO-3306. In summary,this effective approach offers new insights into the molecular landscape of human liver cancers,and the identification of the C2 subtype and its unique response to the treatment pave the way for the creation of more effective,personalized therapeutic strategies. The online version contains supplementary material available at 10.1186/s12967-024-05870-9. 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

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here,we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes,14 potent neutralizing antibodies were identified,with the most potent one,BD-368-2,exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2,respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally,the 3.8 {\AA} cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover,we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether,we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases. View Publication

The placenta and umbilical cord are pre-eminent candidate sources of mesenchymal stem cells (MSCs). However,placenta-derived MSCs (P-MSCs) showed greater proliferation capacity than umbilical cord-derived MSCs (UC-MSCs) in our study. We investigated the drivers of this proliferation difference and elucidated the mechanisms of proliferation regulation. Proteomic profiling and Gene Ontology (GO) functional enrichment were conducted to identify candidate proteins that may influence proliferation. Using lentiviral or small interfering RNA infection,we established overexpression and knockdown models and observed changes in cell proliferation to examine whether a relationship exists between the candidate proteins and proliferation capacity. Real-time quantitative polymerase chain reaction,western blot analysis,and immunofluorescence assays were conducted to elucidate the mechanisms underlying proliferation. Six candidate proteins were selected based on the results of proteomic profiling and GO functional enrichment. Through further validation,yes-associated protein 1 (YAP1) and $\beta$-catenin were confirmed to affect MSCs proliferation rates. YAP1 and $\beta$-catenin showed increased nuclear colocalization during cell expansion. YAP1 overexpression significantly enhanced proliferation capacity and upregulated the expression of both $\beta$-catenin and the transcriptional targets of Wnt signaling,CCND1,and c-MYC,whereas silencing $\beta$-catenin attenuated this influence. We found that YAP1 directly interacts with $\beta$-catenin in the nucleus to form a transcriptional YAP/$\beta$-catenin/TCF4 complex. Our study revealed that YAP1 and $\beta$-catenin caused the different proliferation capacities of P-MSCs and UC-MSCs. Mechanism analysis showed that YAP1 stabilized the nuclear $\beta$-catenin protein,and also triggered the Wnt/$\beta$-catenin pathway,promoting proliferation. View Publication

PURPOSE A major clinical problem in the treatment of breast cancer is the inherent and acquired resistance to antiestrogen therapy. In this study,we sought to determine whether antiprogestin treatment,used as a monotherapy or in combination with antiestrogen therapy,induced growth arrest and active cell death in antiestrogen-resistant breast cancer cells. EXPERIMENTAL DESIGN MCF-7 sublines were established from independent clonal isolations performed in the absence of drug selection and tested for their response to the antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780 (fulvestrant),and the antiprogestin mifepristone (MIF). The cytostatic (growth arrest) effects of the hormones were assessed with proliferation assays,cell counting,flow cytometry,and a determination of the phosphorylation status of the retinoblastoma protein. The cytotoxic (apoptotic) effects were analyzed by assessing increases in caspase activity and cleavage of poly(ADP-ribose) polymerase. RESULTS All of the clonally derived MCF-7 sublines expressed estrogen receptor and progesterone receptor but showed a wide range of antiestrogen sensitivity,including resistance to physiological levels of 4-OHT. Importantly,all of the clones were sensitive to the antiprogestin MIF,whether used as a monotherapy or in combination with 4-OHT. MIF induced retinoblastoma activation,G(1) arrest,and apoptosis preceded by caspase activation. CONCLUSIONS We demonstrate that: (a) estrogen receptor(+)progesterone receptor(+),4-OHT-resistant clonal variants can be isolated from an MCF-7 cell line in the absence of antiestrogen selection; and (b) MIF and MIF plus 4-OHT combination therapy induces growth arrest and active cell death of the antiestrogen-resistant breast cancer cells. These preclinical findings show potential for a combined hormonal regimen of an antiestrogen and an antiprogestin to combat the emergence of antiestrogen-resistant breast cancer cells and,ultimately,improve the therapeutic index of antiestrogen therapy. View Publication

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8alpha expression and their localization in lymphoid organs. Several reports suggest that CD8alpha(+) and CD8alpha(-) DC subsets could be functionally different. In this study,using a panel of MHC class I- and/or class II-restricted peptides,we analyzed CD4(+) and CD8(+) T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First,we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4(+) T cell priming. Second,we showed that in vivo activation of CD4(+) T cells by CD8alpha(+) or CD8alpha(-) DC,injected i.v.,leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8alpha(-) subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8alpha(+) subset. However,IL-5 was produced by CD4(+) T cells activated by both DC subsets. When both CD4(+) and CD8(+) T cells were primed by DC injected i.v.,a similar pattern of cytokines was observed,but,under these conditions,Th1 cytokines were mainly produced by CD8(+) T cells,while Th2 cytokines were produced by CD4(+) T cells. Thus,this study clearly shows that CD4(+) T cell responses do not influence the development of specific CD8(+) T cell cytotoxic responses induced either by CD8alpha(+) or CD8alpha(-) DC subsets. View Publication

In humans the majority of endothelial cells (EC) constitutively express MHC class II Ags. We know that in vitro ECs can activate CD45RO(+) B7-independent CD4(+) T cells to proliferate and produce IL-2. The in vivo correlate of this T cell response is not known,and here we have explored whether endothelial expression of MHC class II Ags affects the transendothelial migration of alloreactive CD4(+) CD45RO(+) B7-independent T cells. Alloreactive CD4(+) T cell clones and lines were generated against HLA-DR11,DR13,DR4,and DR1 MHC Ags,and their rates of migration across untreated EC line Eahy.926 (MHC class II negative) or Eahy.926 transfected with CIITA (EahyCIITA) to express DR11 and DR13 were investigated. The migrations of EahyCIITA-specific T cell clones and lines were retarded in a DR-specific manner,and retardation was reversed in the presence of mAb to DR Ag. When investigating the ability of T cells to proliferate in response to EahyCIITA before and after transmigration,migrated cells were still able to proliferate,but the frequency of EahyCIITA-specific cells was much reduced compared with that of nonmigrated cells. The use of fluorescently labeled T cells revealed that specific cells become trapped within the endothelial monolayer. Pretreatment of EahyCIITA with IFN-gamma restored the ability of DR11- or DR13-specific T cells to transmigrate and proliferate,thus abrogating DR-specific retardation. We conclude that cognate interaction between T cells and endothelial MHC class II initiates a stop signal possibly similar to an immunological synapse,but this is overcome in an inflammatory milieu. View Publication

BACKGROUND: Resistance to imatinib monotherapy frequently emerges in advanced stages of chronic myelogenous leukemia (CML),supporting the rationale for combination drug therapy. In the present study,the activities of the farnesyltransferase inhibitors (FTIs) L744,832 and LB42918,as single agents and in combination with imatinib,were investigated in different imatinib-sensitive and -resistant BCR-ABL-positive CML cells. MATERIALS AND METHODS: Growth inhibition of the cell lines and primary patient cells was assessed by MTT assays and colony-forming cell assays,respectively. Drug interactions were analyzed according to the median-effect method of Chou and Talalay. The determination of apoptotic cell death was performed by annexin V/propidium iodide staining. RESULTS: Combinations of both FTIs with imatinib displayed synergism or sensitization (potentiation) in all the cell lines tested. In primary chronic phase CML cells,additive and synergistic effects were discernible for the combination of imatinib plus L744,832 and imatinib plus LB42918,respectively. Annexin V/propidium iodide staining showed enhancement of imatinib-induced apoptosis with either drug combination,both in imatinib-sensitive and -resistant cells. CONCLUSION: The results indicated the potential of L744,832 and LB42918 as combination agents for CML patients on imatinib treatment. View Publication