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Communications Biology Jun 2024Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of renal cancer, accounting for over 75% of cases. The asymptomatic nature of the disease contributes...
Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of renal cancer, accounting for over 75% of cases. The asymptomatic nature of the disease contributes to late-stage diagnoses and poor survival. Highly vascularized and immune infiltrated microenvironment are prominent features of ccRCC, yet the interplay between vasculature and immune cells, disease progression and response to therapy remains poorly understood. Using droplet-based single-cell RNA sequencing we profile 50,236 transcriptomes from paired tumor and healthy adjacent kidney tissues. Our analysis reveals significant heterogeneity and inter-patient variability of the tumor microenvironment. Notably, we discover a previously uncharacterized vasculature subpopulation associated with epithelial-mesenchymal transition. The cell-cell communication analysis reveals multiple modes of immunosuppressive interactions within the tumor microenvironment, including clinically relevant interactions between tumor vasculature and stromal cells with immune cells. The upregulation of the genes involved in these interactions is associated with worse survival in the TCGA KIRC cohort. Our findings demonstrate the role of tumor vasculature and stromal cell populations in shaping the ccRCC microenvironment and uncover a subpopulation of cells within the tumor vasculature that is associated with an angiogenic phenotype.
Topics: Humans; Carcinoma, Renal Cell; Kidney Neoplasms; Single-Cell Analysis; Tumor Microenvironment; Gene Expression Profiling; Phenotype; Gene Expression Regulation, Neoplastic; Endothelial Cells; Transcriptome; Epithelial-Mesenchymal Transition; Male; Female
PubMed: 38942917
DOI: 10.1038/s42003-024-06478-x -
Journal of Oral Biosciences Jun 2024This study aimed to investigate the regulatory mechanisms governing dental mesenchymal cell commitment during tooth development, focusing on odontoblast differentiation...
Exploring the Role of DNMT1 in Dental Papilla Cell Fate Specification during Mouse Tooth Germ Development through Integrated Single-Cell Transcriptomics and Bulk RNA Sequencing.
OBJECTIVES
This study aimed to investigate the regulatory mechanisms governing dental mesenchymal cell commitment during tooth development, focusing on odontoblast differentiation and the role of epigenetic regulation in this process.
METHODS
We performed single-cell RNA sequencing (scRNA-seq) of dental cells from embryonic day 14.5 (E14.5) mice to understand the heterogeneity of developing tooth germ cells. Computational analyses including gene regulatory network (GRN) assessment were conducted. We validated our findings using immunohistochemistry (IHC) and in vitro loss-of-function analyses using the DNA methyltransferase 1 (DNMT1) inhibitor Gsk-3484862 in primary dental mesenchymal cells (DMCs) isolated from E14.5 mouse tooth germs. Bulk RNA-seq of Gsk-3484862-treated DMCs was performed to identify potential downstream targets of DNMT1.
RESULTS
scRNA-seq analysis revealed diverse cell populations within the tooth germs, including epithelial, mesenchymal, immune, and muscle cells. Using single-cell regulatory network inference and clustering (SCENIC), we identified Dnmt1 as a key regulator of early odontoblast development. IHC analysis showed the ubiquitous expression of DNMT1 in the dental papilla and epithelium. Bulk RNA-seq of cultured DMCs showed that Gsk-3484862 treatment upregulated odontoblast-related genes, whereas genes associated with cell division and the cell cycle were downregulated. Integrated analysis of bulk RNA-seq data with scRNA-seq SCENIC profiles was used to identify the potential Dnmt1 target genes.
CONCLUSIONS
Dnmt1 may negatively affect odontoblast commitment and differentiation during tooth development. These findings contribute to a better understanding of the molecular mechanisms underlying tooth development and future development of hard-tissue regenerative therapies.
PubMed: 38942194
DOI: 10.1016/j.job.2024.06.010 -
EBioMedicine Jun 2024Pancreatic ductal adenocarcinoma (PDAC) is a tumour entity with unmet medical need. To assess the therapeutic potential of oncolytic virotherapy (OVT) against PDAC,...
BACKGROUND
Pancreatic ductal adenocarcinoma (PDAC) is a tumour entity with unmet medical need. To assess the therapeutic potential of oncolytic virotherapy (OVT) against PDAC, different oncolytic viruses (OVs) are currently investigated in clinical trials. However, systematic comparisons of these different OVs in terms of efficacy against PDAC and biomarkers predicting therapeutic response are lacking.
METHODS
We screened fourteen patient-derived PDAC cultures which reflect the intra- and intertumoural heterogeneity of PDAC for their sensitivity to five clinically relevant OVs, namely serotype 5 adenovirus Ad5-hTERT, herpes virus T-VEC, measles vaccine strain MV-NIS, reovirus jin-3, and protoparvovirus H-1PV. Live cell analysis, quantification of viral genome/gene expression, cell viability as well as cytotoxicity assays and titration of viral progeny were conducted. Transcriptome profiling was employed to identify potential predictive biomarkers for response to OV treatment.
FINDINGS
Patient-derived PDAC cultures showed individual response patterns to OV treatment. Twelve of fourteen cultures were responsive to at least one OV, with no single OV proving superior or inferior across all cultures. Known host factors for distinct viruses were retrieved as potential biomarkers. Compared to the classical molecular subtype, the quasi-mesenchymal or basal-like subtype of PDAC was found to be more sensitive to H-1PV, jin-3, and T-VEC. Generally, expression of viral entry receptors did not correlate with sensitivity to OV treatment, with one exception: Expression of Galectin-1 (LGALS1), a factor involved in H-1PV entry, positively correlated with H-1PV induced cell killing. Rather, cellular pathways controlling immunological, metabolic and proliferative signaling appeared to determine outcome. For instance, high baseline expression of interferon-stimulated genes (ISGs) correlated with relative resistance to oncolytic measles virus, whereas low cyclic GMP-AMP synthase (cGAS) expression was associated with exceptional response. Combination treatment of MV-NIS with a cGAS inhibitor improved tumour cell killing in several PDAC cultures and cells overexpressing cGAS were found to be less sensitive to MV oncolysis.
INTERPRETATION
Considering the heterogeneity of PDAC and the complexity of biological therapies such as OVs, no single biomarker can explain the spectrum of response patterns. For selection of a particular OV, PDAC molecular subtype, ISG expression as well as activation of distinct signaling and metabolic pathways should be considered. Combination therapies can overcome resistance in specific constellations. Overall, oncolytic virotherapy is a viable treatment option for PDAC, which warrants further development. This study highlights the need for personalised treatment in OVT. By providing all primary data, this study provides a rich source and guidance for ongoing developments.
FUNDING
German National Science Foundation (Deutsche Forschungsgemeinschaft, DFG), German Cancer Aid (Deutsche Krebshilfe), German National Academic Scholarship Foundation (Studienstiftung des deutschen Volkes), Survival with Pancreatic Cancer Foundation.
PubMed: 38941955
DOI: 10.1016/j.ebiom.2024.105219 -
Reproductive Biology Jun 2024Mesenchymal cells within theplacental villi play a crucial role in shaping the morphology of branching structures and driving the development of blood vessels. However,...
Mesenchymal cells within theplacental villi play a crucial role in shaping the morphology of branching structures and driving the development of blood vessels. However, the markers and functions of placental villous pericytes (PVPs) as distinct subgroups of placental villous mesenchymal cells, remain unclear. Therefore, in this study, the markers and functions of PVPs were investigated. Single-cell sequencing data from the first-trimester placental villi was obtained and the Seurat tool was used to identify PVP markers. Gene Ontology (GO) analysis of specific genes was performed using the DAVID database. The Cellchat tool was employed to investigate the interaction signals between the PVPs and other cells. Expression of the PVP markers was confirmed using immunofluorescence. Presence of extracellular vesicles in the placental villous mesenchyme and PVPs was examined by transmission electron microscopy. Our findings revealed that renin (REN) and amphiregulin (AREG)-positive fibroblasts in the placental villi specifically expressed several classic pericyte markers. In the first trimester, certain conserved functions of pericytes were observed and they displayed tissue-specific functions such as in the integrin-mediated signaling pathway and extracellular exosomes. Moreover, the placental villous mesenchyme was found to be rich in extracellular vesicles. AREG is specifically transcribed in the first trimester PVPs, however, its protein was located in syncytiotrophoblasts. These insights contribute to a comprehensive understanding of early placental development and offer new therapeutic targets for placenta-derived pregnancy complications.
PubMed: 38941941
DOI: 10.1016/j.repbio.2024.100919 -
Phytomedicine : International Journal... Jun 2024Colorectal cancer (CRC) is one of the most common causes of cancer-related mortality and significantly impairs quality of life. Astragali Radix-Curcumae Rhizoma (AC) is...
BACKGROUND
Colorectal cancer (CRC) is one of the most common causes of cancer-related mortality and significantly impairs quality of life. Astragali Radix-Curcumae Rhizoma (AC) is widely employed in the treatment of CRC in Chinese medicine, but the precise mechanisms remain unclear.
PURPOSE
This study aimed to elucidate the mechanisms by which AC inhibits CRC progression.
METHODS
The active components of AC were identified using UPLC-MS/MS analysis. An orthotopic transplantation colorectal tumor model was established in BALB/c mice using the CT26-Lucifer cell line to evaluate the effects of AC. Tumor volumes were monitored using IVIS imaging technology. Histological examination of tumor morphology was performed with hematoxylin and eosin (H&E) staining. Transcriptomic sequencing of mouse tumor samples was conducted to identify critical pathways and molecular targets. The impact of AC on cell viability and migration was assessed using CCK-8 and wound healing assays, respectively. To investigate the effects of AC on CRC cells, an in vitro hypoxic model was established using cobalt chloride (CoCl), a hypoxia inducer. HIF-2α overexpression was achieved by constructing stable lentiviral vectors. Key targets identified from RNA-seq, such as c-Myc, Ki-67, β-catenin, cleaved caspase 3, CD133, and CD44, were evaluated using western blotting, qRT-PCR, and immunofluorescence assays. Epithelial-Mesenchymal Transition (EMT) and spheroid cloning assays were employed to evaluate phenotypic changes in cancer stem cells.
RESULTS
Twelve components of AC were identified. AC effectively inhibited CRC progression in vivo. Transcriptomic analysis highlighted hypoxic signaling as a significantly enriched pathway, implicating its role in suppressing CRC progression by AC. In the hypoxic model, AC inhibited the proliferation and migration of CRC cells in vitro. Furthermore, AC reduced cancer stemness by downregulating stemness markers, inhibiting EMT, and decreasing tumor sphere formation. The downregulation of hypoxic responses and the shift in stemness by AC involved attenuation of HIF-2α and WNT/β-catenin signaling.
CONCLUSION
This study provides the first evidence that AC reduces the stemness of CRC and the inhibition of the transition of CRC to stem-like cells by AC is closely related to the downregulation of the HIF-2α/β-catenin pathway, especially under hypoxic conditions.
PubMed: 38941816
DOI: 10.1016/j.phymed.2024.155824 -
Poultry Science Jun 2024The late embryonic development of the liver, a major metabolic organ, remains poorly characterized at single cell resolution. Here, we used single-nucleus RNA-sequencing...
The late embryonic development of the liver, a major metabolic organ, remains poorly characterized at single cell resolution. Here, we used single-nucleus RNA-sequencing (snRNA-seq) to characterize the chicken liver cells at 2 embryonic development time points (E14 and D1). We uncovered 8 cell types including hepatocytes, endothelial cells, hepatic stellate cells, erythrocytes, cholangiocytes, kupffer cells, mesothelial cells, and lymphocytes. And we discovered significant differences in the abundance of different cell types between E14 and D1. Moreover, we characterized the heterogeneity of hepatocytes, endothelial cells, and mesenchymal cells based on the gene regulatory networks of each clusters. Trajectory analyses revealed 128 genes associated with hepatocyte development and function, including apolipoprotein genes involved hepatic lipid metabolism and NADH dehydrogenase subunits involved hepatic oxidative phosphorylation. Furthermore, we identified the differentially expressed genes (DEGs) between E14 and D1 at the cellular levels, which contribute to changes in liver development and function. These DEGs were significantly enriched in PPAR signaling pathways and lipid metabolism related pathways. Our results presented the single-cell mapping of chick embryonic liver at late stages of development and demonstrated the metabolic changes across the 2 age stages at the cellular level, which can help to further study the molecular development mechanism of embryonic liver.
PubMed: 38941785
DOI: 10.1016/j.psj.2024.103979 -
Reproduction (Cambridge, England) Jun 2024This study investigated the methylation patterns of H3K4me3 and H3K9me3, as well as the mRNA expression of genes encoding the epigenetic regulators KDM1AX1, KDM1AX2, and...
This study investigated the methylation patterns of H3K4me3 and H3K9me3, as well as the mRNA expression of genes encoding the epigenetic regulators KDM1AX1, KDM1AX2, and KDM3A in goat preantral follicles developed in vivo (Uncultured control) or after in vitro culture for 7 days in either the absence (α-MEM+) or presence of conditioned medium (α-MEM+ + CM) from Wharton's jelly mesenchymal stem cells (WJ-MSCs). In the in vivo setting, all follicular categories exhibited similar H3K4me3 and H3K9me3 patterns, and transcripts of KDM1AX1, KDM1AX2, and KDM3A were detected in all samples. During in vitro culture, α-MEM+ + CM enhanced several important aspects. It increased the percentage of normal growing follicles, oocyte diameters across all categories, stromal cell density, and the H3K4me3 methylation pattern in preantral follicles. Simultaneously, it decreased the levels of reduced thiols and reactive oxygen species in the spent media, diminished the presence of lipofuscin aggresomes, lowered granulosa cell apoptotic rates, and reduced the H3K9me3 methylation pattern in preantral follicles. In conclusion, the findings from this study provide compelling evidence that supplementing the in vitro culture medium (α-MEM+) with CM from WJ-MSCs has a protective effect on goat preantral follicles. Notably, CM supplementation preserved follicular survival, as evidenced by enhanced follicular and oocyte growth and increased stromal cell density when compared to the standard culture conditions in the α-MEM+ medium. Furthermore, CM reduced oxidative stress and apoptosis and promoted alterations in H3K4me3 and H3K9me3 patterns.
PubMed: 38941180
DOI: 10.1530/REP-23-0483 -
Chemistry & Biodiversity Jun 2024Delayed healing of chronic wounds results in amputation and mortality rates in serious cases. The present study examines the merged wound-restorative efficacy of...
Delayed healing of chronic wounds results in amputation and mortality rates in serious cases. The present study examines the merged wound-restorative efficacy of injectable bone marrow-derived mesenchymal stem cells (BMMSCs) and topical Callyspongia sp. extract in immunocompromised rats. HR-LC-MS analysis of Callyspongia sp. extract tentatively identified twenty-nine compounds (1-29) and highlighted its richness in fatty acids and terpenoids, known for their wound regenerating efficacies. The wound closure was greatly prominent in the BMMSCs/Callyspongia sp. group in contrast to the control group (p < 0.001). The RT-PCR gene expression emphasized these results by attenuating the oxidative, inflammatory, and immunity markers, further confirmed by histopathological findings. Additionally, in silico modeling was particularly targeting matrix metalloproteinase-9 (MMP9), a key player in wound healing processes. Computational analysis revealed that compounds 18 and 19 potentially modulate MMP9 activity. The combination of BMMSCs and topical Callyspongia sp. extract holds a promise for regenerative therapy constituting a drastic advance in the wound cure of immunocompromised patients, eventually further safety assessments and clinical trials are required.
PubMed: 38941178
DOI: 10.1002/cbdv.202400682 -
Cancer Science Jun 2024Osimertinib induces a marked response in non-small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) gene mutations. However, acquired...
Osimertinib induces a marked response in non-small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) gene mutations. However, acquired resistance to osimertinib remains an inevitable problem. In this study, we aimed to investigate osimertinib-resistant mechanisms and evaluate the combination therapy of afatinib and chemotherapy. We established osimertinib-resistant cell lines (PC-9-OR and H1975-OR) from EGFR-mutant lung adenocarcinoma cell lines PC-9 and H1975 by high exposure and stepwise method. Combination therapy of afatinib plus carboplatin (CBDCA) and pemetrexed (PEM) was effective in both parental and osimertinib-resistant cells. We found that expression of thrombospondin-1 (TSP-1) was upregulated in resistant cells using cDNA microarray analysis. We demonstrated that TSP-1 increases the expression of matrix metalloproteinases through integrin signaling and promotes tumor invasion in both PC-9-OR and H1975-OR, and that epithelial-to-mesenchymal transition (EMT) was involved in H1975-OR. Afatinib plus CBDCA and PEM reversed TSP-1-induced invasion ability and EMT changes in resistant cells. In PC-9-OR xenograft mouse models (five female Balb/c-Nude mice in each group), combination therapy strongly inhibited tumor growth compared with afatinib monotherapy (5 mg/kg, orally, five times per week) or CBDCA (75 mg/kg, intraperitoneally, one time per week) + PEM (100 mg/kg, intraperitoneally, one time per week) over a 28-day period. These results suggest that the combination of afatinib plus CBDCA and PEM, which effectively suppresses TSP-1 expression, may be a promising option in EGFR-mutated NSCLC patients after the acquisition of osimertinib resistance.
PubMed: 38941131
DOI: 10.1111/cas.16199 -
Stem Cell Reviews and Reports Jun 2024Intravenous infusion has been used as the method of cell delivery in many preclinical studies as well as in some early clinical trials. Among its advantages are broad...
Intravenous infusion has been used as the method of cell delivery in many preclinical studies as well as in some early clinical trials. Among its advantages are broad distribution, ability to handle a large-volume infusion, and ease of access. Progenitor cells used in cell-based therapy act through their secretomes, rather than their ability to differentiate into lineage-specific cell type. Since not all progenitor cells have similar secretome potency, the innate abilities of the secretome of cells used in clinical trials will obviously dictate their effectiveness. We previously found that cardiac neonatal mesenchymal stromal cells (nMSCs) are more effective in repairing the infarcted myocardium compared to adult mesenchymal stromal cells (aMSCs) due to their robust secretome (Sharma et al Circulation Research 120:816-834, 2017). In this study, we explored the efficacy of intravenous (IV) delivery of nMSCs for myocardial recovery. Six-week-old male Brown Norway rats underwent acute MI by ligation of the left anterior descending artery, followed by IV infusion of cell dose 5 × 10 nMSCs/rat body weight (kg) or saline on days 0 and 5. We found that cardiac parameters in the rodent ischemia model improved 1 month after nMSCs infusion, and the result is comparable with the intramyocardial injection of nMSCs. Tracking the infused cells in target organ revealed that their movement after IV delivery was mediated by the cell surface receptor CD44. Systemic injection of nMSCs stimulated immunomodulatory responses specifically by increasing FoxP3 T-regulatory cell influenced anti-inflammatory macrophages (M2) in heart. These data demonstrate that nMSCs promote immunogenic tolerance via CD44-driven T-reg/M2 stimulation that helps nMSCs for longer viability in the injured myocardium for better functional recovery. Our data also demonstrate a rationale for a clinical trial of IV infusion of nMSCs to promote cardiac function improvement in the ischemic patients.
PubMed: 38941039
DOI: 10.1007/s12015-024-10750-2