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Epithelial-to-mesenchymal transition (EMT),cancer stem cells (CSCs),and colorectal cancer (CRC) therapy resistance are closely associated. Prior reports have demonstrated that sphingosine-1-phosphate (S1P) supports stem cells and maintains the CSC phenotype. We hypothesized that the EMT inducer SNAI1 drives S1P signaling to amplify CSC self-renewal capacity and chemoresistance. CRC cell lines with or without ectopic expression of SNAI1 were used to study the role of S1P signaling as mediators of cancer stemness and 5-fluorouracil (5FU) chemoresistance. The therapeutic ability of sphingosine kinase 2 (SPHK2) was assessed using siRNA and ABC294640,a SPHK2 inhibitor. CSCs were isolated from patient-derived xenografts (PDXs) and assessed for SPHK2 and SNAI1 expression. Ectopic SNAI1 expressing cell lines demonstrated elevated SPHK2 expression and increased SPHK2 promoter activity. SPHK2 inhibition with siRNA or ABC294640 ablated in vitro self-renewal and sensitized cells to 5FU. CSCs isolated from CRC PDXs express increased SPHK2 relative to the non-CSC population. Combination ABC294640/5FU therapy significantly inhibited tumor growth in mice and enhanced 5FU response in therapy-resistant CRC patient-derived tumor organoids (PDTOs). SNAI1/SPHK2 signaling mediates cancer stemness and 5FU resistance,implicating S1P as a therapeutic target for CRC. The S1P inhibitor ABC294640 holds potential as a therapeutic agent to target CSCs in therapy refractory CRC. View Publication

Down syndrome predisposes individuals to haematological abnormalities,such as increased number of erythrocytes and leukaemia in a process that is initiated before birth and is not entirely understood 1 – 3 . Here,to understand dysregulated haematopoiesis in Down syndrome,we integrated single-cell transcriptomics of over 1.1 million cells with chromatin accessibility and spatial transcriptomics datasets using human fetal liver and bone marrow samples from 3 fetuses with disomy and 15 fetuses with trisomy. We found that differences in gene expression in Down syndrome were dependent on both cell type and environment. Furthermore,we found multiple lines of evidence that haematopoietic stem cells (HSCs) in Down syndrome are ‘primed’ to differentiate. We subsequently established a Down syndrome-specific map linking non-coding elements to genes in disomic and trisomic HSCs using 10X multiome data. By integrating this map with genetic variants associated with blood cell counts,we discovered that trisomy restructured regulatory interactions to dysregulate enhancer activity and gene expression critical to erythroid lineage differentiation. Furthermore,as mutations in Down syndrome display a signature of oxidative stress 4,5,we validated both increased mitochondrial mass and oxidative stress in Down syndrome,and observed that these mutations preferentially fell into regulatory regions of expressed genes in HSCs. Together,our single-cell,multi-omic resource provides a high-resolution molecular map of fetal haematopoiesis in Down syndrome and indicates significant regulatory restructuring giving rise to co-occurring haematological conditions. Subject terms: Haematopoietic stem cells,Leukaemia,Haematopoiesis,Haematological diseases,Aneuploidy View Publication

Autosomal recessive osteopetrosis (ARO) is a rare genetic disease,characterized by increased bone density due to defective osteoclast function. Most of the cases are due to TCIRG1 gene mutation,leading to severe bone phenotype and death in the first years of life. The standard therapy is the hematopoietic stem cell transplantation (HSCT),but its success is limited by several constraints. Conversely,gene therapy (GT) could minimize the immune-mediated complications of allogeneic HSCT and offer a prompt treatment to these patients. The Tcirg1 -defective oc/oc mouse model displays a short lifespan and high bone density,closely mirroring the human condition. In this work,we exploited the oc/oc neonate mice to optimize the critical steps for a successful therapy. First,we showed that lentiviral vector GT can revert the osteopetrotic bone phenotype,allowing long-term survival and reducing extramedullary haematopoiesis. Then,we demonstrated that plerixafor-induced mobilization can further increase the high number of HSPCs circulating in peripheral blood,facilitating the collection of adequate numbers of cells for therapeutic purposes. Finally,pre-transplant non-genotoxic conditioning allowed the stable engraftment of HSPCs,albeit at lower level than conventional total body irradiation,and led to long-term survival and correction of bone phenotype,in the absence of acute toxicity. These results will pave the way to the implementation of an effective GT protocol,reducing the transplant-related complication risks in the very young and severely affected ARO patients. View Publication

Automation and quality control (QC) are critical in manufacturing safe and effective cell and gene therapy products. However,current QC methods,reliant on molecular staining,pose difficulty in in-line testing and can increase manufacturing costs. Here we demonstrate the potential of using label-free ghost cytometry (LF-GC),a machine learning-driven,multidimensional,high-content,and high-throughput flow cytometry approach,in various stages of the cell therapy manufacturing processes. LF-GC accurately quantified cell count and viability of human peripheral blood mononuclear cells (PBMCs) and identified non-apoptotic live cells and early apoptotic/dead cells in PBMCs (ROC-AUC: area under receiver operating characteristic curve = 0.975),T cells and non-T cells in white blood cells (ROC-AUC = 0.969),activated T cells and quiescent T cells in PBMCs (ROC-AUC = 0.990),and particulate impurities in PBMCs (ROC-AUC ≧ 0.998). The results support that LF-GC is a non-destructive label-free cell analytical method that can be used to monitor cell numbers,assess viability,identify specific cell subsets or phenotypic states,and remove impurities during cell therapy manufacturing. Thus,LF-GC holds the potential to enable full automation in the manufacturing of cell therapy products with reduced cost and increased efficiency. Subject terms: Biotechnology,Cell biology,Immunology,Biomedical engineering View Publication

Paulownin,a natural compound derived from Paulownia tomentosa wood,exhibits various physiological functions,including anti-bacterial and anti-fungal effects. However,the impact of paulownin on natural killer (NK) cell immune activity remains largely unknown. In this study,we investigated the effect of paulownin on NK cell activity both in vitro and in vivo,and explored its potential mechanisms. NK-92 cells were used for in vitro experiments and a BALB/c mouse model with B16F10 cells injected subcutaneously were used for in vivo anti-tumor analysis. We found that paulownin enhanced the cytolytic activity of NK-92 cells against leukemia,human colon,and human lung cancer cell lines. Paulownin treatment increased the expression of the degranulation marker protein CD107a and cytolytic granules,including granzyme B and perforin in NK-92 cells. Moreover,these enhancements of cytotoxicity and the expression of cytolytic granules induced by paulownin were also observed in human primary NK cells. Signaling studies showed that paulownin promoted the phosphorylation of JNK. The increased perforin expression and elevated cytotoxic activity induced by paulownin were effectively inhibited by pre-treatment with a JNK inhibitor. In vivo studies demonstrated that the administration of paulownin suppressed the growth of B16F10 melanoma cells allografted into mice. Paulownin administration promoted the activation of NK cells in the spleen of mice,resulting in enhanced cytotoxicity against YAC-1 cells. Moreover,the anti-tumor effects of paulownin were reduced upon the depletion of NK cells. Therefore,these results suggest that paulownin enhances NK cell cytotoxicity by activating the JNK signaling pathway and provide significant implications for developing new strategies for cancer immunotherapy. View Publication

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD,even before symptom onset. Because these early disease phenotypes remain incompletely understood,we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients,presymptomatic C9ORF72-HRE (C9-HRE) carriers,and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition,single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids,including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further,molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly,organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology,whereas the extent to which more downstream cellular defects,as found in C9-ALS/FTD models,were detected varied for the different presymptomatic C9-HRE cases. Together,these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies. The online version contains supplementary material available at 10.1186/s40478-024-01857-1. View Publication

High-grade serous ovarian cancer remains a poorly understood disease with a high mortality rate. Although most patients respond to cytotoxic therapies,a majority will experience recurrence. This may be due to a minority of drug-resistant cancer stem-like cells (CSC) that survive chemotherapy and are capable of repopulating heterogeneous tumors. It remains unclear how CSCs are supported in the tumor microenvironment (TME) particularly during chemotherapy exposure. Tumor-associated macrophages (TAM) make up half of the immune population of the ovarian TME and are known to support CSCs and contribute to cancer progression. TAMs are plastic cells that alter their phenotype in response to environmental stimuli and thus may influence CSC maintenance during chemotherapy. Given the plasticity of TAMs,we studied the effects of carboplatin on macrophage phenotypes using both THP1- and peripheral blood mononuclear cell (PBMC)–derived macrophages and whether this supports CSCs and ovarian cancer progression following treatment. We found that carboplatin exposure induces an M1-like proinflammatory phenotype that promotes SOX2 expression,spheroid formation,and CD117 + ovarian CSCs,and that macrophage-secreted CCL2/MCP-1 is at least partially responsible for this effect. Depletion of TAMs during carboplatin exposure results in fewer CSCs and prolonged survival in a xenograft model of ovarian cancer. This study supports a role for platinum-based chemotherapies in promoting a transient proinflammatory M1-like TAM that enriches for CSCs during treatment. Improving our understanding of TME responses to cytotoxic drugs and identifying novel mechanisms of CSC maintenance will enable the development of better therapeutic strategies for high-grade serous ovarian cancer. Significance: We show that chemotherapy enhances proinflammatory macrophage phenotypes that correlate with ovarian cancer progression. Given that macrophages are the most prominent immune cell within these tumors,this work provides the foundation for future translational studies targeting specific macrophage populations during chemotherapy,a promising approach to prevent relapse in ovarian cancer. View Publication

A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). C9orf72 repeat expansions are currently identified with long-range PCR or Southern blot for clinical and research purposes,but these methods lack accuracy and sensitivity. The GC-rich and repetitive content of the region cannot be amplified by PCR,which leads traditional sequencing approaches to fail. We turned instead to PacBio single-molecule sequencing to detect and size the C9orf72 repeat expansion without amplification. We isolated high molecular weight genomic DNA from patient-derived iPSCs of varying repeat lengths and then excised the region containing the C9orf72 repeat expansion from naked DNA with a CRISPR/Cas9 system. We added adapters to the cut ends,capturing the target region for sequencing on PacBio’s Sequel,Sequel II,or Sequel IIe. This approach enriches the C9orf72 repeat region without amplification and allows the repeat expansion to be consistently and accurately sized,even for repeats in the thousands. Key features • This protocol is adapted from PacBio’s previous “no-amp targeted sequencing utilizing the CRISPR-Cas9 system.” • Optimized for sizing C9orf72 repeat expansions in patient-derived iPSCs and applicable to DNA from any cell type,blood,or tissue. • Requires high molecular weight naked DNA. • Compatible with Sequel I and II but not Revio. View Publication

In recent years,Raman spectroscopy has garnered growing interest in the field of biomedical research. It offers a non-invasive and label-free approach to defining the molecular fingerprint of immune cells. We utilized Raman spectroscopy on optically trapped immune cells to investigate their molecular compositions. While numerous immune cell types have been studied in the past,the characterization of living human CD3/CD28-stimulated T cell subsets remains incomplete. In this study,we demonstrate the capability of Raman spectroscopy to readily distinguish between naïve and stimulated CD4 and CD8 cells. Additionally,we compared these cells with monocytes and discovered remarkable similarities between stimulated T cells and monocytes. This paper contributes to expanding our knowledge of Raman spectroscopy of immune cells and serves as a launching point for future clinical applications. View Publication

Dimethylsulfoxide (DMSO) has conventionally been used for cell cryopreservation both in research and in clinical applications,but has long-term cytotoxic effects. Trehalose,a natural disaccharide,has been proposed as a non-toxic cryoprotectant. However,the lack of specific cell membrane transporter receptors inhibits transmembrane transport and severely limits its cryoprotective capability. This research presents a method to successfully deliver trehalose into mesenchymal stem cells (MSCs) using ultrasound in the presence of microbubbles. The optimised trehalose concentration was shown to be able to not only preserve membrane integrity and cell viability but also the multipotency of MSCs,which are essential for stem cell therapy. Confocal imaging revealed that rhodamine-labelled trehalose was transported into cells rather than simply attached to the membrane. Additionally,the membranes were successfully preserved in lyophilised cells. This study demonstrates that ultrasonication with microbubbles facilitated trehalose delivery,offering promising cryoprotective capability without the cytotoxicity associated with DMSO-based methods. Subject terms: Membrane biophysics,Biomedical engineering View Publication

Immune checkpoint blockade (ICB) has made remarkable achievements,but newly identified armored and cold tumors cannot respond to ICB therapy. The high prevalence of concomitant medications has huge impact on immunotherapeutic responses,but the clinical effects on the therapeutic outcome of armored and cold tumors are still unclear. In this research,using large-scale transcriptomics datasets,the expression and potential biological functions of angiotensin II receptor 1 (AGTR1),the target of angiotensin receptor blocker (ARB),were investigated. Next,the roles of ARB in tumor cells and tumor microenvironment cells were defined by a series of in vitro and in vivo assays. In addition,the clinical impacts of ARB on ICB therapy were assessed by multicenter cohorts and meta-analysis. AGTR1 was overexpressed in armored and cold tumors and associated with poor response to ICB therapy. ARB,the inhibitor for AGTR1,only suppressed the aggressiveness of tumor cells with high AGTR1 expression,which accounted for a very small proportion. Further analysis revealed that AGTR1 was always highly expressed in cancer-associated fibroblasts (CAFs) and ARB inhibited type I collagen expression in CAFs by suppressing the RhoA-YAP axis. Moreover,ARB could also drastically reverse the phenotype of armored and cold to soft and hot in vivo,leading to a higher response to ICB therapy. In addition,both our in-house cohorts and meta-analysis further supported the idea that ARB can significantly enhance ICB efficacy. Overall,we identify AGTR1 as a novel target in armored and cold tumors and demonstrate the improved therapeutic efficacy of ICB in combination with ARB. These findings could provide novel clinical insight into how to treat patients with refractory armored and cold tumors. View Publication

Reduction of adult hippocampal neurogenesis is an early critical event in Alzheimer’s disease (AD),contributing to progressive memory loss and cognitive decline. Reduced levels of the nucleoporin 153 (Nup153),a key epigenetic regulator of NSC stemness,characterize the neural stem cells isolated from a mouse model of AD (3×Tg) (AD-NSCs) and determine their altered plasticity and gene expression. Nup153-regulated mechanisms contributing to NSC function were investigated: (1) in cultured NSCs isolated from AD and wild type (WT) mice by proteomics; (2) in vivo by lentiviral-mediated delivery of Nup153 or GFP in the hippocampus of AD and control mice analyzing neurogenesis and cognitive function; (3) in human iPSC-derived brain organoids obtained from AD patients and control subjects as a model of neurodevelopment. Proteomic approach identified Nup153 interactors in WT- and AD-NSCs potentially implicated in neurogenesis regulation. Gene ontology (GO) analysis showed that Nup153-bound proteins in WT-NSCs were involved in RNA metabolism,nuclear import and epigenetic mechanisms. Nup153-bound proteins in AD-NSCs were involved in pathways of neurodegeneration,mitochondrial dysfunction,proteasomal processing and RNA degradation. Furthermore,recovery of Nup153 levels in AD-NSCs reduced the levels of oxidative stress markers and recovered proteasomal activity. Lentiviral-mediated delivery of Nup153 in the hippocampal niche of AD mice increased the proliferation of early progenitors,marked by BrdU/DCX and BrdU/PSANCAM positivity and,later,the integration of differentiating neurons in the cell granule layer (BrdU/NeuN + cells) compared with GFP-injected AD mice. Consistently,Nup153-injected AD mice showed an improvement of cognitive performance in comparison to AD-GFP mice at 1 month after virus delivery assessed by Morris Water Maze. To validate the role of Nup153 in neurogenesis we took advantage of brain organoids derived from AD-iPSCs characterized by fewer neuroepithelial progenitor loops and reduced differentiation areas. The upregulation of Nup153 in AD organoids recovered the formation of neural-like tubes and differentiation. Our data suggest that the positive effect of Nup153 on neurogenesis is based on a complex regulatory network orchestrated by Nup153 and that this protein is a valuable disease target. The online version contains supplementary material available at 10.1186/s13287-024-03805-1. View Publication