scholarly journals Single-molecule imaging and microfluidic platform reveal molecular mechanisms of leukemic cell rolling

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Bader Al Alwan ◽  
Karmen AbuZineh ◽  
Shuho Nozue ◽  
Aigerim Rakhmatulina ◽  
Mansour Aldehaiman ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Leukemic Cell ◽  
Initial Step ◽  
Leukemic Cells ◽  
Large Area ◽  
Hematopoietic Stem ◽  
Cell Rolling ◽  
Selectin Ligands ◽  
Ligand Interactions

AbstractHematopoietic stem/progenitor cell (HSPC) and leukemic cell homing is an important biological phenomenon that occurs through key interactions between adhesion molecules. Tethering and rolling of the cells on endothelium, the crucial initial step of the adhesion cascade, is mediated by interactions between selectins expressed on endothelium to their ligands expressed on HSPCs/leukemic cells in flow. Although multiple factors that affect the rolling behavior of the cells have been identified, molecular mechanisms that enable the essential slow and stable cell rolling remain elusive. Here, using a microfluidics-based single-molecule live cell fluorescence imaging, we reveal that unique spatiotemporal dynamics of selectin ligands on the membrane tethers and slings, which are distinct from that on the cell body, play an essential role in the rolling of the cell. Our results suggest that the spatial confinement of the selectin ligands to the tethers and slings together with the rapid scanning of a large area by the selectin ligands, increases the efficiency of selectin-ligand interactions during cell rolling, resulting in slow and stable rolling of the cell on the selectins. Our findings provide novel insights and contribute significantly to the molecular-level understanding of the initial and essential step of the homing process.

scholarly journals Single-Molecule Imaging and Microfluidic Platform Reveal Molecular Mechanisms of Leukemic Cell Rolling

2020 ◽  
Author(s):  
Bader Al Alwan ◽  
Karmen AbuZineh ◽  
Shuho Nozue ◽  
Aigerim Rakhmatulina ◽  
Mansour Aldehaiman ◽  
...  
Keyword(s):  
Adhesion Molecules ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Molecular Level ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Large Area ◽  
Hematopoietic Stem ◽  
Cell Rolling ◽  
Selectin Ligands

Hematopoietic stem/progenitor cell (HSPC) and leukemic cell homing is an important biological phenomenon that occurs through key interactions between adhesion molecules. Tethering and rolling of the cells on endothelium, the crucial initial step of the adhesion cascade, is mediated by interactions between selectins expressed on endothelium to their ligands expressed on HSPCs/leukemic cells in flow. Although multiple factors that affect the rolling behavior of the cells have been identified, molecular mechanisms that enable the essential slow and stable cell rolling remain elusive. To investigate the molecular mechanisms of cell rolling, an experimental platform that enables molecular level characterization of the adhesion molecules in the context of space, time, and force is required. Here, using a microfluidics-based single-molecule live cell fluorescence imaging, we reveal that unique spatiotemporal dynamics of selectin ligands on the membrane tethers and slings, which are distinct from that on the cell body, play an essential role in the rolling of the cell. Our results suggest that the spatial confinement of the selectin ligands to the tethers and slings together with the rapid scanning of a large area by the selectin ligands, increases the efficiency of selectin-ligand interactions during cell rolling, resulting in slow and stable rolling of the cell on the selectins. Our findings provide novel insights and contribute significantly to the molecular-level understanding of the initial and essential step of the homing process.

scholarly journals Human erythroleukemia genetics and transcriptomes identify master transcription factors as functional disease drivers

Blood ◽  
2020 ◽  
Vol 136 (6) ◽  
pp. 698-714 ◽  
Author(s):  
Alexandre Fagnan ◽  
Frederik Otzen Bagger ◽  
Maria-Riera Piqué-Borràs ◽  
Cathy Ignacimouttou ◽  
Alexis Caulier ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Molecular Mechanisms ◽  
Hematologic Malignancy ◽  
Ectopic Expression ◽  
Leukemic Cells ◽  
Erythroid Progenitors ◽  
Molecular Subgroup ◽  
Aberrant Expression ◽  
In Vivo Models ◽  
Hematopoietic Stem

Abstract Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

A Novel Ring-Substituted Diindolylmethane 1,1-bis [3′-(5-methoxyindolyl)]-1-(p-t-butylphenyl) Methane Abrogates ERK Activation and Induces Apoptosis in Acute Myeloid Leukemia (AML).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3399-3399
Author(s):  
Rooha Contractor ◽  
Ismael J. Samudio ◽  
Zeev Estrov ◽  
David Harris ◽  
James A. McCubrey ◽  
...  
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Caspase 9 ◽  
Apoptotic Pathway ◽  
Erk Activation ◽  
New Class ◽  
Induced Apoptosis ◽  
Extracellular Regulated Kinase

Abstract We investigated the antileukemic activity and molecular mechanisms of action of a newly synthesized ring-substituted diindolylmethane (DIM) derivative, named, 1,1-bis [3′-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane (DIM #34), in myeloid leukemic cells. DIM #34 inhibited leukemic cell growth via induction of apoptosis. DIM #34 inhibited clonogenic growth and induced apoptosis of AML CD34+ progenitor cells but spared normal progenitors. DIM #34 induced loss of mitochondrial membrane potential, which was accompanied by the release of cytochrome c into the cytosol and early cleavage of caspase-9 followed by the cleavage of caspases -8, and -3. Bcl-2 overexpression and caspase-9-deficient cells were partially protected against DIM #34-induced apoptosis, suggesting activation of the intrinsic apoptotic pathway. DIM #34 induced Bax cleavage, and Bax knockout cells were partially resistant to cell death. Furthermore, DIM #34 transiently inhibited the phosphorylation and the activity of the extracellular-regulated kinase (ERK) and abrogated Bcl-2 phosphorylation. Because other methylene substituted DIM analogs transactivate the nuclear receptor PPARγ, we studied the role of PPARγ in apoptosis induction. Although the co-treatment of cells with a selective PPARγ antagonist T007, and a low dose of DIM #34 partially diminished apoptosis, apoptosis was not inhibited at higher concentrations of DIM #34, suggesting the involvement of both, receptor-dependent and independent mechanisms. Co-treatment with RXR- and RAR-ligands enhanced DIM #34-induced cell death. Together, these findings showed that substituted DIMs represent a new class of compounds that selectively induce apoptosis in AML cells through interference with ERK and activation of PPARγ signaling pathways.

Expression and Function of the CXCR7 Chemokine Receptor in Leukemias

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2423-2423
Author(s):  
Sergej Konoplev ◽  
Hongbo Lu ◽  
Michael A Fiegl ◽  
Zhihong Zeng ◽  
Wenjing Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
And Function

Abstract Background: Bone marrow produced stromal-derived factor-1a (SDF-1a) is a key chemokine involved in chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. While the majority of well-defined functions of SDF-1a are mediated via its receptor CXCR4, recent studies have characterized CXCR7 as an alternative receptor capable of binding SDF-1a. Although the functions of CXCR7 are still incompletely understood, the receptor was reported to promote migration and adhesion in certain cell types and function as a pro-survival factor in breast cancer cells. CXCR7 expression and function in human leukemia cells has not been characterized. In this study, we examined CXCR7 expression in leukemia cell lines and primary samples from patients with acute lymphoblastic leukemia (ALL) and utilized a small molecule inhibitor of CXCR7 to probe CXCR7’s function. Materials and methods: CXCR4 and CXCR7 expression was determined by flow cytometry, real-time PCR (RT-PCR) and immunocytochemistry (ICC) in leukemic cell lines including AML (OCI-AML2, OCI-AML3, HL60, U937 NB4, Molm13), ALL (REH, Raji, RS4; 11, Nalm6, Molt4) and CML (KBM5, K562) cells. In primary ALL patient samples, CD34+CD19+ gating was applied to detect CXCR7 expression on pre-B leukemic cells by flow cytometry. The migration of leukemic cells towards SDF-1a was studied using a transwell system. CXCR4 inhibitor AMD3100 was purchased from Sigma, and CXCR7 inhibitor CCX-733 was provided by ChemoCentryx Inc., Mountain View, CA. Results: CXCR4 was found to be ubiquitously expressed on the cell surface of all leukemic cell lines tested. CXCR7 mRNA and protein expression was detectable only in Burkitt lymphoma Raji cells, as analyzed by flow cytometry (clone 11G8, R&D systems), RT-PCR and ICC. Curiously, CXCR7 expression was significantly induced in MOLM13 cells under hypoxic (6% O2) conditions (p=0.01). Low levels of surface CXCR7 were found in 8 of the 9 primary ALL samples by flow cytometry. To determine the respective roles of CXCR4 and CXCR7 in migration of leukemic cells, we utilized CXCR4 inhibitor AMD3100 and CXCR7 inhibitor CCR733 in Raji (CXCR7 positive) and RS4;11 (CXCR7 negative) cells. AMD3100 at 25μM significantly inhibited SDF-1a induced migration (from 38.5% to 12%); CCR733 at 10μM also inhibited SDF-1a induced migration (from 38.5% to 24%) and the combination of AMD3100 and CCR733 resulted in 81% inhibition of migration (from 38.5% to 7.2%). AMD3100 blocked SDF-1a induced migration of CXCR4+CXCR7− RS4;11 cells (from 36.5% to 15.8%), while CCR733 had no effect (36.5% and 39.2%). In conclusion, these studies demonstrate functional expression of the SDF-1 receptor CXCR-7 on Raji and primary ALL cells and suggest that CXCR7 plays an active role in the migration of leukemic cells. CXCR-7 may serve as an alternative receptor to CXCR4. Studies addressing the role of CXCR7 in adhesion, SDF-1a-mediated signaling and survival of leukemic cells are in progress.

Essential Role for the MAML1 Co-Activator In T-ALL

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2501-2501
Author(s):  
Chunxia Cao ◽  
Liang Tian ◽  
Jian-Liang Li ◽  
James D. Griffin ◽  
Suming Huang ◽  
...  
Keyword(s):  
Cell Growth ◽  
Notch Signaling ◽  
Leukemic Cell ◽  
Genetic Alterations ◽  
Leukemic Cells ◽  
Cell Renewal ◽  
Hematopoietic Stem ◽  
Growth And Survival ◽  
Oncogenic Pathways ◽  
Cell Growth And Survival

Abstract Abstract 2501 T cell acute lymphoblastic leukemia (T-ALL) is the most common malignancy in children and accounts for nearly one third of all pediatric cancers. In this type of leukemia, lymphoid progenitor cells that are responsible for the generation of mature lymphocytes become genetically altered, leading to deregulated proliferation, survival, and clonal expansion. Two common genetic alterations frequently associated with this disease are mutations in the NOTCH1 cell-surface receptor and aberrant expression of the TAL1 transcription factor, with each abnormality detected in more than half of human T-ALL patients. The mutations in the NOTCH1 gene result in the aberrant activation of Notch signaling, a highly conserved signal transduction pathway that is critical for lymphocyte growth, maturation and survival. The constitutive activation of Notch signaling induces leukemia in mouse models and is required for human T-ALL leukemic cell growth and survival. On the other hand, TAL1 is required for the functions of hematopoietic stem cells and is essential for the generation of the erythroid and myeloid lineages. The ectopic activation of the TAL1 gene deregulates normal hematopoietic stem cell renewal and differentiation, leading to leukemia in cooperation with other oncogenes. Therefore, Notch and TAL1 oncogenic activities are critical for the initiation and maintenance of T-ALL. In this study, we investigated the role of a transcriptional co-activator, MAML1, in regulating NOTCH1 and TAL1 transforming activities in leukemic cells. In addition to its known function in co-activating Notch signaling, we found that MAML1 is a novel interacting partner for TAL1. MAML1 also enhanced TAL1 transcriptional activities, suggesting a role for MAML1 in TAL1-regulated transcription and leukemogenesis. A subset of T-ALL leukemic cells exhibit aberrant activation in both the NOTCH1 and TAL1 activities; thus, it suggests that these two genetic alterations cooperate in promoting leukemic cell growth and survival. Indeed, we found that the combined inhibition of both the pathways (via the pharmacological blockade of Notch signaling and shRNA-mediated TAL1 knockdown) results in synergistic responses in leukemic cells that carry genetic alterations in both the NOTCH1 and TAL1 genes, indicating that the two pathways synergize in promoting T-ALL. Since MAML1 appears to be a common key regulator for both TAL1 and Notch1 pathways, we next determined whether MAML1 expression level affects leukemic cell growth and survival. Gene knockdown studies suggest that MAML1 is essential for leukemic cell growth and survival by possibly regulating NOTCH1 and TAL1-mediated transcription. Overall, our data reveals a novel common regulatory mechanism for both NOTCH1 and TAL1 oncogenic pathways, and suggest that the manipulation of MAML1 expression or functional activities will affect leukemia initiation and progression. Therefore, our current studies focus on assessing the MAML1 co-activator as a target for these two oncogenic pathways. Disclosures: Griffin: Novartis: Consultancy, Research Funding.

scholarly journals Spatial Confinement and Temporal Dynamics of Selectin Ligands Enable Stable Hematopoietic Stem Cell Rolling

2020 ◽  
Author(s):  
Bader Al Alwan ◽  
Karmen AbuZineh ◽  
Shuho Nozue ◽  
Aigerim Rakhmatulina ◽  
Mansour Aldehaiman ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Temporal Dynamics ◽  
Spatial Confinement ◽  
Hematopoietic Stem ◽  
Cell Rolling ◽  
Selectin Ligands

Mobilization and Chemosensitization of AML with the CXCR4 Antagonist Plerixafor (AMD3100): A Phase I/II Study of AMD3100+MEC in Patients with Relapsed or Refractory Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1944-1944 ◽  
Author(s):  
Geoffrey L. Uy ◽  
Michael P. Rettig ◽  
Kyle M. McFarland ◽  
Lindsay M. Hladnik ◽  
Shashikant Kulkarni ◽  
...  
Keyword(s):  
Phase I ◽  
Leukemic Cell ◽  
Fold Increase ◽  
Complete Response ◽  
Peripheral Circulation ◽  
Cxcr4 Expression ◽  
Leukemic Cells ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Dose Levels

Abstract The interaction of leukemic blasts with the bone marrow microenvironment is postulated to be an important mediator of chemoresistance in AML. Although a number of receptor/ligand pairs have been implicated, the CXCR4/SDF-1 axis functions as the principal regulator of homing and retention of both normal and malignant hematopoietic cells in the marrow. Plerixafor (AMD3100) is a bicyclam molecule which reversibly blocks CXCR4 binding to SDF-1 and is being developed clinically as a mobilization agent for hematopoietic stem cell transplantation. Preclinical data from our group has demonstrated that in murine models, plerixafor can disrupt the interaction of leukemic cells with the marrow microenvironment and sensitize blasts to the effect of chemotherapy. Based on these data, we have initiated a phase I/II study in patients with relapsed or refractory AML in which plerixafor is administered prior to salvage chemotherapy. Subjects were required to have AML which is primary refractory to at least 2 induction regimens, in 1st relapse with an initial remission duration of < 12 months, in 1st relapse having failed ≥ 1 salvage regimens, or in 2nd relapse or higher. Plerixafor is administered by SQ injection followed by a 24 hour observation period to analyze its effects on leukemic cell mobilization. Then plerixafor is given daily 4 hours prior to chemotherapy consisting of mitoxantrone 8mg/m2/d, etoposide 100 mg/m2/d and cytarabine 1000 mg/m2/d x 5 days. To date, 19 patients have been treated at 3 dose levels of plerixafor: 80, 160 and 240 mcg/kg/day. We find that plerixafor can modestly mobilize leukemic cells (~ 2-fold increase) into the peripheral circulation at a peak of 6–8 hours after administration. FISH studies performed from informative samples demonstrates that this mobilization occurs equally in both non-leukemic and leukemic populations. While CXCR4 expression is increased on the surface of mobilized blasts, no clear relationship has been observed between CXCR4 expression or plerixafor dose and mobilization. At the 80 and 160 mcg/kg dose levels, a complete response (CR+CRi) was observed in 2 of 6 patients (33%). At the plerixafor 240 mcg/kg dose level, a complete response (CR+CRi) was achieved in 6 of 8 evaluable patients (75%) in this historically chemorefractory population. Plerixafor was well tolerated with no evidence of hyperleukocytosis or significant delays in neutrophil recovery (median 30 days, range 24–40). Based on this encouraging evidence of safety and efficacy, expansion of a phase II cohort is ongoing.

In Vivo Mobilization of Leukemic Human Precursor-B-ALL Cells by the CXCR4-Antagonist AMD3100 Is Via Secretion of SDF-1 and Synergistically by Catecholamine Action.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1920-1920
Author(s):  
Eike C. Buss ◽  
Alexander Kalinkovich ◽  
Amir Schajnovitz ◽  
Orit Kollet ◽  
Ayelet Dar ◽  
...  
Keyword(s):  
Cell Line ◽  
Progenitor Cells ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Irradiation Damage ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Beta 2 ◽  
Cell Mobilization

Abstract Introduction Mobilization of leukemic cells from the bone marrow (BM) to the circulation in order to better kill them with DNA damaging chemotherapy agents is emerging as a new experimental therapeutic intervention, however the mechanism is not entirely clear. Currently CXCR4-antagonists such as the mobilizing agent AMD3100 (AMD) are becoming available for clinical usage. The aim of this study is to explore mechanisms of human precursor-B-ALL cell mobilization from the BM in a functional, pre-clinical immune deficient mouse model. Methodology Immunodeficient mice were stably engrafted with the childhood pre-B-ALL leukemic cell line G2 (4 weeks after transplantation in NOD/SCID mice) and with primary childhood precursor-B-ALL cells from 4 patients (4-8 weeks after transplantation in NOD/SCID IL2R {gamma} null and NOD/SCID/B2m(null) mice). Two of the patients had a translocation (t4;11) (pro-B-ALL). All human leukemias were engrafted without prior irradiation of the mice. This approach prevents possible irradiation damage to the host microenvironment and thereby leads to a model which better mimics growth of human leukemias. To accommodate for differences in the level of leukemic BM engraftment (FACS analysis for huCD45+ cells), we assessed the leukemia mobilization level by calculating a leukemia mobilization index: WBC x % leukemic cells in the PB / % leukemic cells in the BM. Results and Discussion Treatment with AMD leads to a significant mobilization of all transplanted leukemias with a mobilization level of between 3 – 8 times above baseline. As we recently showed for mobilization of normal murine progenitors, AMD induces a strong release of SDF-1 from the BM (Dar et al. ASH 2006). To examine if this is also instrumental for the leukemia mobilization process, we inhibited SDF-1 action by injection of neutralizing CXCR4 antibodies (clone 12G5) in leukemic chimeras. This led to an abrogation of AMD-induced leukemia mobilization. Pointing towards the same mechanism, 3 daily injections of fucoidan, a known SDF-1 releasing agent, also led to significant leukemia mobilization in G2 and precursor-B-ALL chimeras. Recently we demonstrated that human hematopoietic stem and progenitor cells express receptors for catecholamines, such as dopamine and epinephrine (Epi) and that treatment with catecholamines leads to mobilization of murine progenitor cells (Spiegel et al. Nat. Immunol. 2007). Accordingly, we examined the effect of neurotransmitters. First, we found that the G2 cell line and all 4 examined precursor-BALL samples express the catecholamine receptors D3, D5 and beta-2. The expression is dynamic, as it was, in part, increased after engraftment of immunodeficient mice. Treatment of chimeras with high doses of Epi alone led to leukemia mobilization in vivo similar to AMD-induced mobilization. In combination with AMD, lower doses of norepinephrine increased leukemia obilization synergistically and significantly, resulting in dramatic leukemia mobilization up to 20 times above baseline. Unexpectedly and in contrast to normal cells, treatment of chimeras with the beta-2 agonist clenbuterol was accompanied by inhibition of AMD-induced mobilization of leukemic cells. These observations suggest similarities and differences in the activation of catecholamine receptors in the mobilization process of normal and leukemic cells. Conclusions Our results show that SDF-1 has a crucial role in AMD-induced leukemic cell mobilization. Human leukemias can be mobilized by catecholamine action synergistically with AMD in immunodeficient mice. This approach could be potentially used for future mobilization protocols of leukemia in combination with established chemotherapy to improve eradication of minimal residual disease of leukemia.

Competition In Engraftment of Normal Hematopoietic Stem Cells and Leukemic Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4836-4836
Author(s):  
Gyeongsin Park ◽  
Michael Heuser ◽  
Tobias Berg ◽  
R. Keith Humphries
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Leukemic Cell ◽  
Fixed Number ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Abstract Abstract 4836 Engraftment is a process including homing to bone marrow, implantation and proliferation. Implantation implies interactions with specialized microenvironments, niches, in which hematopoietic stem cells (HSCs) live and are regulated. Studies have demonstrated the possibility that leukemic stem cells (LSCs) interact with niches in a similar manner to HSCs. We investigated whether HSCs and LSCs compete with each other in their engraftment. We employed a mouse transplantation assay with unmanipulatated bone marrow cells (BMCs) as a source of normal HSCs and LSCs generated by transduction of BMCs with Meningioma 1 (MN1), a potent oncogene causing myeloid leukemia in mice. In irradiated recipients (750 cGy), cotransplantation of leukemic cells (1×105) with various numbers of BMCs (1×105, 1×106 and 1×107) demonstrated that the engraftment level of leukemic cells is influenced by BMCs in a dose dependant manner (5.2%, 41.3% and 82.2% at 2-weeks; 52.3%, 69.5% and 86.9% at 4weeks; mice died before the 5 weeks bleeding, 94.9% and 97.5% at 5weeks, respectively). Cotransplantation of various numbers of leukemic cells (1×104, 1×105 and 1×106) with a fixed number of BMCs (1×106) demonstrated a similar pattern of leukemic engraftment (7.0%, 59.5% and 87.1% at 2weeks; 62.0%, 85.7% at 4 weeks, and mice died before the four week bleeding, respectively). To further elucidate the competition between HSCs and LSCs, we transplanted the cells at different time intervals. Transplantation of normal BMCs (1×106) 2 days prior to transplantation of LSCs (1×105) resulted in much reduced levels of leukemic engraftment compared to that seen in mice simultaneously transplanted (3.5% vs 59.5% at 2 weeks; 73.1% vs 85.76% at 4weeks). This competitive suppression of leukemic engraftment was further enhanced by transplanting larger numbers of normal BMCs (2×107) as little as 12 hours prior LSC transplantation (5×105) compared to simultaneous injection (0% vs 7.26% at 2weeks, 0.9% vs 35.3% at 3 weeks, and 6.0% vs 60.6% at 4 weeks). When BMCs (1×105) or leukemic cells (1×105) were transplanted at equal doses of 1×105 together with normal helper cells (1×106) the leukemic cells expanded 280-fold compared to only 7.3 fold for normal BMCs at 2 weeks (total cell count from two femurs and two tibias per 1×105 transplanted cells). Thus the competitive suppression of leukemic cell growth seen upon sequential transplantation of normal BMCs is not readily explained by enhanced kinetics of normal BMC growth but rather by competition at the level of initial engraftment. In conclusion, our data demonstrate that there is a competition between normal and leukemic cells during the engraftment process, suggesting niche competition of HSCs and LSCs. Disclosures: No relevant conflicts of interest to declare.

Distinct Effects of SCF and Hes1 On Normal Hematopoietic Stem and Progenitor Cells During Leukemic Cell Expansion in a T-ALL Mouse Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1216-1216
Author(s):  
Chen Tian ◽  
Zhipan Cao ◽  
Qiao Li ◽  
Jinhong Wang ◽  
Zhenyu Ju ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Real Time ◽  
Hematopoietic Cells ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Rt Pcr ◽  
Hematopoietic Stem

Abstract Abstract 1216 During leukemia development, emerging leukemic cells out-compete normal hematopoietic cells and become predominant in the body. How hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) respond to the growth of leukemic cell population is an important, yet less investigated area. Our previous study demonstrated differential effects of a leukemic environment on normal HSCs and HPCs in the Notch1-induced T-ALL mouse model (Hu X, et al. Blood 2009). We found that normal HSCs were better preserved in the leukemic bone marrow in part due to increased quiescence of the HSCs and in contrast, HPCs were exhausted during the expansion of leukemic cells. Our current work is aimed to further explore the molecular mechanisms concerning the distinct impacts of leukemic environment on normal HSCs and HPCs in the T-ALL mouse model. Given the previous report by others showing that increased secretion of stem cell factor (SCF) by myeloid leukemia cells played an important role in inducing normal HSCs/HPCs out of their niche and thus allowing leukemic cells to occupy the niche in the human-NOD/SCID xeno-graft model (Sipkins DA et al, Science 2008), we first examined the expression of SCF by ELISA, Western blot and real-time RT PCR in both normal hematopoietic and leukemic cell fractions in the Notch1-induced T-ALL mouse model as previously reported. We found that while expression of SCF in peripheral blood (PB) or bone marrow (BM) was increased in the leukemic mice, both mRNA and protein levels of SCF in normal hematopoietic cells were higher than that in leukemic cells, thereby suggesting that elevated SCF might be mainly secreted by non-leukemic cells in the leukemic hosts of our model. Further assessments on the role of SCF in leukemogenesis with the mice specifically deficient in SCF in different niche cell types are currently under investigation in our laboratory. In order to define potential mediators in HSCs in response to leukemic cell growth, a microarray study on normal HSCs isolated from T-ALL leukemic mice and the control mice was conducted. Gene expression profiling showed significantly differed expression of 169 genes (127 up and 42 down). Especially, real-time RT PCR confirmed an increase of Hes1, p21, Fbxw11, IL-18R1 and Itgb3, and a decrease of CXCR4 and Mmp2. Interestingly, the expression of Hes1 and its target gene, p21 were elevated in normal HSCs but not in HPCs, letting us to hypothesize that Hes1 might be in part mediate the different responses of HSCs and HPCs to the T-ALL leukemic environment. To test this hypothesis, we ectopically expressed Hes1 in normal hematopoietic cells and then examined their functions under the leukemic condition. BM cells from B6.SJL mice were transduced with either MSCV-Hes1-IRES-GFP or control MSCV-GFP vector. After transduction, Hes1-GFP+or control-GFP+cells were co-transplanted with the Notch1-induced T-ALL cells into lethally irradiated C57BL/6J recipients. The engrafted cells from the leukemic BM were analyzed and Hes1-GFP+or control-GFP+cells were sorted for functional assessments. Interestingly, although over-expression of Hes1 inhibited the growth of colony forming cell (CFC) in vitro, it could potentiate the long-term repopulating cells by maintaining more cells in the quiescent (G0) state in vivo. Taken together, our current study supports a role of Hes1 in mediating the distinct responses of normal HSCs and HPCs to the T-ALL leukemic environment. Disclosures: No relevant conflicts of interest to declare.

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