Adhesion Molecule ICAM-4 Participates in Erythroblastic Island Formation.

Abstract Erythroblasts proliferate, differentiate and enucleate within erythroblastic islands, three dimensional structures comprised of developing erythroblasts surrounding a central macrophage. Collective evidence suggests that erythroblastic islands are highly specialized bone marrow subcompartments where adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. ICAM-4, a recently characterized member of the immunoglobulin superfamily, is expressed early in erythroid differentiation. This adhesion molecule interacts with multiple integrin binding partners, including alpha4beta1 and alphaV integrins (alphaVbeta1, alphaVbeta3 and alphaVbeta5). Since erythroblasts express alpha4beta1 and ICAM-4 and macrophages exhibit alphaV, ICAM-4 is an attractive candidate for mediating erythroblast-erythroblast and erythroblast-macrophage attachments. A molecular model of ICAM-4 derived from the crystal structure of closely related ICAM-2 presents the extracellular region of ICAM-4 as two Ig-like domains comprised of A,B,C,D,E,F, and G strands. Employing targeted mutagenesis of surface-exposed amino acid residues, we earlier identified a patch or footprint that mediates adhesion to alphaV integrins comprised of three A strand residues and five G strand residues on N-terminal domain 1. To explore whether ICAM-4 attachments are active in erythroblastic islands we first developed a quantitative live cell assay for reforming islands from single cell suspensions of freshly harvested mouse bone marrow. Islands and their cellular components were identified and quantitated by three-color immunofluorescent microscopy employing fluoresceinated erythroid-specific TER119 antibody, macrophage-specific F4/80 antibody and a DNA probe. To determine the amount of variation in number of islands reformed from a single cell suspension of 1 x 105 cells, we counted islands at the beginning and conclusion of experiments on five different mice. The island numbers were very reproducible and equaled 1000 +/− 158. We then tested the effects of two synthetic peptides that we have previously shown block ICAM-4/alphaV adhesion: peptides FWV and ATSR, corresponding to sequences of the A and G strands of ICAM-4 domain 1, respectively. Both peptides caused a marked, concentration dependent decrease in the percentage of islands formed. 2mM ATSR inhibited island formation by 75% while 2mM FWV inhibited island formation by 70%. In marked contrast, a strand D control peptide had minimal to no effect on island formation. Our data strongly suggest that erythroblast ICAM-4 binding to macrophage alphaV is critical for erythroblastic island formation. We postulate that this newly identified receptor-counterreceptor interaction may be important not only for adhesive integrity of the island structure but also for initiating intracellular signaling essential for normal erythroid terminal differentiation.

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Targeted Gene Deletion Demonstrates That Adhesion Molecule ICAM-4 Is Critical for Erythroblastic Island Formation.

Blood ◽

10.1182/blood.v106.11.1661.1661 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1661-1661

Author(s):

Gloria Lee ◽

Annie Lo ◽

Sarah Short ◽

Tosti Mankelow ◽

Stephen Parsons ◽

...

Keyword(s):

Bone Marrow ◽

Intracellular Signaling ◽

Marked Decrease ◽

Immunoglobulin Superfamily ◽

Mouse Bone Marrow ◽

Wild Type ◽

Erythroid Progenitors ◽

Island Formation ◽

Erythroblastic Island

Abstract Erythroid progenitors proliferate, differentiate and enucleate within specialized bone marrow subcompartments, termed erythroblastic islands, which are comprised of developing erythroblasts surrounding a central macrophage. Growing evidence suggests that within erythroblastic islands adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. We are exploring the potential function of erythroid ICAM-4, a recently characterized member of the immunoglobulin superfamily, in erythroblastic island formation. We earlier identified α4β1 and αV integrins as ICAM-4 binding partners. Since erythroblasts express α4β1 and ICAM-4 and macrophages exhibit αV, ICAM-4 is an attractive candidate for mediating erythroblast-erythroblast and erythroblast-macrophage attachments. Indeed, two synthetic peptides that block ICAM-4/αV adhesion caused a marked decrease in the percentage of islands formed. To more definitively test whether ICAM-4 attachments are active in erythroblastic islands we generated ICAM-4 knockout mice and compared the capacity of single cell suspensions from freshly harvested ICAM-4 null and wild type bone marrow to form erythroblastic islands in vitro, using a reproducible live cell island reconstitution assay that we have established. Islands and their cellular components were identified and quantitated by three-color immunofluorescent microscopy employing fluoresceinated erythroid-specific TER119 antibody, macrophage-specific F4/80 antibody and a DNA probe. Strikingly, we observed a 47% decrease in the percentage of islands formed from bone marrow of ICAM-4 null mice compared to wild type littermates (n=10 and n=10, respectively). We also studied the ability of ICAM-4 null erythroblasts to form islands in vivo by analyzing intact islands freshly harvested from mouse bone marrow. Similar to the in vitro data we found a marked decrease in the percentage of islands formed in the bone marrow of ICAM-4 null mice compared to wild type littermates. The null mice had 44% fewer islands than wild type mice. Taken together, the results of this phenotypic analysis provide convincing evidence that ICAM-4 is one of the adhesion molecules critical for erythroblastic island formation. We postulate that this newly identified erythroblast receptor may be important not only for adhesive integrity of the island structure but also for initiating intracellular signaling essential for normal erythroid terminal differentiation.

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Targeted gene deletion demonstrates that the cell adhesion molecule ICAM-4 is critical for erythroblastic island formation

Blood ◽

10.1182/blood-2006-03-006759 ◽

2006 ◽

Vol 108 (6) ◽

pp. 2064-2071 ◽

Cited By ~ 82

Author(s):

Gloria Lee ◽

Annie Lo ◽

Sarah A. Short ◽

Tosti J. Mankelow ◽

Frances Spring ◽

...

Keyword(s):

Adhesion Molecule ◽

Molecular Mechanisms ◽

Intercellular Adhesion Molecule ◽

Immunoglobulin Superfamily ◽

Erythroid Progenitors ◽

Island Formation ◽

Knock Out ◽

Erythroblastic Island ◽

Adhesive Interactions ◽

Knock Out Mice

AbstractErythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule 4 (ICAM-4), an immunoglobulin superfamily member, participates in island formation. Earlier, we identified αV integrins as ICAM-4 counterreceptors. Because macrophages express αV, ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knock-out mice and developed quantitative, live cell techniques for harvesting intact islands and for re-forming islands in vitro. We observed a 47% decrease in islands reconstituted from ICAM-4 null marrow compared to wild-type marrow. We also found a striking decrease in islands formed in vivo in knock-out mice. Further, peptides that block ICAM-4/αV adhesion produced a 53% to 57% decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage αV functions in island integrity. Importantly, we documented that αV integrin is expressed in macrophages isolated from erythroblastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/αV adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis.

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Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow

10.1101/2021.08.06.455158 ◽

2021 ◽

Author(s):

Zixian Liu ◽

Jinhong Wang ◽

Miner Xie ◽

Peng Wu ◽

Yao Ma ◽

...

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Pcr Analysis ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Colony Assay ◽

Megakaryocyte Progenitors ◽

Cell Colony

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in C57BL/6 mice. Single-cell transplantation and single-cell colony assay showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average eight megakaryocytes per colony) than did previously reported megakaryocyte progenitors (MkPs). Single-cell RNA-sequencing supported that these MgPs lie between HSCs and MkPs along the megakaryocyte differentiation pathway. Single-cell colony assay and single-cell RT-PCR analysis suggested the coexpression of CD41 and Pf4 is associated with megakaryocyte colony-forming activity. Single-cell colony assay of a small number of cells generated from single HSCs in culture suggested that MgPs are not direct progeny of HSCs. In this study, we propose a differentiation model in which HSCs give rise to MkPs through MgPs.

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

Blood ◽

10.1182/blood.v128.22.430.430 ◽

2016 ◽

Vol 128 (22) ◽

pp. 430-430 ◽

Cited By ~ 1

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.

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

10.21203/rs.3.rs-138736/v1 ◽

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.

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Distinct Transforming and Leukemogenic Activities of Tel-Arg and Tel-Abl Oncogenes in Mice: Possible Negative Role for the Transforming Activity in C-Terminus of Arg

Blood ◽

10.1182/blood.v112.11.2845.2845 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2845-2845

Author(s):

Keiko Okuda ◽

Nari Harakawa ◽

Richard A VanEtten ◽

Nihal Patel ◽

Naochika Domae ◽

...

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

Tyrosine Kinase ◽

Intracellular Signaling ◽

Lymphoblastic Leukemia ◽

Kinase Domain ◽

Actin Binding ◽

Mouse Bone Marrow ◽

C Terminus ◽

Transforming Activity

Abstract ARG (ABL2) is a member of ABL tyrosine kinase gene family that is highly homologous to c-ABL (ABL1) in overall domain structure (SH3-SH2-SH1) and amino acid sequence. ARG has recently been implicated in the pathogenesis of human acute leukemia though t(1;12) translocations that fuse a transcription factor gene, ETV6/TEL, to ARG (Iijima et al., Blood2000;95:2126). The resulting TEL-ARG fusion tyrosine kinase is similar in structure to the TEL-ABL fusion found in some acute leukemia and atypical CML patients, and, like TEL-ABL, can transform Ba/F3 cells and fibroblasts in vitro and activate a similar set of intracellular signaling pathways (Iijima et al., Oncogene2002;21:4374). To assess the leukemogenic activity of TEL-ARG, we co-expressed TEL-ARG with GFP in mouse bone marrow using a retroviral bone marrow transduction/transplantation strategy. Whereas TEL-ABL induces rapidly fatal myelolproliferative disease (MPD) in recipient mice (Million et al., Blood2000;96:664), recipients of TEL-ARG-transduced BM did not develop overt MPD, but succumbed instead to long-latency (30–45 weeks) T-cell acute lymphoblastic leukemia/lymphoma characterized by modest leukocytosis and a malignant pleural effusion composed of Thy-1+B220- tumor cells. To study the molecular basis of the marked difference in the leukemogenic activity of TEL-ARG and TEL-ABL, we produced TEL-ARG mutants that swapped the kinase domain or C-terminus of ARG with the corresponding domain in ABL. The mutants were introduced into Ba/F3 cells by retroviral transduction and transduced cells selected for equal expression of the fusion protein by flow sorting for populations with equivalent intensity of GFP fluorescence. All chimeric proteins were expressed and showed equivalent levels of auto-phosphorylation by western blot analysis of the sorted cells. However, the quantitative transforming activity of TEL-ARG in Ba/F3 cells, measured by the number of days required to achieve measurable cell growth following IL-3 deprivation, was significantly lower than for TEL-ABL (25 ± 4.3 days for TEL-ARG vs. 1 to 2 days for TEL-ABL). A TEL-ARG mutant containing the ABL kinase domain was similar to TEL-ARG in this assay, but replacing the ARG C-terminal domain with that of ABL increased Ba/F3 transformation to levels equivalent to TEL-ABL. To further dissect the functional domains that are responsible for this effect, a new series of mutants containing internal deletions in ARG C-terminus of TEL-ARG [aa.826–976 (Delta Box1), aa.977–1213 (Delta Box2), aa 1214–1316 (Delta Box3), aa.1317–1465 (Delta Box4)] were generated. Regarding to their function, only Box2 and Box4 were reported as F- or F,G- actin binding domain. From a preliminary study using the cell lines which express each of the mutants, Delta Box 1 clones obtained an accelerated proliferation which compared with that of Tel-Abl, suggesting that there is a disadvantage for cell proliferation by ARG c-terminus. These results indicate that distinct bio-phenotype associated with Abl family tyrosine kinase is the most likely regulated by their c-terminus and the c-terminus of Arg contains functional subdomain that impairs growth signal induced by Abl family tyrosine kinase.

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Isolation of the stromal-vascular fraction of mouse bone marrow markedly enhances the yield of clonogenic stromal progenitors

Blood ◽

10.1182/blood-2011-08-372334 ◽

2012 ◽

Vol 119 (11) ◽

pp. e86-e95 ◽

Cited By ~ 22

Author(s):

Colby Suire ◽

Nathalie Brouard ◽

Karen Hirschi ◽

Paul J. Simmons

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Stromal Vascular Fraction ◽

Cell Suspensions ◽

Mouse Bone Marrow ◽

Multilineage Differentiation ◽

Low Oxygen ◽

Low Incidence

Abstract The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of mouse bone marrow stromal cells (BMSCs), a common problem being contamination with hematopoietic cells. Taking advantage of burgeoning evidence demonstrating the perivascular location of stromal cell stem/progenitors, we hypothesized that a potential reason for the low yield of mouse BMSCs is the flushing of the marrow used to remove single-cell suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of BMSCs physically associated with the abluminal surface of blood vessels. Herein, we describe a simple methodology based on preparation and enzymatic disaggregation of intact marrow plugs, which yields distinct populations of both stromal and endothelial cells. The recovery of CFU-F obtained by pooling the product of each digestion (1631.8 + 199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32 + 1.9) by at least 2 orders of magnitude (P < .001; N = 8) with an accompanying 113.95-fold enrichment of CFU-F frequency when plated at low oxygen (5%). Purified BMSC populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and from freshly prepared single-cell suspensions. Furthermore, this population demonstrates robust multilineage differentiation using standard in vivo and in vitro bioassays.

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GM-CSF Impairs Erythropoiesis by Disrupting Erythroblastic Island Formation via Macrophages

10.21203/rs.3.rs-1057729/v1 ◽

2021 ◽

Author(s):

Weijie Cao ◽

Wenjuan Fan ◽

Fang Wang ◽

Yinyin Zhang ◽

Guanghua Wu ◽

...

Keyword(s):

Adhesion Molecule ◽

Inflammatory Diseases ◽

Mouse Bone Marrow ◽

Adhesion Molecule Expression ◽

Supporting Function ◽

Gm Csf ◽

Erythroblastic Island ◽

Novel Strategy

Abstract Anemia is a significant complication of chronic inflammation and may be related to dysregulated activities among erythroblastic island (EBI) macrophages. GM-CSF was reported to be upregulated and attracted as a therapeutic target in many inflammatory diseases. Among EBIs, we found that the GM-CSF receptor is preferentially and highly expressed among EBI macrophages but not among erythroblasts. GM-CSF treatment significantly decreases human EBI formation in vitro by decreasing the adhesion molecule expression of CD163. RNA-sequence analysis suggests that GM-CSF treatment impairs the supporting function of human EBI macrophages during erythropoiesis. GM-CSF treatment also polarizes human EBI macrophages from M2-like type to M1-like type. In addition, GM-CSF decreases mouse bone marrow (BM) erythroblasts as well as EBI macrophages, leading to a reduction in EBI numbers. In defining the molecular mechanism at work, we found that GM-CSF treatment significantly decreases the adhesion molecule expression of CD163 and Vcam1 in vivo. Importantly, GM-CSF treatment also decreases the phagocytosis rate of EBI macrophages in mouse BM as well as decreases the expression of the engulfment-related molecules Mertk, Axl, and Timd4. In addition, GM-CSF treatment polarizes mouse BM EBI macrophages from M2-like type to M1-like type. Thus, we document that GM-CSF impairs EBI formation in mice and humans. Our findings support that targeting GM-CSF or reprogramming EBI macrophages might be a novel strategy to treat anemia resulting from inflammatory diseases.

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The L1 adhesion molecule is a cellular ligand for VLA-5.

The Journal of Cell Biology ◽

10.1083/jcb.131.6.1881 ◽

1995 ◽

Vol 131 (6) ◽

pp. 1881-1891 ◽

Cited By ~ 97

Author(s):

M Ruppert ◽

S Aigner ◽

M Hubbe ◽

H Yagita ◽

P Altevogt

Keyword(s):

Adhesion Molecule ◽

Bone Marrow Cells ◽

Cell Types ◽

Immunoglobulin Superfamily ◽

Mouse Bone Marrow ◽

Neural Cells ◽

Dependent Manner ◽

Binding Assays ◽

Fibronectin Receptor ◽

Heat Stable Antigen

The L1 adhesion molecule is a member of the immunoglobulin superfamily shared by neural and immune cells. In the nervous system L1 can mediate cell binding by a homophilic mechanism. To analyze its function on leukocytes we studied whether L1 could interact with integrins. Here we demonstrate that VLA-5, an RGD-specific fibronectin receptor on a wide variety of cell types, can bind to murine L1. Mouse ESb-MP cells expressing VLA-5 and L1 could be induced to aggregate in the presence of specific mAbs to CD24 (heat-stable antigen), a highly and heterogeneously glycosylated glycophosphatidylinositol-linked differentiation antigen of hematopoietic and neural cells. The aggregation was blocked by both mAbs to L1 and VLA-5, respectively. Aggregation was blocked also by a synthetic RGD-containing peptide derived from the Ig-domain VI of the L1 protein. ESb-MP subclones with low L1 expression could not aggregate. In heterotypic binding assays mouse bone marrow cells could adhere in an L1-dependent fashion to platelets that expressed VLA-5. Also purified L1 coated to polystyrene beads could bind to platelets. The binding of L1-beads was again inhibited by mAbs to L1 and VLA-5, by soluble L1 and the L1-RGD peptide in a dose-dependent manner. Thymocytes or human Nalm-6 tumor cells expressing VLA-5 could adhere to affinity-purified L1 and to the L1-derived RGD-containing peptide coated to glass slides. The adhesion was strongly enhanced in the presence of Mn(2+)-ions and blocked by mAbs to VLA-5. We also demonstrate a direct L1-VLA-5 protein interaction. Our results suggest a novel binding pathway, in which the VLA-5 integrin binds to L1 on adjacent cells. Given its rapid downregulation on lymphocytes after induction of cell proliferation, L1 may be important in integrin-mediated and activation-regulated cell-cell interactions.

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Network-Based Discovery of Prognostic Markers in Pediatric AML by Multi-Dimensional Single Cell Mass Cytometry

Blood ◽

10.1182/blood.v120.21.1411.1411 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1411-1411

Author(s):

Karen Sachs ◽

Garry P. Nolan ◽

Wendy J. Fantl ◽

Erin F. Simonds ◽

Sean C. Bendall ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Clinical Outcome ◽

Single Cell ◽

Intracellular Signaling ◽

Cell Mass ◽

Signaling Networks ◽

Surface Markers ◽

Mass Cytometry ◽

Pediatric Aml

Abstract Abstract 1411 Background: Currently available indicators of risk stratification for acute myeloid leukemia (AML) approximate a patient's true prognosis (Rubnitz, J.E., et al., Lancet Oncol, 2010). While signaling networks are central to biological systems they do not lend themselves to easy characterization. As such, network characteristics are not incorporated into risk stratification. There exists a need for computational techniques that enable elucidation of a patient's global signaling state. Flow cytometry is an essential tool for classifying AML surface markers by surface marker expression. Previous work has shown that intracellular signaling responses can be predictive of clinical outcome (Irish et al., Cell 2004; Kornblau et al., Clin. Cancer Res. 2010, Kornblau et al. Blood Cancer J 2011). We hypothesized that there may exist additional layers of information in both cell phenotypes and signaling responses which were not recognized in the low-parameter systems used in these studies, and which, upon characterization, may yield prognostic benefit. Methods: 31-parameter single cell mass cytometry was used to measure the simultaneous co-expression of 16 surface markers and 15 intracellular signaling epitopes in pediatric AML diagnosis bone marrow samples (n=15) and healthy adult bone marrow controls (n=3). Signaling dynamics were measured under 18 stimulation conditions including cytokines and small molecule kinase inhibitors. Single cell resolution enables statistical extraction of correlative relationships which exist among the measured variables. Network inference elucidated the regulatory signaling structure in each patient (Sachs et al. Science 2005, Itani et al. JMLR 2008), and a statistical tool called a decision tree was used to predict the patient's relapse status (Figure 1). Results: The analysis selects network features predictive of clinical outcome, here, relapse status. Of the network edges with a strong statistical signal, the most informative were the relationships between pS6 and pSTAT1, pAKT and cleaved Caspase3, pAMPK and pRb, pRb and pP38, pAMPK and pErk and pCREB and pCbl. Despite the small dataset size, the decision tree correctly classified >85% of the patients, an improvement over a random model which returns 50% accuracy. (The predicted value was excluded from training to avoid “memorization” by the classification model). For a small subset of the patients (3) who relapsed, paired relapse samples were also available. Strikingly, of the network features which consistently differed between the diagnostic sample and its paired relapse counterpart, two overlapped with those found to have predictive information in the above: pAMPK – pRb and pRb – pP38. These results implicate a coordination of metabolism, cell cycle and survival Conclusions: Statistical classification typically relies on large numbers of profiled samples; thus the ability to predict relapse status from diagnostic samples in this small pilot study is a strong indication of the utility of this approach. Predictive accuracy relied on network features and was not achieved using univariate measurements alone. Furthermore, the features extracted by the predictive model determine which correlations help reveal the prognostic fate of a tumor at diagnosis, potentially providing mechanistic insight. These correlative relationships corroborate the roles of metabolism, survival and cell cycle in AML. The application of 31-parameter mass cytometry at the single cell level provided a level of detail that enabled characterization of signaling relationships at high resolution. This proof of principle study will inform future studies of AML signaling networks and prognostic biomarkers. Disclosures: No relevant conflicts of interest to declare.

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