scholarly journals Single-cell landscape of bone marrow metastases in human neuroblastoma unraveled by deep multiplex imaging

2020 ◽  
Author(s):  
Daria Lazic ◽  
Florian Kromp ◽  
Michael Kirr ◽  
Filip Mivalt ◽  
Fikret Rifatbegovic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Single Cells ◽  
Therapy Response ◽  
Disseminated Tumor Cells ◽  
Bone Marrow Niche ◽  
Inhibitory Protein ◽  
Apoptotic Pathway ◽  
Human Neuroblastoma ◽  
Bone Marrow Metastases

ABSTRACTBone marrow commonly serves as a metastatic niche for disseminated tumor cells (DTCs) of solid cancers in patients with unfavorable clinical outcome. Single-cell assessment of bone marrow metastases is essential to decipher the entire spectrum of tumor heterogeneity in these cancers, however, has previously not been performed.Here we used multi-epitope-ligand cartography (MELC) to spatially profile 20 biomarkers and assess morphology in DTCs as well as hematopoietic and mesenchymal cells of eight bone marrow metastases from neuroblastoma patients. We developed DeepFLEX, a single-cell image analysis pipeline for MELC data that combines deep learning-based cell and nucleus segmentation and overcomes frequent challenges of multiplex imaging methods including autofluorescence and unspecific antibody binding.Using DeepFLEX, we built a single-cell atlas of bone marrow metastases comprising more than 35,000 single cells. Comparisons of cell type proportions between samples indicated that microenvironmental changes in the metastatic bone marrow are associated with tumor cell infiltration and therapy response. Hierarchical clustering of DTCs revealed multiple phenotypes with highly diverse expression of markers such as FAIM2, an inhibitory protein in the Fas apoptotic pathway, which we propose as a complementary marker to capture DTC heterogeneity in neuroblastoma.The presented single-cell atlas provides first insights into the heterogeneity of human bone marrow metastases and is an important step towards a deeper understanding of DTCs and their interactions with the bone marrow niche.

scholarly journals Landscape of Bone Marrow Metastasis in Human Neuroblastoma Unraveled by Transcriptomics and Deep Multiplex Imaging

Cancers ◽  
2021 ◽  
Vol 13 (17) ◽  
pp. 4311
Author(s):  
Daria Lazic ◽  
Florian Kromp ◽  
Fikret Rifatbegovic ◽  
Peter Repiscak ◽  
Michael Kirr ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Tumor Cells ◽  
Bone Marrow Involvement ◽  
Solid Cancer ◽  
Bone Marrow Niche ◽  
Single Cell Level ◽  
Bone Marrow Metastasis ◽  
Cell Level ◽  
Human Neuroblastoma

While the bone marrow attracts tumor cells in many solid cancers leading to poor outcome in affected patients, comprehensive analyses of bone marrow metastases have not been performed on a single-cell level. We here set out to capture tumor heterogeneity and unravel microenvironmental changes in neuroblastoma, a solid cancer with bone marrow involvement. To this end, we employed a multi-omics data mining approach to define a multiplex imaging panel and developed DeepFLEX, a pipeline for subsequent multiplex image analysis, whereby we constructed a single-cell atlas of over 35,000 disseminated tumor cells (DTCs) and cells of their microenvironment in the metastatic bone marrow niche. Further, we independently profiled the transcriptome of a cohort of 38 patients with and without bone marrow metastasis. Our results revealed vast diversity among DTCs and suggest that FAIM2 can act as a complementary marker to capture DTC heterogeneity. Importantly, we demonstrate that malignant bone marrow infiltration is associated with an inflammatory response and at the same time the presence of immuno-suppressive cell types, most prominently an immature neutrophil/granulocytic myeloid-derived suppressor-like cell type. The presented findings indicate that metastatic tumor cells shape the bone marrow microenvironment, warranting deeper investigations of spatio-temporal dynamics at the single-cell level and their clinical relevance.

scholarly journals The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors

Blood ◽  
1995 ◽  
Vol 85 (9) ◽  
pp. 2422-2435 ◽  
Author(s):  
EK Waller ◽  
J Olweus ◽  
F Lund-Johansen ◽  
S Huang ◽  
M Nguyen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Progenitor Cells ◽  
Cell Sorting ◽  
Single Cells ◽  
Light Scatter ◽  
Hematopoietic Progenitor ◽  
Individual Culture ◽  
Hla Dr ◽  
Growth Properties

There is a long-standing controversy as to whether a single bone marrow (BM)-derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence- activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38- , HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM.

Phenotypic and Genotypic Diversity of Human Neuroblastoma Studied in Three IGR Cell Line Models Derived from Bone Marrow Metastases

1999 ◽  
Vol 112 (2) ◽  
pp. 124-129 ◽  
Author(s):  
Alexander Valent ◽  
Jean Bénard ◽  
Anne-Marie Vénuat ◽  
Jacqueline Da Silva ◽  
Annette Duverger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Genotypic Diversity ◽  
Human Neuroblastoma ◽  
Bone Marrow Metastases

Proteome Analyses and Single-Cell RNA Sequencing Reveal Age-Dependent Re-Wiring of Central Carbon Metabolism in Myeloid-Biased Subsets of Human Hematopoietic Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 873-873
Author(s):  
Marco L Hennrich ◽  
Natalie Romanov ◽  
Patrick Horn ◽  
Samira Jaeger ◽  
Volker Eckstein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Carbon Metabolism ◽  
Human Subjects ◽  
Central Carbon Metabolism ◽  
Glycolytic Enzymes ◽  
Bone Marrow Niche ◽  
Mrna Levels ◽  
Hematopoietic Stem ◽  
Central Carbon

Abstract Background: Diminishing potential to replace damaged tissues is a hallmark for aging of somatic stem cells, but the mechanisms leading to aging remain elusive. Applying comprehensive proteomics studies on human hematopoietic stem and progenitor cells (HPCs) as well as 5 other cell populations that constitute the bone marrow niche, we have acquired a systems understanding of the mechanisms involved in aging of HPCs. Methods and materials: We present a proteome-wide atlas of age-associated alterations in HPCs along with 5 other cell populations including mesenchymal stromal cells (MSC) that comprise the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified was assessed in a cohort of 59 human subjects from different age groups. In selected samples, transcriptomics, metabolomics, and single cell RNA-Sequencing studies were simultaneously performed. Results: As the HPCs become older, one of the most prominent changes is the increase in abundance of proteins involved in the pathways for central carbon metabolism. This change is found only in HPCs and is reminiscent of the Warburg effect where glycolytic intermediates are rerouted towards pentose phosphate shunt and anabolism. Transcriptomic data confirm the increase in abundance of mRNA for the respective glycolytic enzymes in older HPCs. Metabolomics analyses provide further proofs demonstrating the trend towards anabolism upon aging. Altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation at the expense of lymphoid development. Simultaneously, significant and complementary alterations in the bone marrow niche are observed. Whereas key factors responsible for homing, egress and adhesion of HPCs, e.g. SDF1/CXCL12, VCAM1, FN1, integrins α4, αL, β1 andβ2 decrease in abundance in MSCs, soluble factors responsible for myeloid differentiation, e.g.TGF-beta1, increase in abundance in the cellular niche with age. Transcriptomic analyses of single-sorted HPCs have demonstrated unequivocally that the mRNA levels of age-dependent increase in glycolytic enzymes are expressed at significantly higher levels in myeloid-biased versus than those in lymphoid-biased HPCs, whereas age-unaffected enzymes have similar mRNA levels in both subsets. The increase in abundance of glycolytic enzymes is hence linked with skewing of myeloid-biased HPCs in older human subjects. Conclusion: We have generated a comprehensive atlas of alterations in proteome landscapes of human HPCs and niche cells in bone marrow upon aging. In addition to findings that recapitulate the results derived from murine studies, the major novelties are (a) the alterations in central carbon metabolism in aging human HPCs, (b) the complementary decrease in levels of adhesive molecules and respective ligands in MSCs and HPCs. Single-cell studies have demonstrated that the age-related increase in abundance of the glycolytic enzymes is linked to the myeloid-biased HPCs. These data represent a valuable resource and serve as a basis for development of strategies targeting metabolic changes to enhance HPC regeneration. Disclosures No relevant conflicts of interest to declare.

Distinct Stress Responses of Bone Marrow Stromal Cell Subgroups Defined by Unbiased Single-Cell Mass-Cytometry Analysis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 430-430 ◽  
Author(s):  
Nicolas Severe ◽  
Murat Karabacak ◽  
Ninib Baryawno ◽  
Karin Gustafsson ◽  
Youmna Sami Kfoury ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Stromal Cell ◽  
Marrow Stromal Cells ◽  
Osteoblastic Cells ◽  
Mouse Bone Marrow ◽  
Bone Marrow Niche ◽  
Mass Cytometry

Abstract The bone marrow niche is a heterogeneous tissue comprised of multiple cell types that collectively regulate hematopoiesis. It is thought to be a critical stress sensor, integrating information at the level of the organism down to signals at the level of the single cell. In so doing, the niche orchestrates hematopoietic stem and progenitor cell (HSPC) responses to organismal stress. However, most studies of the niche have depended on genetic marker or deletion studies that inherently limit analysis to the selected indicator genes or cells. While this has greatly enhanced our understanding of bone marrow function, it does not permit systems level evaluation of subgroups of cells and their relative response to a particular challenge. We therefore sought a less biased strategy to study bone marrow stromal cells and the cytokines they elaborate under homeostatic and stress conditions. We used Mass-Cytometry (CyTOF) to resolve protein levels at single cell resolution in mouse bone marrow. We established a panel of 36 antibodies: 20 surface and intracellular phenotypic markers, 12 cytokines regulating hematopoiesis, 1 marker of proliferation, 1 marker for DNA damage, 1 viability marker and 1 nucleated cell marker. We intentionally selected antibodies that recognize antigens already defined by others as bone marrow stromal markers. Freshly isolated non-hematopoietic cells from long bones and pelvis were analyzed and clustered into subgroups based on their protein expression signature. We applied k-means clustering using common markers to group bone marrow stromal cells into phenotypical subtypes. At steady state, analysis of over 20.000 mouse bone marrow stromal single-cells negative for the hematopoietic markers CD45 and Ter119 revealed 4 large clusters: an endothelial population expressing CD31, Sca1 and CD105, a mesenchymal stromal cell population expressing Sca1, CD140a, Nestin and LeptinR, a bone marrow stromal progenitor population expressing CD105, CD271 and Runx2 and a mature bone cell population expressing Osteocalcin and CD140a. Within these clusters, sub-populations were evident by adding CD106, CD90, CD73, Embigin, CD29, CD200, c-Kit and CD51. In total, 28 distinct populations of bone marrow stromal cells were identified based on their phenotypic signature. Only one cluster of cells was negative for all the markers we selected. Therefore, the complex heterogeneity of the bone marrow niche cells can be resolved to 28 subpopulations by single-cell protein analysis. Assessing the response of these groups to systemic challenges of medical relevance, we evaluated cells prior to whole body lethal irradiation (9.5Gy), one hour and one day later (the time of transplantation) and 3 days after irradiation (2d post transplantation) with and without transplanted cells. Notably, LeptinR+CD106+Sca1+ cells putatively essential for hematopoiesis and stem cell support were highly sensitive to and largely killed by irradiation. In contrast, endothelial cells and osteoblastic cells were resistant to irradiation. In particular, osteoblastic cells expressing osteocalcin (GFP+), embigin, NGFR and CD73 increased their expression of multiple hematopoietic cytokines including SDF-1, kit ligand, IL-6, G-CSF and TGF-b one day after irradiation. These data indicate that LeptinR+CD106+Sca1 stromal cells are unlikely to participate in HSPC engraftment post-irradiation while a subset of osteoblastic cells are. Unbiased single cell analysis can resolve subsets of bone marrow cells that respond differently to organismal stress. This method enables comprehensively quantifying subpopulation changes with specific challenges to begin defining the systems biology of the bone marrow niche. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mesenchymal Stromal Cells in the Bone Marrow Niche Consist of Multi-Populations With Distinct Transcriptional and Epigenetic Properties

2021 ◽  
Author(s):  
Sanshiro Kanazawa ◽  
Hironori Hojo ◽  
Shinsuke Ohba ◽  
Junichi Iwata ◽  
Makoto Komura ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Single Cell ◽  
Expression Profiles ◽  
Marker Genes ◽  
Mouse Bone Marrow ◽  
Bone Marrow Niche ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Abstract Although multiple studies have investigated the mesenchymal stem and progenitor cells (MSCs) that give rise to mature bone marrow, high heterogeneity in their morphologies and properties causes difficulties in molecular separation of their distinct populations. In this study, by taking advantage of the resolution of the single cell transcriptome, we analyzed Sca-1 and PDGFR-α fraction in the mouse bone marrow tissue. The single cell transcriptome enabled us to further classify the population into seven populations according to their gene expression profiles. We then separately obtained the seven populations based on candidate marker genes, and specified their gene expression properties and epigenetic landscape by ATAC-seq. Our findings will enable to elucidate the stem cell niche signal in the bone marrow microenvironment, reconstitute bone marrow in vitro, and shed light on the potentially important role of identified subpopulation in various clinical applications to the treatment of bone- and bone marrow-related diseases.

scholarly journals Single Cell RNA-Seq Reveals Deranged Developmental Hierarchy with Coexisting Oncogenic States and Immune Evasion Programs in ETP T-ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3953-3953
Author(s):  
Amy Guillaumet-Adkins ◽  
Praveen Anand ◽  
Huiyoung Yun ◽  
Yotam Drier ◽  
Anna Rogers ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Single Cells ◽  
Pearson Correlation ◽  
Normal Donor ◽  
Developmental Hierarchy

Introduction: Early T-cell precursor acute lymphoblastic leukemia (ETP T-ALL) is a distinct subtype of T-ALL characterized by higher rates of relapse and induction failure. Large-scale genetic sequencing studies have identified frequently mutated oncogenes and gene fusions in ETP T-ALL, while bulk transcriptome analyses have revealed expression features resembling myeloid precursors and myeloid malignancies. However, the contributions of intra-tumoral functional heterogeneity and microenvironment to tumor biology and treatment failure remain unknown. Methods: We performed full-length single-cell RNA-sequencing of 5,077 malignant and normal immune cells from bone marrow or blood from five patients with relapsed/refractory ETP T-ALL (based on immunophenotyping, all with NOTCH1 mutations), before and after targeted therapy against NOTCH1. These patients were enrolled on a phase I trial with the γ-secretase inhibitor (GSI) BMS-906024 (NCT01363817). Expression of selected genes was validated by RT-PCR, flow cytometry and immunohistochemistry. Results: Single cell transcriptome analyses revealed a deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cells implying ineffectual commitment to either lymphoid or myeloid lineage. Most ETP T-ALL cells co-expressed HSC (hematopoietic stem cell), CMP (common myeloid progenitor) and CLP (common lymphoid progenitor) signatures simultaneously (Pearson correlation: CLP-CMP: R= 0.41, p < 2.2e-16; HSC-CLP: R= 0.53; p < 2.2e-16; HSC-CMP: R = 0.39, p <2.2e-16). Only a fraction of cells (less than 15%) demonstrated mutually exclusive CLP or HSC signatures. In contrast, CLP, CMP and HSC signatures were not co-expressed and always negatively correlated in normal bone marrow cells (CLP-CMP: R= -0.11, p < 2.2e-16; HSC-CLP: R= -0.38; p < 2.2e-16; HSC-CMP: R = -0.67, p <2.2e-16). Direct targeting of NOTCH1 as the driving oncogene has shown disappointing results in the clinical setting due to the rapid development of resistance. PI3K activation has been shown as a genetic mechanism of Notch resistance, however it is unclear if transcriptional rewiring can give rise to PI3K dependent cells after Notch inhibition. To address this question, we predicted the activity of signaling pathways in single cells after Notch inhibitor treatment using PROGENy. Most single cells demonstrated loss of Notch signaling. PI3K signaling activity was the most anti-correlated signaling pathway to Notch signaling (Pearson correlation: R= -0.51, p < 2.2e-16). Of note, this population preexisted at a frequency of ~30% in the untreated population, coexisting with cells with high Notch activation. Analysis of the immune microenvironment revealed an oligoclonal T-cell population in ETP T-ALL compared to normal donor T-cells. CD8+ T-cells from ETP patients expressed markers of T-cell exhaustion (PDCD1, TIGIT, LAG3, HAVCR2). Analyses of expression levels of the respective ligands on leukemic blasts and the predicted interaction with their receptors on endogenous CD8+ T-cells demonstrated the highest interaction score between HAVCR2 and its ligand LGALS9. LGALS9 was universally expressed in all leukemic cells, which was confirmed by flow cytometry staining in leukemic blasts and IHC staining in bone marrow of 8 patients with ETP T-ALL and 7 patients with T-ALL. T-ALL supernatant increased expression levels of the exhaustion markers HAVCR2,TIGIT and decreased effector marker GZMB in polyclonal activated normal donor CD8+ T-cells (RT-PCR). This effect was abrogated by neutralizing LGALS9 and could be rescued with recombinant LGALS9. Conclusion: We identified deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cell states and ineffectual commitment to either lymphoid or myeloid lineage in ETP T-ALL. Leukemic blasts demonstrate preexisting heterogeneity of diverse oncogenic states as evidenced by opposing PI3K and Notch activity, suggesting possible novel combination therapies. Notch inhibition abolishes the Notch high state without effecting the PI3K active state. Finally, we demonstrate a possible role for HAVCR2-LGALS9 interactions in causing CD8+ T-cell dysfunction in ETP T-ALL patients, which may provide a novel therapeutic strategy in this disease. Disclosures Silverman: Takeda: Consultancy; Servier: Consultancy, Research Funding. Lane:AbbVie: Research Funding; Stemline Therapeutics: Research Funding; N-of-One: Consultancy. DeAngelo:Glycomimetics: Research Funding; Amgen, Autolus, Celgene, Forty-seven, Incyte, Jazzs, Pfizer, Shire, Takeda: Consultancy; Blueprint: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Abbvie: Research Funding. Lohr:Celgene: Research Funding; T2 Biosystems: Honoraria.

scholarly journals Single-Cell RNA-Seq Reveals the Differentiation Hierarchy of Normal Human Bone Marrow and a Distinct Transcriptome Signature of Monosomy 7 Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 574-574
Author(s):  
Xin Zhao ◽  
Shouguo Gao ◽  
Xingmin Feng ◽  
Delong Liu ◽  
Sachiko Kajigaya ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Single Cell ◽  
Signaling Pathway ◽  
Copy Number ◽  
Research Funding ◽  
Single Cells ◽  
Myeloid Cells ◽  
Healthy Controls ◽  
Monosomy 7

Abstract Monosomy 7 is a frequent cytogenetic abnormality in hematopoietic malignancies and a general indicator of poor prognosis. Due to lack of distinct cell surface markers between monosomy 7 cells and normal cells, it is not feasible to physically separate aneuploid from diploid cells. We performed single-cell RNA-seq (scRNA-seq), which allows the entire transcriptome of large numbers of single cells to be assayed in an unbiased way, to investigate hematopoietic differentiation of normal and aneuploid human hematopoietic cells. Bone marrow samples were collected from four patients (P1-P4) with myelodysplastic syndrome, and four healthy volunteers. Conventional cytogenetics showed -7/7q- in bone marrow cells from P1, P3 and P4, and dup(1)(q21q32) in cells from P2; retrospectively, P2 was found positive for monosomy 7 as well as trisomy 8 by fluorescence in situ hybridization. Fresh CD34+CD38- and CD34+CD38+cells were sorted by flow cytometry and then subjected to Fluidigm C1 Single-Cell Auto Prep System for scRNA-seq. After excluding cells with low transcriptome coverage, 326 cells from P1 and P2 (analysis is in progress for P3 and P4), and 391 cells from healthy subjects were analyzed by comparison of transcriptomes from 17,071 genes. Nonlinear dimension reduction and visualization were achieved using t-distributed Stochastic Neighbor Embedding (tSNE). Cells from healthy controls clustered into seven subgroups based on their gene expression pattern, and each group could be associated with a previously reported hematopoietic cell type by known marker genes (Laurenti E, Nat Immunol, 2013). These cell types included hematopoietic stem cell (HSC), multilymphoid progenitor (MLP), granulocyte-monocyte progenitor (GMP), Pro-B cell (ProB), earliest thymic progenitor (ETP), and megakaryocytic-erythroid progenitor (MEP) (Fig 1a). Individual cells from healthy controls were ordered by Monocle software based on their expression profile similarity to uncover a differentiation hierarchy. A two-branch trajectory of development from HSC was revealed, with one branch progressing towards erythroid cell and the other to lymphoid/myeloid cells (Fig 1b). This pattern differs from the classic hematopoietic model, but is consistent with reports claiming existence of early-lymphoid-biased progenitors that retain myeloid but not erythroid potential (Doulatov S, Nat Immunol, 2010), and of dominance of multipotent and unipotent progenitors over scarce oligopotent progenitors in the adult marrow hematopoietic hierarchy (Notta F, Science, 2016). We compared single cells from patients and healthy controls for regional and chromosomal copy number differences in gene expression. We identified subclonal populations of cells from patients that showed decreased expression of chromosome 7 genes (60% in P1, and 55% in P2; Fig 1c and 1d), and increased expression of chromosome 8 (77% in P2) and chromosome 1 long arm genes (P2), at FDR=0.05 estimated with cells from health donors. Gene Ontology enrichment analysis using topGO indicated that cells with low global expression of chromosome 7 genes had dysregulated expression of immune related genes, including B cell receptor signaling pathway, T cell activation and differentiation, antigen receptor-mediated signaling pathway, as well as signal transduction and Fc-γ Receptor signaling pathway. ScRNA-seq analysis reveals a simple pattern of normal human hematopoietic development and the molecular signature of aneuploid cells from patients with developing "clonal evolution". This powerful method should improve characterization of functional changes in human cells with chromosome abnormalities. Figure 1 a. Single-cell gene expression patterns assigned single cells from healthy controls to seven clusters. 38N: CD34+CD38- population; 38P: CD34+CD38+ population. Different shapes represent cells from different subjects. b. Pseudo-time ordering of cells using Monocle reveals a two-branch stepwise development from stem cells to erythroid or lymphoid/myeloid cells. c. Heatmap of the copy-number variation (CNV) signal normalized against healthy controls shows CNV changes by chromosome (columns) for patients' individual cells (rows). d. Genome-wide gene expression binned per chromosome in single cells from P1, P2 and healthy controls. Chromosomal mapping reads values were median centered. Figure 1. a. Single-cell gene expression patterns assigned single cells from healthy controls to seven clusters. 38N: CD34+CD38- population; 38P: CD34+CD38+ population. Different shapes represent cells from different subjects. b. Pseudo-time ordering of cells using Monocle reveals a two-branch stepwise development from stem cells to erythroid or lymphoid/myeloid cells. c. Heatmap of the copy-number variation (CNV) signal normalized against healthy controls shows CNV changes by chromosome (columns) for patients' individual cells (rows). d. Genome-wide gene expression binned per chromosome in single cells from P1, P2 and healthy controls. Chromosomal mapping reads values were median centered. Disclosures Desierto: GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding. Young:Novartis: Research Funding.

scholarly journals Paired Analyses of AML at Diagnosis and Relapse By Single-Cell RNA Sequencing Identifies Two Distinct Relapse Patterns

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 183-183
Author(s):  
Kai Wu ◽  
Qianyi Ma ◽  
Darren King ◽  
Jun Li ◽  
Sami Malek
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Single Cell ◽  
Bone Marrow Cells ◽  
Single Cells ◽  
Leukemia Cells ◽  
Driver Genes ◽  
Cell Gene Expression ◽  
Normal Human ◽  
Relapse Patterns

Introduction: Despite achievement of complete remission (CR) following chemotherapy, Acute Myelogenous Leukemia (AML) relapses in the majority of adult patients. While relapsed AML is almost always clonally related to the disease at diagnosis, the actual molecular and cellular contributors to chemotherapy resistance and to AML relapse remain incompletely understood. Some molecular determinants of relapse have been identified in genomic, epigenetic and proteomic aberrations, while cellular relapse reservoirs have been identified in leukemia stem cells as well as in more mature leukemic cell compartments. Here, we set out to determine the cellular composition, gene mutation status and gene expression of paired AML specimens procured at diagnosis and at relapse aiming at a better understanding of the AML relapse process. Methods: We employed the drop-seq 3' single cell RNA sequencing (scRNA-seq) method (Macosko 2015) with minor modifications to analyze the mRNA expression in single cells derived from 12 paired AML specimens procured at diagnosis and at relapse from prior CR. We obtained scRNA-seq data on 1000-2000 single cells per sample detecting approximately 2000-3000 unique molecular identifiers (UMIs) and 800-1500 genes per cell. Using WES or panel-based sequencing we determined mutations in known driver genes. Subsequently, we optimized novel methods for detection and mapping of mutated driver genes to individual cells using mutation specific PCR conditions and novel bioinformatics approaches. We annotated scRNA-seq expression profiles of the diagnosis and relapsed AML specimens individually using publicly available data for cell type-specific RNA markers derived from sorted normal cell populations and further compared the scRNA-seq data to scRNA-seq data of 5 pooled normal human bone marrows generated for this study. Results: Through analyses of scRNA-seq data of paired diagnosis and relapse AML specimens via principle components analyses (PCA) or t-distributed stochastic neighbor embedding (t-SNE) we detected varying degrees of separation of cell clusters in all cases analyzed indicative of substantial changes in single cell gene expression between AML diagnosis and relapse. A few of these observed cluster shifts were paralleled by gain or loss of mutated genes (e.g. FLT3-ITD) at relapse while most others lacked obvious clonal genomic markers. Through subsequent comparison of the expression similarities of single AML cells to sorted normal human bone marrow cells we detected two distinct AML relapse patterns: i) a pattern of relapse suggesting simple leukemia regrowth as evidenced by similar proportions of leukemia cells mapping onto discrete normal bone marrow cells (e.g. monocyte-like or GMPs or CMPs), and, ii) a pattern of relapse whereby the gene expression of relapsed cells (but not diagnosis cells) had similarity to normal hematopoietic cells that are conventionally placed more apical in the classical hematopoiesis differentiation cascade (HSCs, MPPs, CMPs; a phenotypic shift to immaturity). In addition, no leukemia sample mapped to just one classically defined bone marrow cell type but instead to multiple cell types, suggesting that most AML leukemia cells harbor aberrant hybrid cell gene expression patterns. Finally, we detected quantitative shifts in T cells and NK cells in some samples at relapse, which will be analyzed in greater detail. Conclusions: The comparative analysis of scRNA-seq data of paired AML specimens procured at diagnosis and relapse, identifies frequent and previously unrecognized changes in gene expression in leukemia cells at relapse. Through a comparison of gene mutation and gene expression at single cell resolution we identify two distinct AML relapse patterns in adult AML. Disclosures No relevant conflicts of interest to declare.

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