技术资料

Persistence of drug-resistant breast cancer stem cells (brCSCs) after a chemotherapeutic regime correlates with disease recurrence and elevated mortality. Therefore,deciphering mechanisms that dictate their drug-resistant phenotype is imperative for designing targeted and more effective therapeutic strategies. The transcription factor SOX2 has been recognized as a protagonist in brCSC maintenance,and previous studies have confirmed that inhibition of SOX2 purportedly eliminated these brCSCs. However,pharmacological targeting of transcription factors like SOX2 is challenging due to their structural incongruities and intrinsic disorders in their binding interfaces. Therefore,transcriptional co-activators may serve as a feasible alternative for effectively targeting the brCSCs. Incidentally,transcriptional co-activators YAP/TAZ were found to be upregulated in CD44 + /CD24 - /ALDH + cells isolated from patient breast tumors and CSC-enriched mammospheres. Interestingly,it was observed that YAP/TAZ exhibited direct physical interaction with SOX2 and silencing YAP/TAZ attenuated SOX2 expression in mammospheres,leading to significantly reduced sphere forming efficiency and cell viability. YAP/TAZ additionally manipulated redox homeostasis and regulated mitochondrial dynamics by restraining the expression of the mitochondrial fission marker,DRP1. Furthermore,YAP/TAZ inhibition induced DRP1 expression and impaired OXPHOS,consequently inducing apoptosis in mammospheres. In order to enhance clinical relevance of the study,an FDA-approved drug verteporfin (VP),was used for pharmacological inhibition of YAP/TAZ. Surprisingly,VP administration was found to reduce tumor-initiating capacity of the mammospheres,concomitant with disrupted mitochondrial homeostasis and significantly reduced brCSC population. Therefore,VP holds immense potential for repurposing and decisively eliminating the chemoresistant brCSCs,offering a potent strategy for managing tumor recurrence effectively. Subject terms: Cancer stem cells,Cancer stem cells View Publication

Telomeres are terminal protective chromosome structures. Genetic variants in genes coding for proteins required for telomere maintenance cause rare,life-threatening Telomere Biology Disorders (TBDs) such as dyskeratosis congenita,aplastic anemia or pulmonary fibrosis. The more frequently used mice strains have telomeres much longer than the human ones which question their use as in vivo models for TBDs. One mice model with shorter telomeres based on the CAST/EiJ mouse strain carrying a mutation in the Terc gene,coding for the telomerase RNA component,has been studied in comparison with C57BL/6J mice,carrying the same mutation and long telomeres. The possible alterations produced in lungs and the haematopoietic system,frequently affected in TBD patients,were determined at different ages of the mice. Homozygous mutant mice presented a very shortened life span,more notorious in the short-telomeres CAST/EiJ strain. The lungs of mutant mice presented a transitory increase in fibrosis and a significant decrease in the relative amount of the alveolar epithelial type 2 cells from six months of age. This decrease was larger in mutant homozygous animals but was also observed in heterozygous animals. On the contrary the expression of the senescence-related protein P21 increased from six months of age in mutant mice of both strains. The analysis of the haematopoietic system indicated a decrease in the number of megakaryocyte-erythroid progenitors in homozygous mutants and an increase in the clonogenic potential of bone marrow and LSK cells. Bone marrow cells from homozygous mutant animals presented decreasing in vitro expansion capacity. The alterations observed are compatible with precocious ageing of lung alveolar cells and the bone marrow cells that correlate with the alterations observed in TBD patients. The alterations seem to be more related to the genotype of the animals that to the basal telomere length of the strains although they are more pronounced in the short-telomere CAST/EiJ-derived strain than in C57BL/6J animals. Therefore,both animal models,at ages over 6–8 months,could represent valuable and convenient models for the study of TBDs and for the assay of new therapeutic products. View Publication

In contrast to mammals,adult zebrafish achieve complete heart regeneration via proliferation of cardiomyocytes. Surprisingly,we found that regenerating cardiomyocytes experience DNA replication stress,which represents one reason for declining tissue regeneration during aging in mammals. Pharmacological inhibition of ATM and ATR kinases revealed that DNA damage response signaling is essential for zebrafish heart regeneration. Manipulation of Bone Morphogenetic Protein (BMP)-Smad signaling using transgenics and mutants showed that BMP signaling alleviates cardiomyocyte replication stress. BMP signaling also rescues neonatal mouse cardiomyocytes,human fibroblasts and human hematopoietic stem and progenitor cells (HSPCs) from replication stress. DNA fiber spreading assays indicate that BMP signaling facilitates re-start of replication forks after replication stress-induced stalling. Our results identify the ability to overcome replication stress as key factor for the elevated zebrafish heart regeneration capacity and reveal a conserved role for BMP signaling in promotion of stress-free DNA replication. Subject terms: Cardiac regeneration,DNA damage and repair,Ageing View Publication

Coronary artery disease (CAD) is linked to atherosclerosis plaque formation. In pro-inflammatory conditions,human Natural Killer (NK) cell frequencies in blood or plaque decrease; however,NK cells are underexplored in CAD pathogenesis,inflammatory mechanisms,and CAD comorbidities,such as human cytomegalovirus (HCMV) infection and diabetes. Analysis of PBMC CITE-seq data from sixty-one CAD patients revealed higher blood NK cell SPON2 expression in CAD patients with higher stenosis severity. Conversely,NK cell SPON2 expression was lower in pro-inflammatory atherosclerosis plaque tissue with an enriched adaptive NK cell gene signature. In CAD patients with higher stenosis severity,peripheral blood NK cell SPON2 expression was lower in patients with high HCMV-induced adaptive NK cell frequencies and corresponded to lower PBMC TGFβ transcript expression with dependency on diabetes status. These results suggest that high NK cell SPON2 expression is linked to atherosclerosis pro-homeostatic status and may have diagnostic and prognostic implications in cardiovascular disease. View Publication

Genetically modified T lymphocytes expressing chimeric antigen receptors (CARs) are becoming increasingly important in the treatment of hematologic malignancies and are also intensively being investigated for other diseases such as autoimmune disorders and HIV. Current CAR T cell therapies predominantly use viral transduction methods which,despite their efficacy,raise safety concerns related to genomic integration and potentially associated malignancies as well as labor- and cost-intensive manufacturing. Therefore,non-viral gene transfer methods,especially mRNA-based approaches,have attracted research interest due to their transient modification and enhanced safety profile. In this study,the optimization of CAR-mRNA for T cell applications is investigated,focusing on the impact of mRNA modifications,in vitro transcription protocols,and purification techniques on the translation efficiency and immunogenicity of mRNA. Furthermore,the refined CAR-mRNA was used to generate transient CAR T cells from acute myeloid leukemia patient samples,demonstrating efficacy in vitro and proof-of-concept for clinically relevant settings. These results highlight the potential of optimized mRNA to produce transient and safe CAR T cells. View Publication

Adipose-derived stromal cells (ASCs) possess significant regenerative potential,playing a key role in tissue repair and angiogenesis. During wound healing,ASC interacts with the extracellular matrix by recognizing arginylglycylaspartic acid (RGD) motifs,which are crucial for mediating these functions. This study investigates how RGD exposure influences ASC behavior,with a focus on angiogenesis. To mimic the wound-healing environment,ASC were cultured in a porcine gelatin sponge,an RGD-exposing matrix. Transcriptomics revealed that ASC cultured in gelatin exhibited an upregulated expression of genes associated with inflammation,angiogenesis,and tissue repair compared to ASC in suspension. Pro-inflammatory and pro-angiogenic factors,including IL-1,IL-6,IL-8,and VEGF,were significantly elevated. Functional assays further demonstrated that ASC-conditioned media enhanced endothelial cell migration,tubulogenesis,and reduced endothelial permeability,all critical processes in angiogenesis. Notably,ASC-conditioned media also promoted vasculogenesis in human vascular organoids. The inhibition of ASC-RGD interactions using the cyclic peptide cilengitide reversed these effects,underscoring the essential role of RGD-integrin interactions in ASC-mediated angiogenesis. These findings suggest that gelatin sponges enhance ASC’s regenerative and angiogenic properties via RGD-dependent mechanisms,offering promising therapeutic potential for tissue repair and vascular regeneration. Understanding how RGD modulates ASC behavior provides valuable insights into advancing cell-based regenerative therapies. View Publication

Lactate dehydrogenase A (LDHA) can regulate tumorigenesis and cancer progression. Nevertheless,whether the regulation of LDHA is involved in the development of gemcitabine resistance in PDAC has not yet been fully elucidated. Increasing studies have shown that cancer acquired drug resistance led to treatment failure is highly attributed to the cancer stem cell (CSC) properties. Therefore,we aim to demonstrate the functions and regulatory mechanisms of LDHA on cancer stem cell (CSC) properties and gemcitabine resistance in PDAC. We investigate the metabolite profiles by liquid chromatography-mass spectrometry between gemcitabine–resistant PDAC and parental PDAC cells. Additionally,gain-of-function and loss-of-function experiments were conducted to examine the roles of LDHA on CSC properties and gemcitabine resistance in the gemcitabine–resistant PDAC and parental PDAC cells. To investigate regulators involved in LDHA-mediated gemcitabine resistance and CSC of pancreatic cancer cells,we further used a combination of the miRNA microarray results and software predictions and confirmed that miR-4259 is a direct target of LDHA by luciferase assay. Furthermore,we constructed serial miR-4259 promoter reporters and searched for response elements using the TESS 2.0/TFSEARCH software to find the transcription factor binding site in the promoter region of miR-4259. We observed that elevated LDHA expression significantly correlates with recurrent pancreatic cancer patients following gemcitabine treatment and with CSC properties. We further identify that FOXO3a-induced miR-4259 directly targets the 3’untranslated region of LDHA and reduced LDHA expression,leading to decreased gemcitabine resistance and a reduction in the CSC phenotypes of pancreatic cancer. Our results demonstrated that LDHA plays a critical role in cancer stemness and gemcitabine resistance of pancreatic cancer,and indicate that targeting the FOXO3a/miR-4259/LDHA pathway might serve as a new treatment for pancreatic cancer patients with a poor response to gemcitabine chemotherapy. The online version contains supplementary material available at 10.1186/s40170-025-00377-3. View Publication

The manufacturing of allogeneic cell therapeutics based on human-induced pluripotent stem cells (hiPSCs) holds considerable potential to revolutionize the accessibility and affordability of modern healthcare. However,achieving the cell yields necessary to ensure robust production hinges on identifying suitable and scalable single-use (SU) bioreactor systems. While specific stirred SU bioreactor types have demonstrated proficiency in supporting hiPSC expansion at L -scale,others,notably instrumented SU multiplate and fixed-bed bioreactors,remain relatively unexplored. By characterizing these bioreactors using both computational fluid dynamics and experimental bioengineering methods,operating ranges were identified for the Xpansion ® 10 and Ascent™ 1 m 2 bioreactors in which satisfactory hiPSC expansion under serum-free conditions was achieved. These operating ranges were shown not only to effectively limit cell exposure to wall shear stress but also facilitated sufficient oxygen transfer and mixing. Through their application,almost 5 × 10 9 viable cells could be produced within 5 days,achieving expansion factors of up to 35 without discernable impact on cell viability,identity,or differentiation potential. Key Points • Bioengineering characterizations allowed the identification of operating ranges that supported satisfactory hiPSC expansion • Both the Xpansion ® 10 multiplate and Ascent™ 1 m 2 fixed-bed reactor accommodated the production of almost 5 × 10 9 viable cells within 5 days • Exposing the hiPSCs to a median wall shear stress of up to 8.2 × 10 −5 N cm −2 did not impair quality The online version contains supplementary material available at 10.1007/s00253-024-13373-2. View Publication

Light chain (AL) amyloidosis is a serious systemic disease caused by the deposition of free misfolded immunoglobulin light chains (LCs) in the form of amyloid fibrils within tissues. Cardiac involvement determines prognosis and mortality. An important cytotoxic impact of amyloidogenic prefibrillar LC oligomers on cardiomyocytes is by now established in isolated rodent cardiomyocytes,simple animal models,or cardiomyocyte-like cell lines. However,the response of human cardiomyocytes to this pathogenic condition is currently unknown. In this work,we have set up a human cellular disease model of AL cardiac amyloidosis (AL-CA) in the form of cardiac spheroids,to study the cytotoxic effects of amyloidogenic LCs with regard to contractile function and calcium handling. To mimic the disease in a reconstituted system,soluble amyloidogenic LCs purified from urine of AL-CA patients were added to a mixture of induced pluripotent stem cell-issued human cardiomyocytes (hiPSC-CM) and human primary cardiac fibroblasts,which resulted in formation of spheroids within 7 days. This procedure ensured a uniform pericellular LC distribution within spheroids. LC-treated hiPSC-CM cultures and LC-containing spheroids presented structural and functional defects including: (1) decreased levels and subcellular disorganization of sarcomeric protein alpha-actinin; (2) abnormal accumulation of calcium handling SERCA2a protein; (3) impaired contractility of spheroids and altered calcium transients. Three independent patient-derived LCs had similar effects,albeit to varying degrees,highlighting the patient-specific properties of this type of amyloids. Taken together,these results indicate that the present cardiac spheroid disease model could be appropriate to the study of cardiac cytotoxicity caused by different amyloidogenic LCs in AL-CA patients,contributing to a better understanding and therapeutic handling of the disease. The online version contains supplementary material available at 10.1038/s41598-024-82442-3. View Publication

Functional cellular heterogeneity in tumours often underlies incomplete response to therapy and relapse. Previously,we demonstrated that the growth of the paediatric brain malignancy,sonic hedgehog subgroup medulloblastoma,is rooted in a dysregulated developmental hierarchy,the apex of which is defined by characteristically quiescent SOX2 + stem-like cells. Integrating gene expression and chromatin accessibility patterns in distinct cellular compartments,we identify the transcription factor Olig2 as regulating the stem cell fate transition from quiescence to activation,driving the generation of downstream neoplastic progenitors. Inactivation of Olig2 blocks stem cell activation and tumour output. Targeting this rare OLIG2-driven proliferative programme with a small molecule inhibitor,CT-179,dramatically attenuates early tumour formation and tumour regrowth post-therapy,and significantly increases median survival in vivo. We demonstrate that targeting transition from quiescence to proliferation at the level of the tumorigenic cell could be a pivotal medulloblastoma treatment strategy. Subject terms: Cancer stem cells,Mechanisms of disease,Cancer therapy View Publication

Chimeric antigen receptor (CAR)-engineered T cell therapy holds promise for treating myeloid malignancies,but challenges remain in bone marrow (BM) infiltration and targeting BM-resident malignant cells. Current autologous CAR-T therapies also face manufacturing and patient selection issues,underscoring the need for off-the-shelf products. In this study,we characterize primary patient samples and identify a unique therapeutic opportunity for CAR-engineered invariant natural killer T (CAR-NKT) cells. Using stem cell gene engineering and a clinically guided culture method,we generate allogeneic CD33-directed CAR-NKT cells with high yield,purity,and robustness. In preclinical mouse models,CAR-NKT cells exhibit strong BM homing and effectively target BM-resident malignant blast cells,including CD33-low/negative leukemia stem and progenitor cells. Furthermore,CAR-NKT cells synergize with hypomethylating agents,enhancing tumor-killing efficacy. These cells also show minimal off-tumor toxicity,reduced graft-versus-host disease and cytokine release syndrome risks,and resistance to allorejection,highlighting their substantial therapeutic potential for treating myeloid malignancies. Subject terms: Cancer therapy,Immunotherapy,Leukaemia View Publication

Dishevelled-associated activator of morphogenesis1 (DAAM1) is a member of the evolutionarily conserved Formin family and plays a significant role in the malignant progression of various human cancers. This study aims to explore the clinical and biological significance of DAAM1 in pancreatic cancer. Multiple public datasets and an in-house cohort were utilized to assess the clinical relevance of DAAM1 in pancreatic cancer. The LinkedOmics platform was employed to perform enrichment analysis of DAAM1-associated molecular pathways in pancreatic cancer. Subsequently,a series of in vitro and in vivo experiments were conducted to evaluate the biological roles of DAAM1 in pancreatic cancer cells and its effects on intratumoral T cells. DAAM1 was found to be upregulated in pancreatic cancer tissues,with higher expression levels observed in tumor cells. Additionally,high expression of DAAM1 was associated with poor prognosis. DAAM1 acted as an oncogene in pancreatic cancer,and its inhibition suppressed tumor cell proliferation,migration,and invasion,while promoted apoptosis. Furthermore,DAAM1 was involved in the JAK1/STAT1 signaling pathway and regulated PD-L1 expression in pancreatic cancer cells. The inhibition of DAAM1 also significantly reduced the exhaustion levels of CD8+ T cells. In conclusion,DAAM1 functions as an oncogene and is immunologically implicated in pancreatic cancer,these findings suggest that DAAM1 may serve as a promising therapeutic target for the clinical management of pancreatic cancer. The online version contains supplementary material available at 10.1186/s12935-024-03631-8. View Publication