技术资料

Mitochondria are central to cellular metabolism. Various fluorescence tools have been developed for imaging the mitochondrial environment. Yet,new reporters and imaging methods for directly reading the mitochondrial status are needed for high spatial-temporal resolution imaging. Here,we introduce PK Mito Deep Red (PKMDR),a low-phototoxicity mitochondrial probe for time-lapse imaging,whose fluorescence lifetime serves as a sensitive indicator of mitochondrial membrane potential (Δψm). The positively charged PKMDR accumulates within mitochondria under a higher Δψm,leading to concentration-induced quenching and a measurable decrease in fluorescence lifetime. Since mitochondrial respiration primarily regulates Δψm,PKMDR’s fluorescence lifetime effectively reports on the status of oxidative phosphorylation. Using PKMDR with fluorescence lifetime imaging microscopy (FLIM),we visualize heterogeneous Δψm across individual cells,organoids,and tissues over time. This method reliably reveals the heterogeneity between metabolically active peripheral mitochondria and relatively inactive perinuclear mitochondria in various cell types. Overall,PKMDR-FLIM is a robust tool for directly visualizing Δψm with high spatiotemporal resolution. Saimi and colleagues present PKMDR,a mitochondrial probe whose fluorescence lifetime serves as an indicator to monitor mitochondrial membrane potential and respiration dynamics with high resolution across cells and tissues. View Publication

Respiratory syncytial virus (RSV) is the most common cause of acute bronchiolitis and pneumonia in children under 5 years of age. RSV infection is characterized by lung inflammation,viral expansion,and airway barrier destruction. The crosstalk between neurons and immune and non-immune cells develops from early life onwards. The lungs and airways are densely innervated by sympathetic neurons where they secrete the neurotransmitter noradrenaline (NA) at homeostasis and following injury in adult mice. NA,once secreted into the lungs,can act on immune and non-immune cells through β-adrenergic receptors (β-ARs). However,it is unknown whether NA-β-AR signaling axis regulates antiviral immunity during early-life infections. In a mouse model of neonatal RSV infection,we find that stimulation of adrenergic signaling via β2-AR agonist increases viral clearance,anti-inflammatory responses,and disease recovery. By contrast,loss of sympathetic neurons and β2-AR increases the RSV viral load and immunopathology in neonates. Stimulating β2-AR signaling has several downstream effects,including reduced airway mucus secretion and protection from endothelial barrier disruption,which together prevent airway blockade,respiratory failure,and facilitate control of RSV infection. Collectively,our studies demonstrate that sympathetic neurons and β2-AR signaling protect neonates from viral expansion and lung barrier breakdown,promoting antiviral defense. RSV is the most common cause of respiratory infections in infants and young children. Here,the authors used a neonatal mouse RSV infection model and uncovered a role of sympathetic neurons and β2-adrenergic signaling in protecting neonates from viral expansion and immunopathology to promote antiviral defense. View Publication

BackgroundDaphnetin has demonstrated various pharmacological activities. The current study evaluated the potential of daphnetin in alleviating unexplained recurrent spontaneous abortion (URSA) and explored underlying mechanisms.MethodsMice with URSA were gavaged with 1 mg/kg,10 mg/kg,and 20 mg/kg of daphnetin,or infected with adeno-associated viruses harboring knockdown of NR4A1 or overexpression of BACH2 before modeling. Human peripheral blood T lymphocytes were induced into CD4+ T cells,followed by lentivirus infection and daphnetin treatment. The influence of daphnetin on CD4+ T cell viability and Treg and Th17 cell differentiation in cells was analyzed. The concentrations of Treg cells-associated cytokines (TGF-β,IL-10) and Th17 cells-associated cytokines (IL-17,IL-23) in the supernatants of CD4+ T cells were assessed. The regulation of NR4A1 on BACH2 was analyzed by ChIP and dual-luciferase assays.ResultsDaphnetin resulted in fewer immature,resorbed,or dead embryos in mice with URSA,with the most pronounced therapeutic effect of 10 mg/kg. Daphnetin attenuated decidual hemorrhage,with a gain in the percentage/number of Treg cells and a loss of the percentage/number of Th17 cells in the spleen and decidual tissues. Daphnetin enhanced the expression of FoxP3,TGF-β,and IL-10,and suppressed the expression of RORγt,IL-17,IL-23,and the contents of TNF-α,IL-6,and IL-1β in CD4+ T cells. Overexpression of BACH2 further alleviated URSA deterioration caused by NR4A1 knockdown. Daphnetin mediated the transcriptional activation of BACH2 by upregulating NR4A1.ConclusionsUpregulation of NR4A1 by daphnetin mediates BACH2 transcription and Th17/Treg cell homeostasis to improve URSA. View Publication

Simple SummaryDespite effective therapies,multiple myeloma remains an incurable disease. Even when achieving deep remissions,almost all patients eventually relapse. Current evaluation of this persistent disease is based on bone marrow evaluation,but this approach has significant drawbacks. Blood-based disease evaluation is less invasive and potentially more comprehensive in evaluation of total tumor mass but lacks sensitivity. It is currently unknown which biomarker in blood is superior. Therefore,we tested three different blood-derived DNA sources: peripheral blood mononuclear cells,enriched circulating tumor cells (CTCs) and cell-free DNA. Enrichment of CTCs,followed by next-generation sequencing,resulted in the highest sensitivity. In patients with detectable disease in their bone marrow,but no detectable CTCs,the first relapse occurred after almost 4 years,while early relapse (<18 months) occurred in 5/12 patients with detectable CTCs. AbstractBackground/Objectives: Minimal residual disease (MRD) persists in most multiple myeloma (MM) patients,causing relapse despite deep remissions. Repeatability of MRD detection in MM bone marrow (BM) samples is limited,underscoring the need for blood-based monitoring approaches that can allow more thorough disease surveillance. Methods: This study compares tumor detection rates in BM-derived DNA with different blood-derived DNA sources,using next-generation sequencing of the immunoglobulin locus (NGS-IG). CD138-targeted immunomagnetic enrichment of circulating tumor cells (CTCs) followed by vacuum evaporation to concentrate DNA was used to optimize the tumor detection rate. Results: Tumor DNA was detected in 76%,88% and 100% of cell-free DNA,peripheral blood-derived mononuclear cells,DNA and enriched CTC-DNA samples of patients with active myeloma,respectively. These data indicate that enriched CTC samples were the most informative for evaluation of disease detection with NGS-IG in patients with active myeloma. MRD detection was performed in paired BM and enriched CTC-DNA samples from 37 patients in remission. MRD positivity was found in the BM of 24 patients,with half of them (12/24) also showing the presence of MRD when enriched CTC-DNA was used. Interestingly,time to progression (TTP) of enriched CTC MRD-negative/BM MRD-positive patients was comparable to that of double MRD-negative (BM and CTC) patients. Moreover,double positive patients showed a trend to earlier relapses. Conclusions: Our data suggest that NGS-IG analysis with enriched CTC-DNA may offer improved predictive abilities for relapse in multiple myeloma compared to the currently used BM-DNA-based tumor detection method. View Publication

The respiratory mucosal system plays a critical role in the pathogenesis of allergic asthma (AA). Currently,therapeutic Fc fusion proteins are as a promising strategy for mucosal vaccine delivery systems. In this work,a plasmid encoding the Mycobacterium tuberculosis ESAT6-Fc fusion protein was successfully constructed,and high-purity ESAT6-Fc fusion protein was subsequently obtained. Administered via intranasal immunization in OVA-induced allergic asthma model mice,ESAT6-Fc fusion protein significantly alleviated airway inflammation and mucus production,and reduced the proportions of Th2 cells,Th17 cells,and eosinophils,while increasing the proportions of Th1 cells with no histopathological changes to major organs. To elucidate the underlying immune regulatory mechanisms of ESAT6,integrated transcriptomic and proteomic analyses were performed,revealing Th1/Th2 cell differentiation and Th17 cell differentiation as the two most significantly enriched pathways at both the gene and protein levels. CD3e (CD3E) and CD3g (CD3G),two essential subunits of the TCR–CD3 complex,were identified as core target factors. The validations from the ESAT6-Fc-treated AA lung tissues,as well as co-cultured TH0 cells from C57BL/6J mice and CD2.4 dendritic cells exposed to the ESAT6-Fc protein,were consistent with the aforementioned findings. ESAT6-Fc exhibits a safe profile with favorable efficacy against OVA-induced AA via intranasal immunization,and ESAT6 ameliorates AA by regulating the differentiation of Th0 cells into Th1 cells,which were closely associated with the down-regulation of CD3e and CD3g expression,presumably leading to the impairment of TCR–CD3 complex assembly. ESAT6-Fc fusion protein demonstrates promise as a potential safe intranasal immunotherapy agent for the treatment of AA. View Publication

BACKGROUND: There is little information on the trajectory and developmental fate of Lin-CD34+DNAM-1bright CXCR4+ progenitors exiting bone marrow during systemic inflammation. OBJECTIVE: To study Lin-CD34+DNAM-1bright CXCR4+ cell circulation in cancer patients,to characterize their entry into involved lung tissue and to characterize their progenies. METHODS: Flow cytometric analysis of PBMC from 18 patients with lung cancer on samples collected immediately before the first and the second treatment was performed to study Lin-CD34+DNAM-1bright CXCR4+ precursors. Precursors were purified (>99%) and cultured in vitro from all patients. Paired PBMC and tissue samples from patients undergoing tumor resection were analyzed by flow cytometry to assess tissue entry and compare phenotype and developmental potential of Lin-CD34+DNAM-1bright CXCR4+ cells in both compartments. RESULTS: Significant circulation of Lin-CD34+DNAM-1bright CXCR4+ precursors was observed 20d after the first treatment. Precursors express CXC3CR1,CXCR3,CXCR1 consistent with travel towards inflamed tissues. Flowcytometric analysis of lung tissue samples showed precursor presence in all patients in tumor and neighboring uninvolved areas. Successful purification and in vitro culture from both blood and lung tissue generates a minor proportion of maturing NK cells (<10%) and a predominant proportion (>85%) of α/β T-progenies with innate-like phenotype expressing NKG2D,NKp30,DNAM-1. Innate-like maturing T-cells in vitro are cytotoxic,can be triggered via NKR/TCR co-stimulation and display broad spectrum Th1,Th2 and Th1/Th17 cytokine production. CONCLUSION: In advanced stage lung cancer CD34+DNAM-1brightCXCR4+ inflammatory precursors increase upon treatment,enter involved tissues,generate functional progenies and may thus represent an additional player contributing to immune balance in the highly SDF-1/CXCR4-biased pro-metastatic tumor microenvironment. View Publication

BACKGROUND: Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However,the response varies depending on the tumor immune microenvironment (TIME). Therefore,there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. METHODS: Here we establish a patient-derived explant culture (PDEC) model of breast cancer,which retains the immune contexture of the primary tumor,recapitulating cytokine profiles and CD8+T cell cytotoxic activity. RESULTS: We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo,discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead,metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I,increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity. CONCLUSIONS: Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME. View Publication

The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude,with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization,these scalable single-cell quantitative expression reporters provide high-contrast readouts,analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development,we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native,perturbed and synthetic CREs at scale,with high sensitivity and at single-cell resolution. View Publication

PURPOSE: Spontaneously occurring glioma in pet dogs is increasingly recognized as a valuable translational model for human glioblastoma. Canine high-grade glioma and human glioblastomas share many molecular similarities,including the accumulation of immunosuppressive regulatory T cells (Tregs) that inhibit anti-tumor immune responses. Identifying in dog mechanisms responsible for Treg recruitment may afford to target the cellular population driving immunosuppression,the results providing a rationale for translational clinical studies in human patients. Our group has previously identified C-C motif chemokine 2 (CCL2) as a glioma-derived T-reg chemoattractant acting on chemokine receptor 4 (CCR4) in a murine orthotopic glioma model. Recently,we demonstrated a robust increase of CCL2 in the brain tissue of canine patients bearing high-grade glioma. METHODS: We performed a series of in vitro experiments using canine Tregs and patient-derived canine glioma cell lines (GSC 1110,GSC 0514,J3T-Bg,G06A) to interrogate the CCL2-CCR4 signaling axis in the canine. RESULTS: We established a flow cytometry gating strategy for identifying and isolating FOXP3+ Tregs in dogs. The canine CD4 + CD25high T-cell population was highly enriched in FOXP3 and CCR4 expression,indicating they are bona fide Tregs. Canine Treg migration was enhanced by CCL2 or by glioma cell line-derived supernatant. Blockade of the CCL2-CCR4 axis significantly reduced migration of canine Tregs. CCL2 mRNA was expressed in all glioma cell lines,and expression increased when exposed to Tregs but not CD4 + helper T-cells. CONCLUSION: Our study validates CCL2-CCR4 as a bi-directional Treg-glioma immunosuppressive and tumor-promoting axis in canine high-grade glioma. View Publication

Nutraceuticals are increasingly investigated for their capacity to modulate oxidative and inflammatory stress,yet preclinical testing still relies largely on immortalized cell lines or animal models that poorly recapitulate human epithelial complexity. To address this gap,we developed an integrated platform based on patient-derived colon organoids generated from non-tumoral mucosa and maintained under proliferative or differentiation conditions to model distinct epithelial states. The system combines millifluidic measurement of individual organoid mass,density,and diameter with bulk RNA sequencing and digital PCR profiling to enable multiparametric characterization. Transcriptional analysis revealed state-specific gene programs and shifts in epithelial and immune-related pathways,while biophysical measurements captured structural remodeling. In this pilot validation,a defined oxidative insult followed by nutraceutical treatment elicited coordinated transcriptional and phenotypic responses. This integrated approach provides a scalable and physiologically relevant framework for functional nutraceutical profiling and mechanistic studies of epithelial stress responses. View Publication

The SIRPα-CD47 "don't eat me" checkpoint axis plays a critical role in shaping antitumor activities of macrophages within the tumor microenvironment (TME). However,targeting this axis with anti-CD47 antibodies to enhance antitumor responses in clinical trials has been challenging. Here,we demonstrated that SIRPA-knockout (KO) iPSC-derived macrophages (iMacs) exhibit superior antitumor activity against various CD47-expressing tumors in vitro when combined with cancer-targeted monoclonal antibodies (mAbs) or chimeric antigen receptors (CARs). Moreover,SIRPA-KO protected macrophages from mAb- and CAR-driven hypophagia,enabling efficient tumoricidal effects even after serial tumor exposures. Retention of phagocytic activities in SIRPA-KO iMacs was associated with heightened surface expression of Fc receptors and GD2-CAR compared to their SIRPA-expressing counterparts. Despite the powerful impact of SIRPA-KO on iMac antitumor activities in vitro,only modest efficacy was observed in human xenograft mouse models of SK-OV3 ovarian carcinoma and CHLA-163 neuroblastoma treated with mAb or CAR-iMac therapy,indicating further engineering or combinatorial therapeutic strategies are needed for potent in vivo antitumor efficacy. Together,these findings identify SIRPα as a regulator in macrophage hypophagia and underscore the advantages of using SIRPA-KO macrophage therapeutic strategies to modulate the SIRPα-CD47 checkpoint to unleash macrophage antitumor activity within the TME. View Publication

Platelet shortages and limited storage stability restrict global platelet transfusion capacity,highlighting the need for efficient in vitro platelet production systems. This study establishes a simple,non-genetic and cost-effective system for efficient in vitro platelet-like particle (PLP) production from human hematopoietic stem/progenitor cells (HSPCs) using a Phase-specific modulation of Hippo-YAP/TAZ signaling modulation. Temporal control of Hippo-YAP/TAZ signaling by lysophosphatidic acid (LPA),an activator of YAP/TAZ activity,significantly enhanced megakaryocyte differentiation,expansion and PLP production,resulting in an approximately 15-fold increase in PLP yield at the end of the procedure. Furthermore,LPA extended the expansion period of HSPC-derived megakaryocytes up to 8 days,resulting in a greater than 20-fold increase in the number of HSPC-derived CD41+ megakaryocytes. Moreover,replacement of expensive commercial recombinant human thrombopoietin (C-rhTPO),one of the major cost-driving components in in vitro PLP production,with recombinant human thrombopoietin produced in Escherichia coli (W-rhTPO) further improved the cost-effectiveness of the procedure. In conclusion,this study demonstrates that temporally controlled Hippo-YAP/TAZ signaling,together with affordable cytokine supplementation,provides a robust and GMP-compatible platform for large-scale PLP manufacturing for future clinical applications. We believe that this system will enable scalable PLP generation,even in resource-constrained settings,to increase human platelet supply for many life-saving therapies in the future. View Publication