Swati S. Bhasin

Chronic wounds, such as diabetic foot ulcers, fail to progress through the normal healing process and impose a significant burden on healthcare systems. While previous single-cell studies have characterized specific wound conditions, a unified understanding of the shared and distinct cellular landscapes across diverse wound microenvironments has been lacking. Therefore, we integrated over 500,541 cells from patients and mice across multiple wound conditions, including acute wound, diabetic foot ulcer, and venous ulcer as well as their healing outcome. Fibroblast-focused analysis identified a bifurcation in differentiation trajectories and identified STAT3 as a potential regulator of a reparative program in chronic wound. Furthermore, we discovered immune dysfunctions in non-healed chronic wounds, contrasting the quiescent memory-like T cells and TIMP1+ macrophages in healed chronic wounds with the exhausted T cells and foamy SPP1+ macrophage enriched in non-healed chronic wounds. Finally, we translated these results into a clinically applicable three-gene signature (CHI3L1, TIMP1, and SPP1) that accurately predicts chronic wound healing. To support wound biology community, we developed WoundSCAtlas, an interactive web resource for exploring diverse wound pathologies. In conclusion, this work provides a comprehensive and cross-species landscape of chronic wound healing, identifying conversed wound outcome-associated molecular programs, predictive biomarker, and interactive data resource.

Pediatric acute myeloid leukemia (pAML) is a heterogeneous malignancy driven by diverse cytogenetic mutations. While identification of cytogenetic lesions improved risk stratification, prognostication remains inadequate with 30% of standard-risk patients experiencing relapse within 5 years. To deeply characterize pAML heterogeneity and identify poor outcome-associated blast cell profiles, we performed an analysis on 708,285 cells from 164 bone marrow biopsies of 95 patients and 11 healthy controls. The longitudinal analysis on cell abundances at the time of disease diagnosis, end of induction, and relapse identified treatment resistant stem-like blast cells specific to RUNX1::RUNX1T1, FLT3-ITD, and CBFB::MYH11 patients that are associated with poor outcomes. Treatment resistant blast cells from RUNX1::RUNX1T1 were found to associate with T cell exhaustion, while those from FLT-ITD utilized enriched antioxidant metabolism to persist through treatment. Interestingly, the analysis also identified novel mast cell-like pAML associated with treatment resistance and poor outcomes. Deconvolution of ex vivo treatment data and subsequent in vitro validation identified bortezomib (RUNX1), ponatinib, and venetoclax (FLT3) as specifically potent against treatment resistant blasts from the respective cytogenetic groups. Our findings indicate immature and mature pAML subtypes are promising biomarkers for enhanced patient risk stratification and identifies targeted agents to increase their clearance after treatment.

Single-cell transcriptome profiling enables unparalleled characterization of the heterogeneous microenvironment of pediatric leukemias. To facilitate comparative analyses and generate pediatric leukemia signatures, we collect, process, and annotate single-cell data comprising over 540,000 cells from 159 different pediatric acute leukemia (myeloid, lymphoid, mixed phenotype lineages) and healthy bone marrow (BM) samples, profiled in our lab and curated from publicly available studies. The analysis identifies a leukemia-enriched signature of nine genes with over-expression in leukemic blast compared to healthy BM cells. This signature is also consistently over-expressed in leukemia samples compared to normal BM in bulk RNA-seq datasets (over 2000 samples). Outcome-based analysis on diagnosis samples using measurable residual disease (MRD) status depicts a significant association of oncogene-induced senescence and g-protein activation pathways with MRD positivity. MRD positivity across pediatric leukemias is also correlated with significant depletion of CD8+ and CD4+ naïve T-cells and M1-macrophages at diagnosis. To enable easy access to this comprehensive pediatric leukemia single-cell atlas, we develop the Pediatric Single-cell Cancer Atlas (PedSCAtlas, https://bhasinlab.bmi.emory.edu/PediatricSCAtlas/). The atlas allows for quick exploration of single-cell data based on genes, cell type composition, and clinical outcomes to understand the cellular landscape of pediatric leukemias.

TAM-family tyrosine kinases (TYRO3, AXL and MERTK) are potential cancer therapeutic targets. In previous studies MERTK inhibition in the immune microenvironment was therapeutically effective in a B-cell acute leukemia (B-ALL) model. Here, we probed anti-leukemia immune mechanisms and evaluated roles for TYRO3 and AXL in the leukemia microenvironment. Host Mertk knock-out or MERTK inhibitor MRX-2843 increased CD8α⁺ dendritic cells (DCs) with enhanced antigen-presentation capacity in the leukemia microenvironment and inhibited leukemogenesis. High MERTK or low DC gene expression were associated with poor prognosis in pediatric ALL patients, indicating the clinical relevance of these findings. MRX-2843 increased CD8⁺ T-cell numbers and prevented induction of exhaustion markers, implicating a DC – T-cell axis. Indeed, combined depletion of CD8α⁺ DCs and CD8⁺ T-cells was required to abrogate anti-leukemia immunity in Mertk^–/– mice. Tyro3^–/– mice were also protected against B-ALL, implicating TYRO3 as an immunotherapeutic target. In contrast to Mertk^–/– mice, Tyro3^–/– did not increase CD8α⁺ DCs with enhanced antigen-presentation capacity and therapeutic activity was less dependent on DCs, indicating a different immune mechanism. Axl^–/– did not impact leukemogenesis. These data demonstrate differential TAM kinase roles in the leukemia microenvironment and provide rationale for development of MERTK and/or TYRO3-targeted immunotherapies.

Pediatric acute myeloid leukemia (pAML) is a heterogeneous malignancy driven by diverse cytogenetic mutations. While risk stratification improved by identifying cytogenetic lesions, prognostication remains inadequate with 30% of standard-risk patients experiencing relapse within 5 years. Single-cell RNA sequencing (scRNAseq) enabled the interrogation of malignant cell heterogeneity in pAML and characterization of the immune microenvironment. Herein we report the largest pAML scRNAseq analysis to date with 708,285 cells from 164 bone marrow biopsies of 95 patients and 11 healthy controls. We uncovered treatment-resistant (TR) subtypes of pAML specific to RUNX1-RUNX1T1, FLT3-ITD, and CBFB-MYH11 patients. The enrichment of TR subtype gene signatures on the TARGET pAML data supported an association with significantly poor outcomes. Intriguingly, in addition to leukemic stem cells, we identified mast cell-like pAML associated with treatment resistance and poor outcomes. Together, immature and mature pAML subtypes are promising biomarkers for identifying patients at increased risk of relapse within cytogenetic categories.

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 P 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.

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.

Acute myeloid leukemia (AML) microenvironment exhibits cellular and molecular differences among various subtypes. Here, we utilize single-cell RNA sequencing (scRNA-seq) to analyze pediatric AML bone marrow (BM) samples from diagnosis (Dx), end of induction (EOI), and relapse timepoints. Analysis of Dx, EOI scRNA-seq, and TARGET AML RNA-seq datasets reveals an AML blasts-associated 7-gene signature (CLEC11A, PRAME, AZU1, NREP, ARMH1, C1QBP, TRH), which we validate on independent datasets. The analysis reveals distinct clusters of Dx relapse- and continuous complete remission (CCR)-associated AML-blasts with differential expression of genes associated with survival. At Dx, relapse-associated samples have more exhausted T cells while CCR-associated samples have more inflammatory M1 macrophages. Post-therapy EOI residual blasts overexpress fatty acid oxidation, tumor growth, and stemness genes. Also, a post-therapy T-cell cluster associated with relapse samples exhibits downregulation of MHC Class I and T-cell regulatory genes. Altogether, this study deeply characterizes pediatric AML relapse- and CCR-associated samples to provide insights into the BM microenvironment landscape.