Supplementary Data from Regulation of Expression of Stromal-Derived Factor-1 Receptors: CXCR4 and CXCR7 in Human Rhabdomyosarcomas

Abstract Rhabdomyosarcomas (RMS) express CXCR4 and CXCR7 receptors that bind prometastatic α-chemokine stromal-derived factor-1 (SDF-1). In this report, we analyzed the activity of both promoters in a model of less metastatic human embryonal-RMS cell line (RD) and more metastatic alveolar-like RMS (RD cells transduced with paired box gene 3/forkhead homologue; PAX3-FKHR fusion gene). First, CXCR4 is barely detectable in RD and becomes upregulated in RD/PAX3-FKHR cells. In contrast, CXCR7 highly expressed in RD becomes downregulated in RD/PAX3-FKHR cells. Next, promoter deletion and mutation studies revealed that whereas (a) expression of CXCR4 in RD and RD/PAX3-FKHR cells required nuclear respiratory factor-1 (NRF-1) binding site and (b) was additionally upregulated by direct interaction of NRF-1 with PAX3-FKHR, CXCR7 promoter activity required a proximal nuclear factor-κB–binding motif. The requirement of these factors for CXCR4 and CXCR7 promoter activities was additionally supported after blocking NRF-1 and nuclear factor-κB. Furthermore, CXCR4 expression in PAX3-FKHR+ RMS cells seems to be enhanced because of the interaction of PAX3-FKHR and NRF-1 proteins in the proximal part of the promoter that prevents access of the negative regulator of transcription YY1 to its binding site. Finally, although hypoxia enhances CXCR4 and CXCR7 promoter activity and receptor expression in RD cells, it inhibits CXCR7 expression in RD/PAX3-FKHR cells. In conclusion, SDF-1 binding receptors CXCR4 and CXCR7 are differently regulated in RMS cells. The upregulation of CXCR4 and downregulation of CXCR7 expression by PAX3-FKHR or hypoxia may give SDF-1 an advantage to better engage the CXCR4 receptor, thus increasing RMS motility. Mol Cancer Res; 8(1); 1–14

We reported that complement (C) becomes activated and cleaved in bone marrow during preconditioning for hematopoietic transplantation and the third C component (C3) cleavage fragments, C3a and desArg C3a, increase responsiveness of hematopoietic stem/progenitor cells (HSPCs) to stromal-derived factor-1 (SDF-1). We also showed that this homing-promoting effect is not C3a receptor (C3aR) dependent. Herein, we report our new observation that transplantation of C3aR −/− HSPCs into lethally irradiated recipients results in: (1) ∼5–7 day delay in recovery of platelets and leukocytes; (2) decrease in formation of day 12 colony-forming units-spleen; and (3) decrease in the number of donor-derived CFU-granulocyte-macrophage progenitors detectable in the bone marrow cavities at day 16 after transplantation. In agreement with the murine data, blockage of C3aR on human umbilical cord blood CD34 + cells by C3aR antagonist SB290157 impairs their engraftment in non-obese diabetic/severe combined immunodeficient mice. However, HSPCs from C3aR −/− mice stimulated by C3a still better responded to SDF-1 gradient, after exposure to C3a, they secrete less matrix metalloprotease-9 and show impaired adhesion to stroma cells. We conclude that C3a, in addition to enhancing responsiveness of HSPCs to SDF-1 gradient in a C3aR independent manner, may also directly modulate HSPC homing by augmenting C3aR-mediated secretion of matrix metalloprotease-9 and cell adhesion.

BACKGROUND:In patients transplanted with cord blood (CB), prolonged thrombocytopenia is a major complication. However, this could be alleviated by supplementing the CB graft with ex vivo-expanded megakaryocytic progenitors (CFU-Meg), provided that the homing properties of these cells are not affected negatively by expansion.METHODS AND RESULTS:We assessed the in vitro homing potential of CFU-Meg progenitors expanded from CB and showed that the combination of thrombopoietin (TPO) with interleukin-3 (IL-3) used for expansion not only results in optimal proliferation of CFU-Meg but also protects these cells from apoptosis. Moreover, we found that ex vivo-expanded CFU-Meg maintained expression of the CXCR4 receptor throughout a 9-day culture and were chemoattracted towards a stromal cell-derived factor-1 (SDF-1) gradient. They also expressed matrix metalloproteinase-9 (MMP-9) and membrane-type (MT) 1-MMP, and transmigrated across the reconstituted basement membrane Matrigel. Finally, we observed that SDF-1 up-regulated the expression of both MMP-9 and MT1-MMP in CB CD34(+) cells and ex vivo-expanded CFU-Meg.DISCUSSION:We suggest that CB-expanded CFU-Meg, in particular those from day 3 of expansion, when their proliferation and in vitro homing potential are maximal, could be employed to supplement CB grafts and speed up platelet recovery in transplant recipients.

Complement cascade (CC) and innate immunity emerge as important and underappreciated modulators of trafficking of hematopoietic stem/progenitor cells (HSPC). Accordingly, we reported that (i) C becomes activated in bone marrow (BM) during G-CSF-induced mobilization by the classical immunoglobulin (Ig)-dependent pathway, and that (ii) C3 cleavage fragments increase the responsiveness of HSPC to an stromal derived factor-1 (SDF-1) gradient. Furthermore, our recent data in immunodeficient mice support the concept that the CC is a major factor modulating egress of HSPC from bone marrow (BM) into peripheral blood (PB). Thus, in light of these findings, mobilization of HSPC could be envisioned as part of an immune response that requires CC activation by the classical Ig-dependent and/or Ig-independent pathways. Hence modulation of CC activation could allow for the development of more efficient mobilization strategies in patients who are poor mobilizers of HSPC.

Background. Fms-related tyrosine kinase 3 (Flt3)-ligand (FL) promotes the proliferation, differentiation, development, and mobilization of hematopoietic cells. We previously found that FL-mobilized hematopoietic stem cells (HSC) engraft efficiently, whereas FL-expanded bone marrow HSC do not. The function of FL-mobilized c-Kit+ Sca-1+Lin− (KSL) subpopulations has not been systematically evaluated. A precise definition of the repopulating ability is needed to define which HSC subpopulations are critical for long-term chimerism and tolerance induction. FL significantly mobilized c-Kithi and c-Kitlo Sca-1+Lin− cells into peripheral blood (PB). Here, we evaluated the influence of Flt3 expression on long-term repopulating ability of HSC subpopulations. Methods. c-Kithi or c-Kitlo KSL cells were sorted from PB of FL-treated green fluorescent protein-positive donors. The function of these cells was evaluated using competitive reconstitution assays, colony-forming units spleen, and colony forming cell assays. The function of c-Kithi CD34−Flt3− KSL, c-Kithi CD34+Flt3− KSL, c-Kithi CD34+Flt3+ KSL were investigated in an in vivo transplantation model. Results. Only FL-mobilized PB c-Kithi KSL cells exhibited high spleen colony-forming unit activity, generated high numbers of both lymphoid and myeloid colonies in vitro, and rescued ablated recipients. FL-mobilization expanded both c-Kithi CD34+Flt3− cells (short-term HSC) and c-Kithi CD34−Flt3− KSL cells (long-term HSC). There was a significant decrease in c-Kithi CD34+Flt3+ KSL late multipotent progenitors in PB. A combination of c-Kithi CD34+Flt3− and c-Kithi CD34−Flt3− KSL cells offered the most effective rescue of ablated recipients. Conclusions. These data suggest that engraftment of purified HSC is influenced by both short- and long-term repopulating populations and that Flt3 expression may be useful for selecting the most critical HSC subpopulations for transplantation.

Poly-(1,6)-beta-D-glucopyranosyl-(1,3)-beta-D-glucopyranose (PGG) beta-glucan is a soluble yeast-derived polysaccharide that has previously been shown to induce hematopoietic progenitor cell (HPC) mobilization. However, the mobilizing mechanism of action remains unknown. Here, we confirmed that PGG beta-glucan alone or in combination with granulocyte colony-stimulating factor (G-CSF) mobilizes HPC into the periphery. Optimal mobilizing effects were seen 24-48 hours after PGG beta-glucan doses of 4.8-9.6 mg/kg. Animals treated with G-CSF and PGG beta-glucan showed a collaborative effect in HPC mobilization compared with G-CSF treatment alone. Additional studies demonstrated that neither complement 3 nor complement receptor 3 played a role in this effect and that PGG beta-glucan treatment did not induce proinflammatory cytokine secretion. However, bone marrow cells from PGG beta-glucan-treated mice secreted abundant matrix metalloproteinase-9 (MMP-9), and PGG beta-glucan-induced HPC mobilization was abrogated in MMP-9 knockout mice. Moreover, we demonstrated that both hematopoietic and nonhematopoietic cells contributed to MMP-9 secretion upon PGG beta-glucan treatment. In addition, HPCs mobilized by PGG beta-glucan had similar levels of engraftment in host and lineage differentiation capability compared with those mobilized by G-CSF. Thus, PGG beta-glucan is an agent that enhances HPC mobilization and may improve the outcome of clinical stem cell transplantation.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescents and children that frequently infiltrates bone marrow (BM). RMS is divided in two sub-types, alveolar-RMS (ARMS) and embryonal-RMS (ERMS). The ARMS subtype is often associated with a more aggressive phenotype, poorer clinical prognosis, and expression of Pax3-FKHR and Pax7-FKHR fusion proteins that function as potent transcriptional activators with enhanced activity as compared to normal Pax3 or Pax7 transcription factors. We reported that the stromal derived factor-1 (SDF-1)-CXCR4 receptor axis plays a crucial role in RMS metastasis to BM (Blood 2002; 100:2597, Cancer Res 2003; 63:7926, Cancer Res. 2007; 67:213). After the recent identification of CXCR7, a new receptor for SDF-1, we became interested in its potential role in metastases of RMS cells. We found that CXCR7 was expressed and functional on all 10 human RMS cell lines investigated in this study. Furthermore, we noticed that while CXCR4 was more involved in regulating RMS chemotaxis, CXCR7 primarily regulated adhesion of RMS cells. Based on this observation, we assessed the mechanisms that regulate expression of these receptors. We noticed that CXCR4 is highly expressed on the more metastatic ARMS while CXCR7 is expressed at higher levels on the less metastatic ERMS cell lines. However, both receptors express several hypoxia-inducible factor (HIF)-1a binding sites, expression of CXCR4 was not affected by exposure to hypoxia, and expression of CXCR7 was even paradoxically downregulated in hypoxic conditions. We also observed that ERMS cells transduced with Pax3-FKHR, a gene typical for ARMS fusion that enhances RMS metastatic behavior, highly upregulated expression of CXCR4. In contrast, expression of CXCR7 was again down-regulated. To learn more on the role of Pax-3-FKHR in the expression of CXCR4, we performed a functional analysis of promoter fragments subcloned into luciferase vector. To our surprise, expression of CXCR4 did not depend on direct binding of Pax3-FKHR to classical Pax3 binding sites in promoter sequences but on direct protein-to-protein interaction with nuclear respiratory factor (NRF)-1 transcription factor. These results suggest that the Pax3-FKHR-NRF-1 complex binds to the NRF-1 binding site and aberrantly activates transcription. In conclusion, both of the SDF-1 binding receptors (CXCR4 and CXCR7) are differently regulated in RMS cells. Since CXCR7 is more important in adhesion and becomes downregulated during hypoxia, this may give an advantage for SDF-1 to signal through CXCR4 receptor to activate pathways critical for chemotactic responses. As a biological consequence, this may increase the metastatic potential of RMS by “mobilizing” RMS cells from the primary tumor to migrate and metastasize. In addition, the novel transcriptional mechanism for the Pax3-FKHR-NRF-1 complex in regulating CXCR4 receptors in RMS cells has been identified.

HSC, sorted as KSL (c-Kit+Sca-1+Lin-) cells, are comprised of primitive long-term repopulating cells and short-term repopulating committed progenitors. Growth factors such as GM-CSF, G-CSF or Flt3-ligand (FL) are utilized to expand and mobilize HSC. We evaluated here the phenotype and function of FL-mobilized HSC. FL significantly expands c-Kithi and c-Kitlo KSL cells in peripheral blood (PB). Only FL-expanded PB c-Kithi KSL cells exhibited high spleen colony-forming unit frequency, generated high numbers of both lymphoid and myeloid colonies in vitro, and rescued ablated recipients. FL expanded two subsets of c-Kithi KSL cells: CD34+flt3- reflective of short-term HSC (ST-HSC) and CD34-flt3- long-term HSC (LT-HSC), while the proportion of c-Kithi CD34+flt3+ KSL cells multipotent progenitors (MPP) was significantly decreased in the PB. When 500 CD34+flt3-, CD34+flt3+ or CD34-flt3- c-Kithi KSL cells were transplanted into ablated syngeneic recipients, all recipients of CD34+flt3+ c-Kithi KSL expired within 16 days (n =5). One of the 5 recipients of c-Kithi CD34+flt3- KSL (ST-HSC) survived over 120 days. The combination of 50 c-Kithi CD34-flt3- KSL cells (LT-HSC) with 500 c-Kithi CD34+flt3- or CD34+flt3+ KSL cells enhanced recipient survival (4/6 recipients of both ST-HSC plus LT-HSC survived over 120 days and some recipients of MPP + LT-HSC survived up to 68 days). These data suggest that flt3 expression may be a useful phenotypic marker for selecting critical stem cell populations to ensure rapid and durable engraftment and confirms that both short and long-term repopulating cells are needed for optimal successful transplantation.

Complement ( C) and innate immunity emerge as important and underappreciated modulators of mobilization of hematopoietic stem/progenitor cells (HSPC). We reported that ( a) C becomes activated in bone marrow ( BM) during granulocyte-colony-stimulating factor (G-CSF)-induced mobilization by the classic immunoglobulin (Ig)-dependent pathway and that (b) C3 cleavage fragments increase the responsiveness of HSPC to a stromal derived factor-1 gradient. Since patients suffering from severe combined immunodeficiency ( SCID) mobilize poorly, we hypothesized that this could be directly linked to the lack of C activating Ig in these patients. In the current study to better elucidate the role of C activation in HSPC mobilization, we mobilized mice that lack Ig (RAG2, SCID, and Jh) by G-CSF or zymosan, compounds that activate C by the classic Ig-dependent and the alternative Ig-independent pathways, respectively. In addition, we evaluated mobilization in C5-deficient animals. Mobilization was evaluated by measuring the number of colony-forming unit-granulocyte macrophage and leukocytes circulating in peripheral blood. We found that ( a) G-CSF- but not zymosan-induced mobilization was severely reduced in RAG2, SCID, and Jh mice; (b) impaired G-CSF- induced mobilization was restored after infusion of purified wild-type Ig; and ( c) mobilization was severely reduced in C5-deficient mice. These data provide strong evidence that the C system plays a pivotal role in mobilization of HSPC and that egress of HSPC from BM occurs as part of an immune response.

Despite advances in stem cell mobilization and techniques, up to 20–25% of patients exhibit poor mobilization and are not able to proceed with auto-transplantation. PGG beta-glucan is a soluble yeast-derived polysaccharide and has been shown previously to induce hematopoietic stem and progenitor cell (HSPC) mobilization. However, the mechanism of action has not been defined. In the current study, we demonstrated that PGG beta-glucan alone was able to mobilize peripheral HSPC at both doses (4.8mg/kg and 9.2mg/kg) after 24 hrs. The combination group (G-CSF/PGG-4.8mg/kg) showed an almost two-fold increase in CFUs compared to the standard of G-CSF alone. Further studies demonstrated that PGG beta-glucan mobilized HSPC via a C or CR3 independent mechanism and did not induce appreciable levels of cytokine secretion. Strikingly, BM cells from PGG beta-glucan mobilized mice secreted abundant matrix metalloproteinase-9 (MMP-9). PGG beta-glucan-induced HSPC mobilization was abrogated in MMP-9 KO mice. Further studies from BM chimeras demonstrated that hematopoietic BM cells and possibly BM endothelial cells stimulated with PGG beta-glucan secreted MMP-9. Taken together, these data suggest that PGG beta-glucan is an agent that enhances HSPC mobilization alone and has a synergistic effect when used in conjunction with G-CSF. This process requires active MMP-9, which results from release of pro-MMP-9 from BM cells.

Background: Peripheral blood stem cell infusion is the preferred method for establishing hematopoiesis in transplantation. Use of G-CSF is now the most commonly used mobilizing agent. Despite advances in stem cell techniques and agents, studies have shown that up to 20–25% of patients exhibit poor mobilization and are not able to proceed with autotransplantation. Strategies to improve mobilization include using chemotherapy alone or in conjunction with growth factor or novel agents such as AMD3100. β-glucan PGG is a soluble yeast beta glucan with a molecular mass of 150kD comprised of a β-D-(1–3)-linked glucopyranosyl backbone with a β-D-(1–6)-linked β(1–3) side chains. In previous studies, β-glucan PGG has been shown to induce hematopoietic stem and progenitor cell (HSPC) mobilization to the periphery. In this study, we examined β-glucan PGG's ability to mobilize HSPC alone and in conjuction with G-CSF and explored its mechanism of action.

We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPC) by i) immunoglobulin (Ig)-dependent pathway and/or by ii) alternative Ig-independent pathway and, as result of this, iii) several potent bioactive CC anaphylatoxins (C3a, desArgC3a, C5a and desArgC5a) are released (Blood 2003; 101,3784; Blood 2004; 103,2071; Blood 2005; 105,40). To learn more on the role of CC and innate immunity in this process, we compared mobilization in mice that possess defects in CC activation by i) classical pathway (C1q−/−, Ig-deficient), ii) both classical and alternative pathway (C2fB−/−) and in animals iii) that do not generate CC-derived anaphylatoxins (C3−/−, C5−/−). For mobilization, we employed G-CSF and zymosan that activate classical and alternative pathways of CC, respectively. First, we found by ELISA that CC activation in fact correlates with the level of HSPC mobilization. Next, studies in mice deficient in CC activation revealed that CC plays both pivotal and pleiotropic roles in this process. Accordingly, while C1q−/− and C3−/− mice turned out to be easy mobilizers, mobilization was very poor in Ig-deficient, C2fB−/− and C5−/− mice that demonstrate that C3 and C5 cleavage fragments differently control the mobilization of HSPC. To explain this at molecular level, we found that C3 cleavage fragments (C3a, desArgC3a) directly interact with HSPC and increase their responsiveness to SDF-1 gradient and thus prevent uncontrolled egress of HSPC from BM. It explains why C1q−/− and C3−/− mice that do not generate C3 cleavage fragments in BM release easily HSPC into circulation. In contrast, C5 cleavage fragments (C5a, desArgC5a) increase permeability of BM-endothelium and thus are crucial for the egress of HSPC from BM to occur. This explains why mice that do not activate efficient CC such as Ig-deficient, C2fB−/− and C5−/− animals are poor mobilizers. We conclude that the mobilization of HSPC is i) dependent on C activation by the classical or alternative pathway and balanced differently by C3 and C5 cleavage fragments that enhance retention or promote egress of HSPC respectively. Thus, modulation of C activation in BM may help to develop new more efficient strategies for both HSPC mobilization and their homing/engraftment.

Recently we identified in murine BM a homogenous population of rare (~0.01% of BMMNC) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1 and highly express Rif-1 telomerase protein (Leukemia 2006; 20,857–869). Direct electronmicroscopical analysis revealed that these cells display several features typical for primary embryonic stem cells such as i) a small size (~2–4 μm in diameter), ii) a large nuclei surrounded by a narrow rim of cytoplasm, and iii) open-type chromatin (euchromatin). These cells isolated freshly from the BM neither grow hematopoietic colonies nor radioprotect lethally irradiated recipients. Recently, however, we noticed that purified VSELs in co-cultures with C2C12 murine sarcoma supportive feeder-layer grow spheres and cells from these VSEL-derived spheres (VSEL-DS) are composed of immature cells with large nuclei containing euchromatin, and similarly as VSELs are CXCR4+SSEA-1+Oct-4+. We found that cells from VSEL-DS after re-plating over C2C12 cells may again (up to 5–7 passages) grow new embryoid-like bodies or if plated into cultures promoting tissue differentiation show pluripotency and expand into cells from all three germ-cell layers. Based on this we tested if CD45− VSEL-DS cells could also differentiate into the hematopoietic lineage. To address this issue we employed similar culture conditions that are employed for hematopoietic differentiation of established embryonic stem cell lines. We noticed that VSEL-DS cells if cultured/passaged in methylocellulose cultures supplemented with hematopoietic growth factors (KL, IL-3, EpO and GM-CSF) give rise to colonies composed of myeloid (CD45+ Gr-1+) and erythroid (Terr-119+) hematopoietic cells. The hematopoietic differentiation of VSEL-DS cells was accompanied by upregulation of mRNA for several genes regulating hematopoiesis (e.g. PU-1, c-myb, LMO2, Ikaros). Based on this we postulate that VSELs that reside in bone marrow may contribute to hematopoiesis. We also provide direct evidence that CD45+ cells may derive from a CD45− population. We postulate that VSELs are closely related to a population of long-term engrafting hematopoietic stem cells and currently we are testing this possibility in animal models.

Proper response of normal stem cells (NSC) to motomorphogens and chemoattractants plays a pivotal role in organ development and renewal/regeneration of damaged tissues. Similar chemoattractants may also regulate metastasis of cancer stem cells (CSC). Growing experimental evidence indicates that both NSC and CSC express G-protein-coupled seven-transmembrane span receptor CXCR4 and respond to its specific ligand alpha-chemokine stromal derived factor-1 (SDF-1), which is expressed by stroma cells from different tissues. In addition, a population of very small embryonic-like (VSEL) stem cells that express CXCR4 and respond robustly to an SDF-1 gradient was recently identified in adult tissues. VSELs express several markers of embryonic and primordial germ cells. It is proposed that these cells are deposited early in the development as a dormant pool of embryonic/pluripotent NSC. Expression of both CXCR4 and SDF-1 is upregulated in response to tissue hypoxia and damage signal attracting circulating NSC and CSC. Thus, pharmacological modulation of the SDF-1-CXCR4 axis may lead to the development of new therapeutic strategies to enhance mobilization of CXCR4+ NSC and their homing to damaged organs as well as inhibition of the metastasis of CXCR4+ cancer cells.

INTRODUCTION:Recently we identified in bone marrow (BM) by employing chemotactic isolation to SDF-1 gradient combined with real time RT-PCR analysis a mobile population of CXCR4+ BM mononuclear cells that express mRNA for various markers of early tissue-committed stem cells (TCSCs). In this study we evaluated whether TCSCs respond to other motomorphogens, such as hepatocyte growth factor (HGF) and leukemia inhibitory factor (LIF).MATERIALS AND METHODS:We again employed chemotactic isolation combined with real-time RT-PCR analysis to assess whether murine and human BM contain TCSCs that respond to HGF and LIF gradients. We also evaluated expressions of HGF and LIF in damaged organs.RESULTS:We noted that the number of TCSCs is highest in BM from young (1- to 2-month-old) mice and decreases in 1-year-old animals. Murine and human TCSCs 1) respond to HGF and LIF gradients in addition to an SDF-1 gradient, 2) reside in populations of BM-derived non-hematopoietic CD45-cells, and 3) are released (mobilized) from BM into the peripheral blood (PB) during tissue injury (e.g. after partial body irradiation).CONCLUSIONS:These findings further support our theory of the BM as a "hideout" for TCSCs and we suggest that their presence in BM tissue should be considered before experimental evidence is interpreted simply as transdifferentiation/plasticity of hematopoietic stem cells. Since we demonstrated that not only SDF-1, but also HGF and LIF are upregulated in damaged tissues, we postulate that CXCR4+ c-Met+ LIF-R+ TCSC could be mobilized from the BM into the PB, from which they are subsequently chemoattracted to damaged organs, where they play a role in tissue repair/regeneration.