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Diabetic foot ulcer (DFU) is a critical complication of diabetes, but the wound microenvironment and its healing process are not completely understood. In this study, we optimized single-cell profiling from sharp debrided ulcers. Our findings demonstrate that healing-DFUs were significantly enriched with distinct fibroblasts expressing genes related to inflammation (CHI3L1, IL6) and extracellular matrix remodeling (ASPN), validating our previous studies on surgically resected ulcers. The race-focused analysis depicted lower expression of key healing-associated genes such as CHIL3L1, MMP11, and SFRP4 in fibroblasts of non-Hispanic Black (NHB) patients compared to White patients. In cellular communication analysis, healing enriched fibroblasts of NHBs exhibited upregulation of signaling pathways such as WNT while those of White showed IGF and MK pathways upregulation. Our findings advocate race as a risk marker of DFU outcomes, likely reflecting underlying disparities in environmental exposures and access to care that profoundly influence healing markers. Using sharp debrided tissues for single-cell assays, this study highlights the need for in-depth investigations into dysregulated wound healing microenvironments of under-represented racial groups.
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The tumor microenvironment (TME) of medulloblastoma (MB) influences progression and therapy response, presenting a promising target for therapeutic advances. Prior single-cell analyses have characterized the cellular components of the TME but lack spatial context. To address this, we performed spatial transcriptomic sequencing on sixteen pediatric MB samples obtained at diagnosis, including two matched diagnosis-relapse pairs. Our analyses revealed inter- and intra-tumoral heterogeneity within the TME, comprised of tumor-associated astrocytes (TAAs), macrophages (TAMs), stromal components, and distinct subpopulations of MB cells at different stages of neuronal differentiation and cell cycle progression. We identified dense regions of quiescent progenitor-like MB cells enriched in patients with high-risk (HR) features and an increase in TAAs, TAMs, and dysregulated vascular endothelium following relapse. Our study presents novel insights into the spatial architecture and cellular landscape of the medulloblastoma TME, highlighting spatial patterns linked to HR features and relapse, which may serve as potential therapeutic targets.
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BACKGROUND: Vein graft failure following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. Although previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on vein graft failure. We, therefore, aimed to elucidate the response of the vein conduit wall during harvest and following implantation, probing the key maladaptive pathways driving graft failure with the overarching goal of identifying therapeutic targets for biologic intervention to minimize these natural responses to surgical vein graft injury. METHODS: Employing a novel approach to investigating vascular pathologies, we harnessed both single-nuclei RNA-sequencing and spatial transcriptomics analyses to profile the genomic effects of vein grafts after harvest and distension, then compared these findings to vein grafts obtained 24 hours after carotid-carotid vein bypass implantation in a canine model (n=4). RESULTS: Spatial transcriptomic analysis of canine cephalic vein after initial conduit harvest and distention revealed significant enrichment of pathways (P<0.05) involved in the activation of endothelial cells (ECs), fibroblasts, and vascular smooth muscle cells, namely pathways responsible for cellular proliferation and migration and platelet activation across the intimal and medial layers, cytokine signaling within the adventitial layer, and ECM (extracellular matrix) remodeling throughout the vein wall. Subsequent single-nuclei RNA-sequencing analysis supported these findings and further unveiled distinct EC and fibroblast subpopulations with significant upregulation (P<0.05) of markers related to endothelial injury response and cellular activation of ECs, fibroblasts, and vascular smooth muscle cells. Similarly, in vein grafts obtained 24 hours after arterial bypass, there was an increase in myeloid cell, protomyofibroblast, injury response EC, and mesenchymal-transitioning EC subpopulations with a concomitant decrease in homeostatic ECs and fibroblasts. Among these markers were genes previously implicated in vein graft injury, including VCAN, FBN1, and VEGFC, in addition to novel genes of interest, such as GLIS3 and EPHA3. These genes were further noted to be driving the expression of genes implicated in vascular remodeling and graft failure, such as IL-6, TGFBR1, SMAD4, and ADAMTS9. By integrating the spatial transcriptomics and single-nuclei RNA-sequencing data sets, we highlighted the spatial architecture of the vein graft following distension, wherein activated and mesenchymal-transitioning ECs, myeloid cells, and fibroblasts were notably enriched in the intima and media of distended veins. Finally, intercellular communication network analysis unveiled the critical roles of activated ECs, mesenchymal-transitioning ECs, protomyofibroblasts, and vascular smooth muscle cells in upregulating signaling pathways associated with cellular proliferation (MDK [midkine], PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor]), transdifferentiation (Notch), migration (ephrin, semaphorin), ECM remodeling (collagen, laminin, fibronectin), and inflammation (thrombospondin), following distension. CONCLUSIONS: Vein conduit harvest and distension elicit a prompt genomic response facilitated by distinct cellular subpopulations heterogeneously distributed throughout the vein wall. This response was found to be further exacerbated following vein graft implantation, resulting in a cascade of maladaptive gene regulatory networks. Together, these results suggest that distension initiates the upregulation of pathological pathways that may ultimately contribute to bypass graft failure and presents potential early targets warranting investigation for targeted therapies. This work highlights the first applications of single-nuclei and spatial transcriptomic analyses to investigate venous pathologies, underscoring the utility of these methodologies and providing a foundation for future investigations.
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Análisis de la Célula Individual , Transcriptoma , Animales , Perros , Masculino , Recolección de Tejidos y Órganos/efectos adversos , Recolección de Tejidos y Órganos/métodos , Femenino , Transducción de Señal , Perfilación de la Expresión Génica/métodosRESUMEN
Multiple Myeloma (MM) remains incurable despite advances in treatment options. Although tumor subtypes and specific DNA abnormalities are linked to worse prognosis, the impact of immune dysfunction on disease emergence and/or treatment sensitivity remains unclear. We established a harmonized consortium to generate an Immune Atlas of MM aimed at informing disease etiology, risk stratification, and potential therapeutic strategies. We generated a transcriptome profile of 1,149,344 single cells from the bone marrow of 263 newly diagnosed patients enrolled in the CoMMpass study and characterized immune and hematopoietic cell populations. Associating cell abundances and gene expression with disease progression revealed the presence of a proinflammatory immune senescence-associated secretory phenotype in rapidly progressing patients. Furthermore, signaling analyses suggested active intercellular communication involving APRIL-BCMA, potentially promoting tumor growth and survival. Finally, we demonstrate that integrating immune cell levels with genetic information can significantly improve patient stratification.
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Background: Vein graft failure (VGF) following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. While previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on VGF. We, therefore, aimed to elucidate the response of the vein conduit wall during harvest and following implantation, probing the key maladaptive pathways driving graft failure with the overarching goal of identifying therapeutic targets for biologic intervention to minimize these natural responses to surgical vein graft injury. Methods: Employing a novel approach to investigating vascular pathologies, we harnessed both single-nuclei RNA-sequencing (snRNA-seq) and spatial transcriptomics (ST) analyses to profile the genomic effects of vein grafts after harvest and distension, then compared these findings to vein grafts obtained 24 hours after carotid-cartoid vein bypass implantation in a canine model (n=4). Results: Spatial transcriptomic analysis of canine cephalic vein after initial conduit harvest and distention revealed significant enrichment of pathways (P < 0.05) involved in the activation of endothelial cells (ECs), fibroblasts (FBs), and vascular smooth muscle cells (VSMCs), namely pathways responsible for cellular proliferation and migration and platelet activation across the intimal and medial layers, cytokine signaling within the adventitial layer, and extracellular matrix (ECM) remodeling throughout the vein wall. Subsequent snRNA-seq analysis supported these findings and further unveiled distinct EC and FB subpopulations with significant upregulation (P < 0.00001) of markers related to endothelial injury response and cellular activation of ECs, FBs, and VSMCs. Similarly, in vein grafts obtained 24 hours after arterial bypass, there was an increase in myeloid cell, protomyofibroblast, injury-response EC, and mesenchymal-transitioning EC subpopulations with a concomitant decrease in homeostatic ECs and fibroblasts. Among these markers were genes previously implicated in vein graft injury, including VCAN (versican), FBN1 (fibrillin-1), and VEGFC (vascular endothelial growth factor C), in addition to novel genes of interest such as GLIS3 (GLIS family zinc finger 3) and EPHA3 (ephrin-A3). These genes were further noted to be driving the expression of genes implicated in vascular remodeling and graft failure, such as IL-6, TGFBR1, SMAD4, and ADAMTS9. By integrating the ST and snRNA-seq datasets, we highlighted the spatial architecture of the vein graft following distension, wherein activated and mesenchymal-transitioning ECs, myeloid cells, and FBs were notably enriched in the intima and media of distended veins. Lastly, intercellular communication network analysis unveiled the critical roles of activated ECs, mesenchymal transitioning ECs, protomyofibroblasts, and VSMCs in upregulating signaling pathways associated with cellular proliferation (MDK, PDGF, VEGF), transdifferentiation (Notch), migration (ephrin, semaphorin), ECM remodeling (collagen, laminin, fibronectin), and inflammation (thrombospondin), following distension. Conclusions: Vein conduit harvest and distension elicit a prompt genomic response facilitated by distinct cellular subpopulations heterogeneously distributed throughout the vein wall. This response was found to be further exacerbated following vein graft implantation, resulting in a cascade of maladaptive gene regulatory networks. Together, these results suggest that distension initiates the upregulation of pathological pathways that may ultimately contribute to bypass graft failure and presents potential early targets warranting investigation for targeted therapies. This work highlights the first applications of single-nuclei and spatial transcriptomic analyses to investigate venous pathologies, underscoring the utility of these methodologies and providing a foundation for future investigations.
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Introduction: Current multistep methods utilized for preparing and cryopreserving single-cell suspensions from blood samples for single-cell RNA sequencing (scRNA-seq) are time-consuming, requiring trained personnel and special equipment, so limiting their clinical adoption. We developed a method, Simple prEservatioN of Single cElls (SENSE), for single-step cryopreservation of whole blood (WB) along with granulocyte depletion during single-cell assay, to generate high quality single-cell profiles (SCP). Methods: WB was cryopreserved using the SENSE method and peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved using the traditional density-gradient method (PBMC method) from the same blood sample (n=6). The SCPs obtained from both methods were processed using a similar pipeline and quality control parameters. Further, entropy calculation, differential gene expression, and cellular communication analysis were performed to compare cell types and subtypes from both methods. Results: Highly viable (86.3 ± 1.51%) single-cell suspensions (22,353 cells) were obtained from the six WB samples cryopreserved using the SENSE method. In-depth characterization of the scRNA-seq datasets from the samples processed with the SENSE method yielded high-quality profiles of lymphoid and myeloid cell types which were in concordance with the profiles obtained with classical multistep PBMC method processed samples. Additionally, the SENSE method cryopreserved samples exhibited significantly higher T-cell enrichment, enabling deeper characterization of T-cell subtypes. Overall, the SENSE and PBMC methods processed samples exhibited transcriptional, and cellular communication network level similarities across cell types with no batch effect except in myeloid lineage cells. Discussion: Comparative analysis of scRNA-seq datasets obtained with the two cryopreservation methods i.e., SENSE and PBMC methods, yielded similar cellular and molecular profiles, confirming the suitability of the former method's incorporation in clinics/labs for cryopreserving and obtaining high-quality single-cells for conducting critical translational research.
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Criopreservación , Leucocitos Mononucleares , Criopreservación/métodos , Control de CalidadRESUMEN
OBJECTIVE: Cancer related fatigue is a distressing condition and correlated with decrease in quality of life of patients with malignant conditions. In continuation of our previous research, we assessed long term anti-fatigue effects of melatonin in patients with the breast cancer. MATERIAL AND METHODS: In this clinical trial, 92 breast cancer patients were randomly assigned to receive either melatonin (18 mg/day) or placebo from 1 week before the adjuvant treatments until 2 years after their completion. The levels of fatigue were assessed before and after intervention using Brief Fatigue Inventory (BFI) and were compared at a significance level of P ≤ .05. RESULTS: The BFI scores were similar between the 2 groups at the baseline (placebo group: 5.56 ± 1.59 and melatonin group: 5.72 ± 1.68, P = .67). After the intervention, not only the mean fatigue score was significantly lower in melatonin group (2.93 ± 1.04 vs 1.99 ± 1.02, P < .001, P ≤ .05), but also a greater reduction in fatigue score in intervention group was evident over time (P ≤ .001). CONCLUSION: Long-term usage of melatonin even after completion of adjuvant therapies in women with breast cancer decreased the levels of fatigue associated with the malignant condition and its treatments. THE TRIAL REGISTRY NAME AND URL, AND REGISTRATION NUMBER: Iranian Registry of Clinical Trials, https://en.irct.ir/trial/62267, IRCT20180426039421N3.
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Neoplasias de la Mama , Melatonina , Humanos , Femenino , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Melatonina/uso terapéutico , Calidad de Vida , IránRESUMEN
A new coronavirus pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been on the rise. This virus is fatal for broad groups of populations, including elderly, men, and patients with comorbidities among which obesity is a possible risk factor. The pathophysiologic connections between obesity/metainflammation and COVID-19 may be directly related to increasing soluble ACE2 (angiotensin-converting enzyme 2) levels which potentiate the viral entrance into the host cells, or indirectly related to dysregulation of immune system, microvascular injury and hypercoagulability. The SARS-CoV-2 S-glycoprotein interacts mainly with ACE2 or possibly DPP4 receptors to enter into the host cells. The host proteases, especially TMPRSS2 (transmembrane protease serine 2), support the fusion process and virus entry. While membranous ACE2 is considered a port of entry to the cell for SARSCoV- 2, it seems that soluble ACE2 retains its virus binding capability and enhances its entry into the cells. Interestingly, ACE2 on cell membrane may have protective roles by diminishing cytokine storm-related injuries to the organs. Applying medications that can reduce soluble ACE2 levels, antagonizing TMPRSS2 or blocking DPP4 can improve the outcomes of COVID-19. Metformin and statins through immunomodulatory activities, Orlistat by reducing viral replication, and thiazolidinediones by upregulating ACE2 expression have potential beneficial effects against COVID-19. However, the combination of dipeptidyl peptidase-4 (DPP4) inhibitors and spironolactone/ eplerenone seems to be more effective by reducing soluble ACE2 level, antagonizing TMPRSS2, maintaining ACE2 on cell membrane and reducing risk of viral entry into the cells.