Extramedullary Bone Marrow Research Articles

Connective tissue growth factor regulates adipocyte differentiation of mesenchymal stromal cells and facilitates leukemia bone marrow engraftment

AbstractMesenchymal stromal cells (MSCs) are a major component of the leukemia bone marrow (BM) microenvironment. Connective tissue growth factor (CTGF) is highly expressed in MSCs, but its role in the BM stroma is unknown. Therefore, we knocked down (KD) CTGF expression in human BM-derived MSCs by CTGF short hairpin RNA. CTGF KD MSCs exhibited fivefold lower proliferation compared with control MSCs and had markedly fewer S-phase cells. CTGF KD MSCs differentiated into adipocytes at a sixfold higher rate than controls in vitro and in vivo. To study the effect of CTGF on engraftment of leukemia cells into BM, an in vivo model of humanized extramedullary BM (EXM-BM) was developed in NOD/SCID/IL-2rgnull mice. Transplanted Nalm-6 or Molm-13 human leukemia cells engrafted at a threefold higher rate in adipocyte-rich CTGF KD MSC-derived EXM-BM than in control EXM-BM. Leptin was found to be highly expressed in CTGF KD EXM-BM and in BM samples of patients with acute myeloid and acute lymphoblastic leukemia, whereas it was not expressed in normal controls. Given the established role of the leptin receptor in leukemia cells, the data suggest an important role of CTGF in MSC differentiation into adipocytes and of leptin in homing and progression of leukemia.

Apoptosis Repressor with Caspase Recruitment Domain (ARC) Increases AML Cell Migration and Adhesion in Vitro and in Vivo by Regulating Leukemia-Stroma Interactions.

Abstract 2626Hematopoietic cells express a wide range of adhesion molecules and bone marrow (BM) stroma cells produce their corresponding ligands. Through these ligand-receptor pairs, hematopoietic cells interact with their BM microenvironment. The same system is hijacked by AML and often adhesion molecules in leukemia cells and/or their ligands in stroma cells are upregulated, promote leukemia-stroma interactions, and protect leukemia cells from therapeutic agents. Understanding the underlying mechanisms is critical for therapeutic strategies aimed at disrupting leukemia-stroma interactions. For example, pharmacological blockage of the CXCR4-SDF1a axis has been shown to result in chemosensitization of AML cells in vitro, in vivo and in clinical trials (Zeng Z et al., Blood 2009; Uy GL et al., Blood 2012; Andreeff M et al., ASCO 2012).ARC (Apoptosis repressor with caspase recruitment domain) is an antiapoptotic protein. We recently determined ARC expression in samples from 511 newly diagnosed AML patients by reverse-phase protein array and reported that ARC is one of the strongest adverse prognostic markers in AML (Carter BZ et al., Blood 2011). In the same sample set, we also probed the expression of more than 200 additional proteins enabling us to correlate ARC expression with the expressions of other proteins. Surprisingly, ARC expression was correlated with multiple proteins involved in cell adhesion and migration. We generated stable ARC overexpressing (O/E) KG-1 cells, ARC knock down (K/D) OCI-AML3 and Molm13 cells, and ARC K/D BM derived mesenchymal stroma cells (MSCs) to investigate ARC’s roles in leukemia-stroma interactions. Expression levels of FAK, integrinb3, fibronectin, and VLA4 were increased in ARC O/E and decreased in ARC K/D cells, compared to controls. CXCR4 mRNA and cell surface CXCR4 protein were higher in ARC O/E KG-1 (3.80- and 1.53-fold, P<0.01; respectively) and lower in ARC K/D cells (OCI-AML3: 0.43- and 0.70-fold, P=0.01, and Molm13: 0.46- and 0.74-fold, P=0.02, respectively), while levels of SDF1a mRNA as well as secreted SDF-1a protein determined by ELISA (P=0.013) were lower in ARC K/D as compared to control MSCs. Interestingly, MSCs co-cultured with AML cells significantly increased SDF1a secretion and this increase was greatly diminished when ARC was knocked down in MSCs.Migration of leukemic cells towards MSCs was significantly higher for ARC O/E and lower for ARC K/D AML cells (P<0.001) and migration of leukemic cells to ARC K/D MSCs was significantly diminished compared to control MSCs for both, OCI-AML3 (P<0.001) and primary AML cells (18.1±0.8% vs. 32.8±0.5%, n=4, P<0.001). In addition, adhesion to MSCs and to VCAM1 coated plates was increased for ARC O/E and decreased for ARC K/D AML cells (P<0.01). Furthermore, adhesion to ARC K/D MSCs was decreased compared to control MSCs for AML cells for both cell lines (KG-1, 43.6±1.2% vs. 58.2±1.7%; OCI-AML3, 34.8±1.0% vs. 48.7±0.7%; Molm13, 9.2±2.2% vs. 16.8±2.7%) and primary patient samples (52.1±1.6% vs. 66.3±6.4%, P=0.02 and 42.1±5.0% vs. 59.6±7.4%, P=0.03).The role of ARC in leukemia-stroma interactions was further assessed using a novel human extramedullary BM model in mice recently developed by our group (Chen Y et al., Blood 2012). Luciferase/GFP Molm13 cells were injected into Nod/ScidIL2Rg−/− mice after extramedullary human BM containing either ARC K/D or control MSCs was established in the flanks of mice. Significantly fewer AML cells engrafted in the human extramedullary BM developed from ARC K/D than from control MSCs as determined by immunohistochemistry staining for human CD45+ cells and quantitative image analysis (P<0.01).In conclusion, we here demonstrate that antiapoptotic ARC regulates migration and adhesion of leukemia cells to BM stroma in vitro and in vivo via activation of multiple mechanisms, not only in AML cells but also in MSCs, thus enhancing its anti-apoptotic activity. Given the fact that both intrinsic apoptosis resistance and extrinsic environmental factors contribute to drug resistance and relapse in AML, ARC may play a central role in AML and be an excellent therapeutic target: ARC inactivation may disrupt leukemia-stroma interactions and increase leukemia cell sensitivity to chemotherapy by increasing susceptibility to apoptosis. We speculate that ARC, a CARD containing protein, acts at least in part via activating NF-kB signaling, which is known to be regulated by CARD proteins. Disclosures:No relevant conflicts of interest to declare.

NF-κB Activation in Mesenchymal Stromal Cells Mediates Leukemia Cell Chemoresistance

Abstract 3518Within the bone marrow (BM) microenvironment, BM mesenchymal stromal cells (BM-MSC) provide leukemia cells with a rich environment that serves as a sanctuary and protects them from chemotherapeutic agents. Interactions between leukemia cells and BM-MSC are thought to change the behavior of both stroma and leukemia cells resulting in an increased resistance to standard drugs. To discover interaction-induced changes in BM-MSCs that might promote microenvironment-mediated chemoresistance we used genome-wide gene expression profiling to study normal-donor BM-MSCs co-cultured with REH cells, a pre-B acute lymphoblastic leukemia (ALL) cell line. Upregulated genes in normal-donor BM-MSCs by co-culture included many cytokines and chemokines and implicated activation of NF-κB in BM-MSCs. Similar results were obtained by co-culturing normal-donor BM-MSCs with OCI-AML3 cells, an acute myeloid leukemia (AML) cell line. Increased expression of NF-κB target genes in BM-MSCs was also induced by co-culture with primary cells from ALL and AML patients, suggesting that NF-κB activation is a common consequence of leukemia-stroma interaction. Blocking canonical-pathway NF-κB activation by overexpressing a super-repressor form of IκBα in BM-MSC, significantly reduced stroma-mediated chemoresistance to vincristine (VCR) of leukemia cells in vitro and in vivo using an extramedullary bone marrow model. When small-molecule IKKβ inhibitors (MLN3316 and CDDO-Me) were used in co-cultures, blocking canonical-pathway NF-κB activation in ALL cell lines (REH or RS4;11) as well as BM-MSCs, the apoptotic effects of VCR on leukemia cells were further increased. Conclusion:results indicate that leukemia cells activate NF-κB in stromal cells and that such activation is in turn essential to promote survival of leukemia cells during chemotherapy. Targeting NF-κB in BM-MSC may ameliorate stroma-mediated chemoresistance and help in eliminating BM-resident leukemia cells. Disclosures:No relevant conflicts of interest to declare.

Human extramedullary bone marrow in mice: a novel in vivo model of genetically controlled hematopoietic microenvironment

AbstractThe interactions between hematopoietic cells and the bone marrow (BM) microenvironment play a critical role in normal and malignant hematopoiesis and drug resistance. These interactions within the BM niche are unique and could be important for developing new therapies. Here, we describe the development of extramedullary bone and bone marrow using human mesenchymal stromal cells and endothelial colony-forming cells implanted subcutaneously into immunodeficient mice. We demonstrate the engraftment of human normal and leukemic cells engraft into the human extramedullary bone marrow. When normal hematopoietic cells are engrafted into the model, only discrete areas of the BM are hypoxic, whereas leukemia engraftment results in widespread severe hypoxia, just as recently reported by us in human leukemias. Importantly, the hematopoietic cell engraftment could be altered by genetical manipulation of the bone marrow microenvironment: Extramedullary bone marrow in which hypoxia-inducible factor 1α was knocked down in mesenchymal stromal cells by lentiviral transfer of short hairpin RNA showed significant reduction (50% ± 6%; P = .0006) in human leukemic cell engraftment. These results highlight the potential of a novel in vivo model of human BM microenvironment that can be genetically modified. The model could be useful for the study of leukemia biology and for the development of novel therapeutic modalities aimed at modifying the hematopoietic microenvironment.

Abstract 2971: Human extramedullary bone and bone marrow in mice: First in vivo model of a genetically controlled hematopoietic environment - Role of CTGF and HIF1-α

Abstract The microenvironment contributes and may regulate cancer development, progression and resistance to treatment. Our group reported first the contribution of bone marrow (BM)-derived mesenchymal stromal cells (MSCs) for tumor development and metastasis. Accumulating evidence has shown that the BM microenvironment also plays a pivotal role in the pathophysiology and propagation of leukemia. We here describe a novel, genetically controlled bone and bone marrow model using human BM derived MSCs and endothelial colony-forming cells (ECFCs) subcutaneously injected into the flanks of NOD/SCID/IL-2rαnull mice, where they developed into bone-like tissues with high osteoblast activity after 10 weeks, with bone structures and typical bone marrow cavities constituting a robust hematopoietic environment. In vivo imaging with Osteosense confirmed the presence of hydroxylapatite, and luciferase imaging of firefly-luciferase labeled human leukemic cells demonstrated the engraftment of human MOLM13/Luc/GFP leukemic cells. The extramedullary leukemic BM was markedly hypoxic, as shown by Pimonidazole staining. Factors critical for MSC to support normal and leukemic hematopoiesis are largely unknown and cannot readily be studied since human MSC do not engraft reliably in xenograft models. We therefore investigated the possibility of genetically modifying MSC in this system and found a significant reduction (50 ± 6%, p&amp;lt;0.001) in leukemial engraftment in extramedullary BM generated with HIF1α knockdown MSCs (1449 ± 194 cells/mm2), compared to vector controls (3037 ± 496 cells/mm2). This finding indicates that HIF1α expression in stromal cells is critical for the engraftment of leukemic cells. Surprisingly, shRNA knockdown of connective tissue growth factor (CTGF) in MSC resulted in the 2-3 fold increased expression of embryonic stem cell markers oct-4, nanog and sox2, increased adipogenic differentiation (PPAR-γ and C/EBβ) and in formation of a more spongy bone. Human leukemic Nalm-6 cells showed 3-fold higher migration to CTGF-KD as compared to control BM, a surprising finding that could mechanistically be related to the 3-fold increased expression of SDF-1 in CTGF-KD-MSC. Conclusion: We have developed a genetically controlled human extra-medullary bone marrow system that allows investigations of the role of specific genes on the homing and expansion of normal and leukemic cells/stem cells in vivo. HIF-1α and CTGF were shown to have distinct and unexpected functions in this system. Results gain further relevance by the recent demonstration of genetic abnormalities in human leukemic bone marrow cells (Andreeff 2008, Blau 2011). The system has allows us to established an in vivo bone and bone marrow model with a genetically controlled human microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2971. doi:1538-7445.AM2012-2971

Heterotopic Implantation of Autologous Bone Marrow in Rock Pigeons (Columba livia): Possible Applications in Avian Bone Grafting

Autologous bone marrow, alone or as a composite marrow graft, has received much attention in various species. To assess the potential osteogenicity of autologous, extramedullary bone marrow implants in an avian model, 24 adult pigeons (Columba livia) were given intramuscular implantations of autologous marrow aspirated from the medial tibiotarsus. Birds were euthanatized at 1, 4, 6, 8, 10, and 12 weeks after surgery to evaluate whether ectopic bone had formed at the implant sites. Primary evaluations by in situ radiography and postmortem histologic examinations showed no evidence of bone formation. Further evaluation with histologic scores and histomorphometry revealed a significantly increased rate of angiogenesis at the implant sites by the sixth and tenth week postimplantation (P < .05). No significant differences between the treatment and control sites were present at any other endpoints. Results of this study show that, although autologous bone marrow lacks heterotopic osteogenic potentials in this avian model, it could still function as a useful adjunct to routine bone grafting techniques because of its unique capabilities to promote early angiogenesis.

Human Extramedullary Bone and Bone Marrow in Mice: First In Vivo Model of a Genetically Controlled Hematopoietic Environment

Abstract 1323The importance of the tumor microenvironment for cancer development, progression and resistance to treatment has recently been recognized. Our group was first to report the contribution of bone marrow (BM) derived mesenchymal stromal cells (MSCs) for tumor development and metastasis. BM is also the dynamic microenvironment (niche) for normal and malignant hematopoietic stem cells (HSC) with high local concentrations of growth factors, chemokines and cytokines. The maintenance of HSCs quiescence and normal hematopoiesis require complex bidirectional interactions between the BM niches and HSCs. Accumulating evidence has shown that the BM microenvironment also plays a pivotal role in the pathophysiology and propagation of leukemia. Leukemia cells undergo spontaneous apoptosis once they are removed from the in vivo microenvironment and placed in suspension cultures without supportive stroma. The understanding of the interactions between leukemic cells and their BM niche is also critically important for leukemia therapy. We here describe a novel artificial bone and bone marrow model mimicking the human hematopoietic microenvironment by using human BM derived MSCs and endothelial colony-forming cells (ECFCs). MSCs and ECFCs were isolated from heparinized human bone marrow or peripheral blood through an initial adhesion step, grown in specific media and then subcutaneously injected into the flanks of the NOD/SCID/IL-2r-gammanull mice, where they developed into bone-like tissues with high osteoblast activity after 10 weeks (Figure 1). Histochemical stains confirmed the bone structures and also showed that these artificial bones contained typical bone marrow cavities constituting a robust hematopoietic environment. In vivo imaging with Osteosense confirmed the presence of hydroxylapatite, and luciferase imaging of firefly luciferase labeled human leukemic cells demonstrated the engraftment of MOLM13/Luc/GFP leukemic cells in the extramedullary BM sites. The extramedullary BM was markedly hypoxic, as shown by Pimonidazole staining, another critical feature of the BM microenvironment. Factors critical for MSC to support the normal and leukemic hematopoiesis are largely unknown and cannot be studied since human MSC do not engraft reliably in xenograft models. We therefore investigated the possibility of genetically modifying MSC in this system and found a significant reduction (50 ± 6%, p<0.001) in MOLM13 cell engraftment in extramedullary BM generated with HIF1-alpha knockdown MSCs (1449 ± 194 cells/mm2), compared to vector controls (3037 ± 496 cells/mm2). This finding indicates that the HIF1-alpha expression in stromal cells is a critical component for the engraftment of leukemic cells in the physiologically hypoxic BM microenvironment. These results, for the first time, establish an in vivo bone and bone marrow model with a genetically controlled human microenvironment. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Connective Tissue Growth Factor (CTGF) Regulates Mesenchymal Stromal Cell De-Differentiation Into Adipocyte Progenitors and Facilitates Leukemic Cell Homing to Extra-Medullary Bone Marrow

Abstract 2391Connective tissue growth factor (CTGF) regulates extracellular matrix production, chemotaxis, cell proliferation and integrin expression. Recent reports suggest that recombinant CTGF transforms mesenchymal stromal cells (MSCs) into fibroblast like cells and inhibits their differentiation potential. We have recently shown that stable knockdown of CTGF expression in bone marrow derived MSCs rendered them quiescent and increased their adipocyte differentiation potential (Battula et al., ASH 2010 abstract 3845, Blood, Vol. 116). Based on these findings, we hypothesized that inhibition of CTGF expression modifies stem cell properties of MSCs and may affect leukemic cell homing. To test this hypothesis, bone marrow derived MSCs were transduced with lentivirus expressing either CTGFshRNA or control vector. Down-regulation of CTGF mRNA by ∼75% was observed in CTGF-KD-MSCs compared to empty vector control-MSCs. To examine the stem cell potential of CTGF-KD-MSCs, embryonic stem cell markers including oct-4, nanog, sox2 were analyzed. Real-time RT-PCR analysis revealed 2–3 fold up regulation of these genes in CTGF-KD-MSCs compared to controls indicating increased stemness. To test if leukemic cells home differentially to CTGF-KD-MSCs, we utilized our recently developed extra-medullary human bone marrow model by implanting a carrier impregnated with BM-MSC mixed with endothelial progenitor cells (EPCs) subcutaneously into NOG mice. These implants generate extra-medullary bone marrow (EXM-BM) in 6–8 weeks and resemble the normal bone marrow microenvironment. In-vivo imaging confirmed that both cell types formed EXM-BM with positive Osteosense binding which reflects the presence of hydroxyapatite. H&E staining of bone sections revealed that CTGF-KD-MSCs derived EXM-BM displayed more spongy bone compared to control-MSCs. To examine the extent of adipocytic differentiation in CTGF-KD-MSCs derived bone pellets, expression of adipocyte-specific markers PPAR-γ and C/EBPα was analyzed. Cells residing in the spongy EXM-BM region expressed high levels of nuclear PPAR-γ and C/EBPα, confirming differentiation into mature adipocytes. These markers were negative in the stromal/endothelial compartment of CTGF-KD-MSCs derived EXM-BM or control EXM-BM. When Nalm-6 (B-ALL cell line) cells expressing firefly luciferase and YFP were transplanted into these mice, leukemic cells migrated 10 fold more avidly towards CTGF-KD-MSCs derived EXM-BM compared to control EXM-BM in the same mouse (n=5). Immunohisto-chemical analysis demonstrated preferential homing of leukemic cells inside spongy regions of the EXM-BM derived from CTGF-KD-MSCs and control-MSCs. To investigate the mechanism behind the leukemic cell homing into CTGF-KD-MSCs derived EXM-BM, expression of stromal cell-derived factor-1α (SDF1α), a major factor involved in leukemic cell homing to bone marrow, was analyzed in both cell types by qRT-PCR. CTGF-KD-MSCs expressed 3-fold higher levels of SDF-1α compared to control cells indicating that SDF1α secretion by CTGF-KD-MSCs derived bone marrow might enhance the leukemia cell homing. In summary, these findings suggest that CTGF plays a crucial role in the regulation of stemness and differentiation of MSCs and affects the homing of leukemic cells to the bone marrow microenvironment. Targeting adipocyte progenitors may reduce the lodging of leukemic cells in the bone marrow niche and enhance chemosensitivity of leukemic cells within the BM microenvironment. Disclosures:No relevant conflicts of interest to declare.