The Chemokine Receptor CXCR-4 Is Expressed on CD34+Hematopoietic Progenitors and Leukemic Cells and Mediates Transendothelial Migration Induced by Stromal Cell-Derived Factor-1

Abstract The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 (fusin, LESTR) are likely to be involved in the trafficking of hematopoietic progenitor and stem cells, as suggested by the reduced bone marrow hematopoiesis in SDF-1–deficient mice and the chemotactic effect of SDF-1 on CD34+ progenitor cells. Migration of leukemic cells might also depend on the expression of chemokine receptors. Therefore, we analyzed expression of CXCR-4 on mobilized normal CD34+ progenitors and leukemic cells. In addition, SDF-1–induced transendothelial migration across a bone marrow endothelial cell layer was assessed in vitro. By flow cytometry, CXCR-4 was found to be expressed in significant amounts on circulating CD34+ hematopoietic progenitor cells, including more primitive subsets (CD34+/CD38− and CD34+/Thy-1+ cells). In accordance with the immunofluorescence data, CD34+ progenitors efficiently migrated across endothelium in response to SDF-1 containing conditioned medium from the stromal cell line MS-5. Leukemic blasts (mostly CD34+) from patients with acute myeloblastic leukemia (AML) expressed variable amounts of CXCR-4, which was functionally active, as demonstrated by a positive correlation between the SDF-1–induced transendothelial migration and the cell surface density of CXCR-4 (r = 0.97). Also recombinant SDF-1β induced migration of CXCR-4–positive leukemic blasts. The effect of both conditioned medium and recombinant SDF-1 was inhibited by a CXCR-4 blocking antibody. In contrast, CD34+ leukemic cell lines (KG1, KG1a, Kasumi-1, MOLM-1) expressed low levels or were negative for CXCR-4, and did not migrate. By reverse transcriptase-polymerase chain reaction (RT-PCR), however, basal levels of CXCR-4 mRNA were also detected in all leukemic cell lines. We conclude that CXCR-4 is expressed on CD34+cells including more primitive, pluripotent progenitors, and may therefore play a role in the homing of hematopoietic stem cells. CXCR-4 expressed in variable amounts on primary AML leukemic cells is functionally active and may be involved in the trafficking of malignant hematopoietic cells.

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The Chemokine Receptor CXCR-4 Is Expressed on CD34+Hematopoietic Progenitors and Leukemic Cells and Mediates Transendothelial Migration Induced by Stromal Cell-Derived Factor-1

Blood ◽

10.1182/blood.v91.12.4523.412k04_4523_4530 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4523-4530 ◽

Cited By ~ 7

Author(s):

Robert Möhle ◽

Frank Bautz ◽

Shahin Rafii ◽

Malcolm A.S. Moore ◽

Wolfram Brugger ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Progenitor Cells ◽

Stromal Cell ◽

Leukemic Cell ◽

Transendothelial Migration ◽

Leukemic Cells ◽

Hematopoietic Progenitor ◽

Leukemic Cell Lines ◽

Stromal Cell Derived Factor

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 (fusin, LESTR) are likely to be involved in the trafficking of hematopoietic progenitor and stem cells, as suggested by the reduced bone marrow hematopoiesis in SDF-1–deficient mice and the chemotactic effect of SDF-1 on CD34+ progenitor cells. Migration of leukemic cells might also depend on the expression of chemokine receptors. Therefore, we analyzed expression of CXCR-4 on mobilized normal CD34+ progenitors and leukemic cells. In addition, SDF-1–induced transendothelial migration across a bone marrow endothelial cell layer was assessed in vitro. By flow cytometry, CXCR-4 was found to be expressed in significant amounts on circulating CD34+ hematopoietic progenitor cells, including more primitive subsets (CD34+/CD38− and CD34+/Thy-1+ cells). In accordance with the immunofluorescence data, CD34+ progenitors efficiently migrated across endothelium in response to SDF-1 containing conditioned medium from the stromal cell line MS-5. Leukemic blasts (mostly CD34+) from patients with acute myeloblastic leukemia (AML) expressed variable amounts of CXCR-4, which was functionally active, as demonstrated by a positive correlation between the SDF-1–induced transendothelial migration and the cell surface density of CXCR-4 (r = 0.97). Also recombinant SDF-1β induced migration of CXCR-4–positive leukemic blasts. The effect of both conditioned medium and recombinant SDF-1 was inhibited by a CXCR-4 blocking antibody. In contrast, CD34+ leukemic cell lines (KG1, KG1a, Kasumi-1, MOLM-1) expressed low levels or were negative for CXCR-4, and did not migrate. By reverse transcriptase-polymerase chain reaction (RT-PCR), however, basal levels of CXCR-4 mRNA were also detected in all leukemic cell lines. We conclude that CXCR-4 is expressed on CD34+cells including more primitive, pluripotent progenitors, and may therefore play a role in the homing of hematopoietic stem cells. CXCR-4 expressed in variable amounts on primary AML leukemic cells is functionally active and may be involved in the trafficking of malignant hematopoietic cells.

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Role of hERG1 K+ Channels as a Positive Regulator In SDF-1alpha-Mediated Proliferation of Leukemia Cells and Leukemia Stem/Progenitor Cells

Blood ◽

10.1182/blood.v116.21.4786.4786 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4786-4786

Author(s):

Fang Zheng ◽

Huiyu Li ◽

Fang Liu ◽

Wen Du ◽

Shiang Huang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Progenitor Cells ◽

Signaling Pathway ◽

Cell Apoptosis ◽

Leukemic Cell ◽

Bone Marrow Microenvironment ◽

Leukemic Cells ◽

K Channels

Abstract Abstract 4786 Background: Mounting evidence that leukemia stem cells (LSCs) occupy and receive important signals from specialized areas (“niches”) that alter the stromal microenvironment and disrupt normal hematopoiesis. The innovative therapeutic strategies focus on targeting of microenvironmental interactions in leukemia. Therefore, it is important to fully elaborate the mechanisms of microenvironment- mediated leukemogenesis. Stromal-cell derived factor-1alpha (SDF-1à) is the main cytokine produced by bone marrow stromal cells. The SDF-1à/CXCR4 axis specifically mediates homing and migration of leukemic blasts. While our previous work has shown that SDF-1à significantly increases hERG1 K+ tail current and a specific hERG1 K+ channels inhibitor significantly blocks SDF-1à- induced migration of leukemic cells. In fact, recent studies suggested that the human ether à-go-go-related gene (HERG) K+ channels are constitutively expressed in AML stem/progenitor cells, and regulate cell proliferation as well as clinical prognosis. Here we investigate the hypothesis that a new leukemic blast–stromal interaction is mediate by hERG1 K+ channels and SDF-1à. Methods: Proliferation assay, apoptosis and cell cycle analysis were used to analyze effects of E-4031(a specific hERG1 K+ channels inhibitor) in the presence of SDF-1à on leukemia cell lines HL-60. RT–PCR and western blot analysis were used to determine changes in herg1 expression and Wnt/β-catenin signaling pathway in response to SDF-1à in the presence and absence of E-4031. Primary leukemias obtained from the bone marrow of de novo AML patients (n=6) at diagnosis. Mononuclear cells were isolated from the samples using Ficoll-Paque density gradient separation, and cultured with SDF-1à in the presence and absence of E-4031. AML colony-forming cell (CFC) assays and flow cytometry were performed to assess the effects of E-4031 in the presence of SDF-1à on LSCs. Results: SDF-1a enhanced cell proliferation in a dose-dependent manner. The maximal increase by 1.6 times was obtained for 100ng/ml. While this effect was impaired by E-4031, which significantly impaired cell proliferation induced by SDF-1a with a concentration of 100ng/mL by (40.3±8.4)%. In addition, E-4031 inhibited SDF-1a-stimulated leukemic cell proliferation by inducing G0/G1 arrest. Cell apoptosis analysis revealed that either E-4031 or SDF-1a has direct effect on HL-60 cell apoptosis. Unexpected, there was no significant synergistic effect upon apoptosis. After exposures to 100ng/ml SDF-1à, hERG1 mRNA and protein levels increased significantly, by approximately 1.5-fold above control levels. Moreover, SDF-1a increased the expression of Wnt/β-catenin target genes, including β-catenin, cyclin-D1, and c-myc. Interestingly, this manner was abolished by E-4031. The presence of progenitor cells was evaluated by plating suspension cells cultured with SDF-1a in CFC assays. E-4031 decreased numbers of CFC in suspension to 77.3%. Upon expansion with SDF-1a, E-4031 resulted in a significant reduction in the number of progenitors to 31.8%. The effects on LSCs were determined on phenotypically described stem cells from AML. Treatment with 1μ M E-4031 for 48 hours inhibited the proliferation of LCSs compared with untreated controls, a mean viability of 11.8% for CD34+CD38- and 10.4% for CD34+CD38+. In contrast, a significant decrease in the viability of stem cells after E-4031 in the present of SDF-1a treatment, with only 9.6% for CD34+CD38- and 9.5% for CD34+CD38+. Conclusions: Initial studies provided evidence that the hERG1 K+ channels and SDF-1 emerged as mediators of stromal/leukemic cell interactions, which largely contribute to the proliferation mediated by the microenvironment. Likewise, other components of bone marrow microenvironment, such as Wnt/β-catenin signaling pathway, may modulate hERG1 K+ channels in leukemic cells. Taken together, these results provided rationale for studies of new molecular events involved in bone marrow microenvironment and leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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The Contribution of Bone Marrow Stromal Cells to Leukemia Evolution: Differential Support of Leukemia Cell Growth at Diagnosis and Remission

Blood ◽

10.1182/blood.v126.23.5419.5419 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5419-5419

Author(s):

Shlomit Yehudai-Reshef ◽

Shira Attias ◽

Tal Gabay ◽

Rawan Sabah ◽

Yael Morgenstern ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Cell Growth ◽

Progenitor Cells ◽

Stromal Cells ◽

Leukemic Cell ◽

Leukemic Cells ◽

Hematopoietic Progenitor ◽

Preferential Growth ◽

Disease Stages

Abstract Introduction: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of the hematopoietic progenitor cells characterized by excessive proliferation of early progenitor cells in the bone marrow (BM) and is hence associated with a variable prognosis. BM niches are specialized types of microenvironment that actively support the growth, maturation and maintenance of hematopoietic stem and progenitor cells. They include a complex network of extracellular matrix proteins, soluble growth factors, and cytokines. Little is known about the leukemic microenvironment or its clinical relevance to AML initiation and propagation. The aim of the study was to define the role of stromal cells in support of leukemic cell growth in active disease as compared to the remission (Rm) state and normal stoma. Thus, we have investigated the expression of various cytokines secreted by stromal cells and involved in hematopoietic progenitor maintenance and proliferation at disease diagnosis (Dx) and Rm. Additionally, we have characterized the growth pattern of leukemic cells on various stroma cells from different sources and disease stages. Methods: Leukemic and stromal cells from BM of 4 patients with AML were collected at Dx. Stromal cells were also collected in Rm. Leukemic cells were enriched for CD34+ using magnetic beads. Patients' own stroma (POS) obtained both at Dx and Rm and the HS-5 cell line (control) were seeded on gelatin-treated plates and were grown with MesenCult medium for 42 days. Morphologic and growth rate changes were monitored every week using inverted microscopy X20. Upon adherence of 40-50% of the stromal cells, CD34+ enriched leukemic cells derived at Dx were seeded on top of POS from both Dx and Rm and were grown together for additional 2 weeks. Co-cultures were harvested and RNA was extracted followed by RT-PCR to amplify the following hematopoietic regulating genes: hepatocyte growth factor (HGF), fibroblast growth factor (FGF) and CXCL12. The results were expressed as arbitrary units normalized to GAPDH gene expression. Results: Expression of CXCL12 and HGF was elevated in a co-culture of leukemic cells and POS obtained at Dx compared to POS from Rm in all 4 patients. Additionally, the co-culture of leukemic cells and POS from Rm showed a higher expression of CXCL12 and HGF as compared to POS alone derived in Rm (Fig. 1). Mean values of cytokine expression were 37.33, 26.44 and 38.62 at Dx and 29.83, 25.55 and 24.55 in Rm for HGF, FGF and CXCL12, respectively. Additionally, a preferential growth advantage of colonies derived from single leukemic cells was observed when these cells were seeded on POS from Dx and relapse (Rx) compared to Rm (Fig. 2A). Moreover, colonies grew mainly in a co-culture with POS while no growth was noted on healthy stromal cells or stroma derived from a patient with myeloma (Fig. 2A) or another AML patient (Fig. 2B). Conclusions: Co-culture of AML cells and POS derived at Dx show a higher expression of CXCL12 and HGF compared to POS obtained in Rm, suggesting favorable microenvironment conditions supporting AML initiation and maintenance. A synergistic effect on expression of CXCL12 and HGF was observed in a co-culture of POS with leukemic cells as compared to POS or cell-line alone. Leukemic cells show preferential growth on POS and no growth on various malignant and normal stromal cells. Leukemic cell growth was diminished in Rm compared to Dx and Rx. Studying a larger number of patients as well as investigating the specific interaction of AML cells with stromal cells derived from various disease stages is needed to confirm the above results. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

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Chemokine stromal cell-derived factor-1α modulates VLA-4 integrin-dependent adhesion to fibronectin and VCAM-1 on bone marrow hematopoietic progenitor cells

Experimental Hematology ◽

10.1016/s0301-472x(00)00668-8 ◽

2001 ◽

Vol 29 (3) ◽

pp. 345-355 ◽

Cited By ~ 90

Author(s):

Andrés Hidalgo ◽

Francisco Sanz-Rodrı́guez ◽

José Luis Rodrı́guez-Fernández ◽

Beatriz Albella ◽

Carolina Blaya ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Stromal Cell ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Stromal Cell Derived Factor

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Enhanced c-Met activity promotes G-CSF–induced mobilization of hematopoietic progenitor cells via ROS signaling

Blood ◽

10.1182/blood-2009-06-230359 ◽

2011 ◽

Vol 117 (2) ◽

pp. 419-428 ◽

Cited By ~ 91

Author(s):

Melania Tesio ◽

Karin Golan ◽

Simona Corso ◽

Silvia Giordano ◽

Amir Schajnovitz ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Bone Marrow ◽

Progenitor Cells ◽

Stromal Cell ◽

Mammalian Target Of Rapamycin ◽

Reactive Oxygen ◽

Target Of Rapamycin ◽

Oxygen Species ◽

Hematopoietic Progenitor ◽

Stromal Cell Derived Factor

Abstract Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor–induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell–derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1+/c-Kit+/Lin− cells and interfered with their enhanced chemotactic migration to stromal cell–derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met–mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.

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Expression of CD271 in Normal and Malignant Hematopoietic Progenitor and Stem Cells.

Blood ◽

10.1182/blood.v108.11.4271.4271 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4271-4271 ◽

Cited By ~ 1

Author(s):

Ying Jiang ◽

Jennifer Powers ◽

George F. Muschler ◽

Jaroslaw P. Maciejewski

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Leukemic Cell ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Leukemic Cell Lines ◽

Specific Subset ◽

Hel Cells ◽

Cell Fractions ◽

And Function

Abstract The relationship between mesenchymal (MSC) and hematopoietic stem cells (HSC) involves close interaction in the marrow microenvironment. They also may share certain phenotypic features and even possibly a multipotent common precursor. CD271 (also known as LNGFR, p75NTR) has been reported to be a suitable marker of resting primitive MSCs. CD271 and its ligands, neurotrophins (NTs, e.g. nerve growth factor) also play important roles in hematopoiesis, such as enhancement of colony formation by human bone marrow progenitor cells. Dysregulated expression or mutations of NT receptors were associated with the initiation and progression of various malignancies. Based on expression of CD271 in the mesenchymal stem cell compartment, we have hypothesized that this antigen may also be present on a specific subset of normal or malignant hematopoietic progenitor and stem cells. First, we investigated the expression and function of CD271 in normal bone marrow. In controls (n=11), expression of surface CD271 was present on 1.67 ± 1.26% and 2.78 ± 4.50% of CD45+ and CD45− cell populations, respectively. Within the immature hematopoietic compartment, CD271 was expressed on 2.16 ± 1.16% cells defined by CD45 and CD34 expression. When the properties of CD271-expressing CD34 cells were studied in methylcellulose colony assays, those sorted for CD271 antigen showed a much lower clonogenic potential as compared to the CD271-CD34+ cell fractions (10.5 ± 2.12 vs. 183.5 ± 17.68). Subsequently, we investigated whether leukemic cell lines express CD271. We have analyzed HL60, U937, UT7, KG1, K562, HEL, MKN95, NB4 and Kasumi cells using flow cytometry. CD271 was found to be strongly expressed (72.8%) on KG1 AML. Smaller but clearly distinct subpopulations of HEL cells (7.8%) and NB4 cells (5.2%, APL cells) expressed CD271 suggesting either aberrant differentiation in some subpopulations of these cells or the presence of an early precursor population. We then assessed whether CD271 is expressed by CD34+ blasts in patients with myelodysplastic syndrome (MDS n=9) and AML (n=9). CD271 antigen was found on blast cells in 5/9 MDS (1 CMML, 1 RARS, 2 MDS-derived sAML, 1 RA) patients (7.16 ± 1.97% of CD271+ cells within blasts) and 3/9 AML patients (9.70 ± 3.65% of CD271+ cells within blast gate) suggesting that this fraction may differ functionally from the remaining majority of blasts. This is the first report that relates CD271 expression to MDS. The properties of a distinct CD34+ cell population expressing CD271 in myeloid malignancies are currently under investigation. Results of these studies may point towards the presence of a specific subset of precursor/stem cells with clinical and prognostic implications.

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FGF2 posttranscriptionally down-regulates expression of SDF1 in bone marrow stromal cells through FGFR1 IIIc

Blood ◽

10.1182/blood-2006-06-028217 ◽

2006 ◽

Vol 109 (4) ◽

pp. 1363-1372 ◽

Cited By ~ 22

Author(s):

Takayuki Nakayama ◽

Noriko Mutsuga ◽

Giovanna Tosato

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Stromal Cell ◽

Marrow Stromal Cells ◽

Hematopoietic Progenitor Cells ◽

Pcr Analysis ◽

Hematopoietic Progenitor ◽

Stromal Cell Derived Factor

AbstractThe chemokine stromal cell–derived factor-1 (SDF-1) is constitutively expressed by bone marrow stromal cells and plays key roles in hematopoiesis. Fibroblast growth factor 2 (FGF2), a member of the FGF family that plays important roles in developmental morphogenic processes, is abnormally elevated in the bone marrow from patients with clonal myeloid disorders and other disorders where normal hematopoiesis is impaired. Here, we report that FGF2 reduces SDF-1 secretion and protein content in bone marrow stromal cells. By inhibiting SDF-1 production, FGF2 compromises stromal cell support of hematopoietic progenitor cells. Reverse-transcriptase–polymerase chain reaction (RT-PCR) analysis revealed that bone marrow stromal cells express 5 FGF receptors (FGFRs) among the 7 known FGFR subtypes. Blocking experiments identified FGFR1 IIIc as the receptor mediating FGF2 inhibition of SDF-1 expression in bone marrow stromal cells. Analysis of the mechanisms underlying FGF2 inhibition of SDF-1 production in bone marrow stromal cells revealed that FGF2 reduces the SDF-1 mRNA content by posttranscriptionally accelerating SDF-1 mRNA decay. Thus, we identify FGF2 as an inhibitor of SDF-1 production in bone marrow stromal cells and a regulator of stromal cell supportive functions for hematopoietic progenitor cells.

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