Marrow Components Research Articles

Angiogenesis inhibition, hypoxia, and targeting the bone marrow microenvironment in multiple myeloma: new strategies and targets

Multiple myeloma (MM) is a hematological B-cell malignancy that has still a fatal prognosis. Although the treatments have improved, one major problem in MM is the clinical resistance to available drugs and combination therapies over time. Novel agents, such as oral proteasome inhibitors, monoclonal antibodies, second generation immunomodulatory drugs and therapies targeting the cell signaling and the tumor microenvironment are in development for the treatment of relapsed/refractory MM. In this review, we refer on the role of new strategies targeting the tumor microenvironment, especially on angiogenesis, hypoxia and other interactions between MM and bone marrow components.

Clinical dilemma: Misdiagnosed…

., we would like to share with you and readers of European Journal of General Dentistry, our experience of a 42‑year‑old male patient, who was clinically misdiagnosed with giant calculus. He gave history of road traffic accident, 12years back causing trauma to the upper left posterior region resulting in loosening of teeth. He had consulted the local dentist for his mobile teeth 5 years back, which were extracted. Since then he had difficulty in eating food from the same area. He was a known case of pemphigus vulgaris and was under treatment. He gave a history of tobacco chewing in the raw form with lime, since 10 years. He had visited the dental college due to pain in left posterior region. The pain aggravates during verbal communication and mastication. His oral hyaline was poor, with generalized stains and multiple carious teeth. Intra‑oral examination revealed edentulous left maxillary arch. An erythematous lesion of approximately 1.5cm × 1 cm in diameter was appreciated in left buccal mucosa in relation to 27, 28 region [Figure 1a and b]. Abrownish mass of varying shades with indentations of teeth mark was observed in relation to 21‑27, having a porous appearance, which was hard in consistency[Figure 1a]. The mass was mobile and appeared to be attached on the anterior side with an orthodontic wire and posteriorly appeared to be attached to the alveolus. Orthopantomograph revealed a radiodense mass in relation to 21‑27[Figure 1c]. Clinically, it was diagnosed as giant calculus and the erythematous lesion was considered to be an inflamed area caused by the giant calculus. The bony hard mass was removed with surgical invention and was sent for histopathological evaluation [Figure 1d]. Microscopically, the Hematoxylin and Eosin stained sections showed necrotic osseous tissue with the absence of marrow components (blood vessels etc.) and empty lacunas with the absence of osteocytes and were suggestive of necrotic bone.Literature search revealed brownish lesions which are hard in consistency in relation to tooth‑bearing areas have been previously misdiagnosed, and or can mimic as neoplastic lesions.

Dual-regulated Lentiviral Vector for Gene Therapy of X-linked Chronic Granulomatosis

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.

The use of a novel bone allograft wash process to generate a biocompatible, mechanically stable and osteoinductive biological scaffold for use in bone tissue engineering.

Fresh-frozen biological allograft remains the most effective substitute for the 'gold standard' autograft, sharing many of its osteogenic properties but, conversely, lacking viable osteogenic cells. Tissue engineering offers the opportunity to improve the osseointegration of this material through the addition of mesenchymal stem cells (MSCs). However, the presence of dead, immunogenic and potentially harmful bone marrow could hinder cell adhesion and differentiation, graft augmentation and incorporation, and wash procedures are therefore being utilized to remove the marrow, thereby improving the material's safety. To this end, we assessed the efficiency of a novel wash technique to produce a biocompatible, biological scaffold void of cellular material that was mechanically stable and had osteoinductive potential. The outcomes of our investigations demonstrated the efficient removal of marrow components (~99.6%), resulting in a biocompatible material with conserved biomechanical stability. Additionally, the scaffold was able to induce osteogenic differentiation of MSCs, with increases in osteogenic gene expression observed following extended culture. This study demonstrates the efficiency of the novel wash process and the potential of the resultant biological material to serve as a scaffold in bone allograft tissue engineering.

Concise Review: From Greenhouse to Garden: The Changing Soil of the Hematopoietic Stem Cell Microenvironment During Development

The hematopoietic system has been intensely studied for many decades. For this reason, it has become the best understood stem cell-derived system that serves as a paradigm for stem cell biology and has found numerous applications in the clinics. While a lot of progress has recently been made in describing the bone marrow components that maintain and control blood stem cell function in the adult, very little is currently known about the regulatory microenvironment in which the first adult-repopulating hematopoietic stem cells are formed during development. Knowledge of these processes is crucial for understanding the basic regulation of hematopoietic stem cell production and behavior and to allow their in vitro expansion and generation from embryonic stem cells or iPS cells for clinical and research purposes. This review summarizes the recent advances that have been made in defining the cellular components, as well as the soluble and physical factors, that are part of the niche involved in regulating hematopoietic stem cell generation in the embryo. The findings are compared with what is known about the adult bone marrow niche to find common pathways for stem cell regulation, but also to highlight processes uniquely required for de novo hematopoietic stem cell generation, as these are the conditions that will need to be recreated for the successful production of blood stem cells in culture.

Adipose‐derived stromal cells grown on a hydroxyapatite scaffold can support hematopoiesis in regenerated bone marrow in vivo

Osteoblastic cells are a key component of the bone marrow (BM) stem cell niche and help regulate hematopoietic stem cells (HSCs). We have previously demonstrated that adipose-derived stromal cells (ADSCs) can differentiate into both osteogenic and chondrogenic cells in vitro. The current study examined whether the anatomical architecture of the BM could be regenerated in vivo by using ADSCs cultured on a hydroxyapatite (HA) scaffold. ADSCs from GFP transgenic mice were cultured in vitro on an HA scaffold. The scaffold with the attached cells was implanted subcutaneously onto the backs of C57/BL6 (Ly5.2) recipient mice. Lineage-negative (Lin-) Ly5.1 BM cells transduced with a lentiviral vector containing the luciferase (Luc) gene were intravenously administered to the recipient mice after lethal irradiation. Eight weeks after BM transplantation, the scaffolds were removed from the first recipient mice and subcutaneously implanted into lethally irradiated second recipient mice. The biodistribution and kinetics of Luc(+) Ly5.1 cells were monitored by bioluminescence imaging and flow cytometry. Luc(+) hematopoietic cells were present in the scaffolds of the secondary implanted mice for at least 8 months. Subcutaneous injection of G-CSF resulted in wide distribution of bioluminescence signals from the original scaffolds to the whole body. Therefore, BM regenerated using ADSCs grown on an HA scaffold can support HSC populations in vivo, suggesting that a functional BM niche is reconstituted. These results may have a significant impact on the development of therapeutic strategies for various hematopoietic diseases.

Identification of the source of elevated hepatocyte growth factor levels in multiple myeloma patients

BackgroundHepatocyte growth factor (HGF) is a pleiotropic cytokine which can lead to cancer cell proliferation, migration and metastasis. In multiple myeloma (MM) patients it is an abundant component of the bone marrow. HGF levels are elevated in 50% of patients and associated with poor prognosis. Here we aim to investigate its source in myeloma.MethodsHGF mRNA levels in bone marrow core biopsies from healthy individuals and myeloma patients were quantified by real-time PCR. HGF gene expression profiling in CD138+ cells isolated from bone marrow aspirates of healthy individuals and MM patients was performed by microarray analysis. HGF protein concentrations present in peripheral blood of MM patients were measured by enzyme-linked immunosorbent assay (ELISA). Cytogenetic status of CD138+ cells was determined by fluorescence in situ hybridization (FISH) and DNA sequencing of the HGF gene promoter. HGF secretion in co-cultures of human myeloma cell lines and bone marrow stromal cells was measured by ELISA.ResultsHGF gene expression profiling in both bone marrow core biopsies and CD138+ cells showed elevated HGF mRNA levels in myeloma patients. HGF mRNA levels in biopsies and in myeloma cells correlated. Quantification of HGF protein levels in serum also correlated with HGF mRNA levels in CD138+ cells from corresponding patients. Cytogenetic analysis showed myeloma cell clones with HGF copy numbers between 1 and 3 copies. There was no correlation between HGF copy number and HGF mRNA levels. Co-cultivation of the human myeloma cell lines ANBL-6 and JJN3 with bone marrow stromal cells or the HS-5 cell line resulted in a significant increase in secreted HGF.ConclusionsWe here show that in myeloma patients HGF is primarily produced by malignant plasma cells, and that HGF production by these cells might be supported by the bone marrow microenvironment. Considering the fact that elevated HGF serum and plasma levels predict poor prognosis, these findings are of particular importance for patients harbouring a myeloma clone which produces large amounts of HGF.

Assessment of an improved bone washing protocol for deceased donor human bone

NHSBT Tissue Services issues bone to surgeons in the UK in two formats, fresh-frozen unprocessed bone from living donors and processed bone from deceased donors. Processed bone may be frozen or freeze dried and all processed bone is currently subjected to a washing protocol to remove blood and bone marrow. In this study we have improved the current bone washing protocol for cancellous bone and assessed the success of the protocol by measuring the removal of the bone marrow components: soluble protein, DNA and haemoglobin at each step in the process, and residual components in the bone at the end of the process. The bone washing protocol is a combination of sonication, warm water washes, centrifugation and chemical (ethanol and hydrogen peroxide) treatments. We report that the bone washing protocol is capable of removing up to 99.85 % soluble protein, 99.95 % DNA and 100 % of haemoglobin from bone. The new bone washing protocol does not render any bone cytotoxic as shown by contact cytotoxicity assays. No microbiological cell growth was detected in any of the wash steps. This process is now in use for processed cancellous bone issued by NHSBT.

Altering the Architecture of Tissue Engineered Hypertrophic Cartilaginous Grafts Facilitates Vascularisation and Accelerates Mineralisation

Cartilaginous tissues engineered using mesenchymal stem cells (MSCs) can be leveraged to generate bone in vivo by executing an endochondral program, leading to increased interest in the use of such hypertrophic grafts for the regeneration of osseous defects. During normal skeletogenesis, canals within the developing hypertrophic cartilage play a key role in facilitating endochondral ossification. Inspired by this developmental feature, the objective of this study was to promote endochondral ossification of an engineered cartilaginous construct through modification of scaffold architecture. Our hypothesis was that the introduction of channels into MSC-seeded hydrogels would firstly facilitate the in vitro development of scaled-up hypertrophic cartilaginous tissues, and secondly would accelerate vascularisation and mineralisation of the graft in vivo. MSCs were encapsulated into hydrogels containing either an array of micro-channels, or into non-channelled ‘solid’ controls, and maintained in culture conditions known to promote a hypertrophic cartilaginous phenotype. Solid constructs accumulated significantly more sGAG and collagen in vitro, while channelled constructs accumulated significantly more calcium. In vivo, the channels acted as conduits for vascularisation and accelerated mineralisation of the engineered graft. Cartilaginous tissue within the channels underwent endochondral ossification, producing lamellar bone surrounding a hematopoietic marrow component. This study highlights the potential of utilising engineering methodologies, inspired by developmental skeletal processes, in order to enhance endochondral bone regeneration strategies.

Potentiation of osteoclastogenesis by adipogenic conversion of bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BMSCs) are the indispensable component of the bone marrow, being the common precursors for adipocytes and osteoblasts. We show here that adipogenic differentiation resulted in increase in the production of adipocyte markers, such as adiponectin,fatty-acid binding proteins (FABP4), peroxisome proliferator-activated receptor γ (PPARγ), as well as the receptor activator of nuclear-κB ligand (RANKL). Co-culture of osteoclast precursors (OCPs) with BMSCs-derived adipocytes significantly enhanced osteoclast differentiation with low-dose RANKL, whose levels alone could not promote osteoclastogenesis. These results demonstrate for the first time that adipogenic differentiation of BMSCs plays a pivotal role in maintaining bone homeostasis.

Immunological dysregulation in multiple myeloma microenvironment.

Multiple Myeloma (MM) is a systemic hematologic disease due to uncontrolled proliferation of monoclonal plasma cells (PC) in bone marrow (BM). Emerging in other solid and liquid cancers, the host immune system and the microenvironment have a pivotal role for PC growth, proliferation, survival, migration, and resistance to drugs and are responsible for some clinical manifestations of MM. In MM, microenvironment is represented by the cellular component of a normal bone marrow together with extracellular matrix proteins, adhesion molecules, cytokines, and growth factors produced by both stromal cells and PC themselves. All these components are able to protect PC from cytotoxic effect of chemo- and radiotherapy. This review is focused on the role of immunome to sustain MM progression, the emerging role of myeloid derived suppressor cells, and their potential clinical implications as novel therapeutic target.

Molecular Mechanisms Of Adipocyte-Leukemia Interactions Revealed By The Quantitative Proteomics Technology: Pro-Survival Function Of Adipocyte-Derived Free Fatty Acids On Acute Monoblastic Leukemia Mitochondrial Biogenesis

Acute monocytic leukemia (AMoL) is a distinct subtype of AML with an average 3-year overall survival of 31% (Tallman, J Clin Oncol 2004), and the majority of patients die from disease progression after relapse. Adipocytes represent an essential component of the adults bone marrow (BM) microenvironment (Battula, Blood 2013, Tabe, Blood 2004, Konopleva, Blood 1999), and promote survival of monoblastic leukemia cells (Tabe, ASH, 2012).In this study, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ, Applied Biosystems) to examine the molecular mechanism of pro-survival properties of BM adipocytes in co-culture with AMoL. The specific pathway alterations were identified by Metacore (GeneGo, St. Joseph, MI). We first confirmed the more prominent protective role of BM-derived adipocytes as compared to BM stromal cells (MSC) on AMoL cells. Co-culture with adipocytes derived from differentiated MSCs significantly protected U937 and primary AMoL cells (n = 5) from serum starvation-induced apoptosis compared to MSCs (U937 p = 0.029, primary samples p = 0.034). Gene expression analysis demonstrated striking upregulation of mRNA expression levels of CD36 (4.1+0.4-fold),FABP4 (569.3+40.1-fold), and PPARG (2.1+0.2-fold) in U937 cells co-cultured with adipocytes compared to the MSC co-cultured cells. Notably, Bcl-2 mRNA expression in U937 cells was significantly upregulated after adipocyte co-culture compared to MSCs (5.4+0.3-fold). We previously reported that fatty acids (FAs) promote leukemic cell survival via leukemia cells metabolic shifting from pyruvate oxidation to fatty acid oxidation for glycolysis, which links to the Bcl-2 anti-apoptotic machinery (Samudio, J Clin Invest. 2010). Mature adipocytes are capable of releasing abundant free fatty acids (Fas), the essential ligands of a nuclear receptor PPARgamma (PPARG), which are internalized by leukemic cells via scavenger receptor CD36 and ligate PPARG through fatty acid binding protein 4 (FABP4). Enhanced transcriptional activity of FA-ligated PPARG may in turn stimulate glucose metabolism, regulate fatty acids storage, maintain mitochondrial membrane potential, and prevents apoptosis by upregulating anti-apoptotic Bcl-2 resulting in further increase of its downstream target genes including CD36 and FABP4. To gain further insights into metabolic alterations induced in adipocyte-leukemia co-cultures, we performed iTRAQ proteomic analysis of U937 cells cultured in the presence of MSCs or adipocytes. A total of 1,610 proteins were detected by this technology, with 106 proteins differentially expressed between co-culture conditions; 50 genes were up-regulated and 56 genes were down-regulated by adipocytes co-culture compared to MSCs. Among the upregulated proteins, FA synthase (p = 0.007) ATP-citrate synthase responsible for cytosolic acetyl-CoA synthesis (p = 0.019); and glyceraldehyde-3-phosphate dehydrogenase (p = 0.028) involved in the activation of glycolysis and gluconeogenesis pathways (p< 0.0001) were identified. Of note, 20% of the upregulated proteins (10/50) were ribosomal proteins, accompanied by an increase in six DNA elongation or replication related proteins. Anti-apoptotic chaperone proteins known to assist ribosome biogenesis, HSP70, HSP90alpha and HSP90beta were also increased by adipocyte co-culture (p<0.01). The mass spectrometry further revealed more prominent repression of Cytochrome c (p=0.01) as well as the proline metabolism pathway (p=0.0011) under adipocyte co-culture conditions, consistent with inhibition of mitochondrial apoptosis of U937 cells. Summaryour results indicate that abundant FAs produced by BM adipocytes support AMoL cell survival via stimulation of PPARG transcription which directly increases the FA metabolism through uptake and translocation of FAs by CD36 and FABP4 in AMoL cells. FA metabolism in turn facilitates supply of Acetyl-CoA via oxidative phosphorylation, which drives the TCA cycle; and enhances ribosomal biogenesis of AMoL cells, resulting in pro-survival signaling in AMoL cells. It is conceivable that increased adipocyte content of the BM in adult AML patients may promote leukemogenesis and negatively affect the responsiveness to chemotherapy. Disclosures:No relevant conflicts of interest to declare.

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.

Mesenchymal Stem Cells in Immune-Mediated Bone Marrow Failure Syndromes

Immune-mediated bone marrow failure syndromes (BMFS) are characterized by ineffective marrow haemopoiesis and subsequent peripheral cytopenias. Ineffective haemopoiesis is the result of a complex marrow deregulation including genetic, epigenetic, and immune-mediated alterations in haemopoietic stem/progenitor cells, as well as abnormal haemopoietic-to-stromal cell interactions, with abnormal release of haemopoietic growth factors, chemokines, and inhibitors. Mesenchymal stem/stromal cells (MSCs) and their progeny (i.e., osteoblasts, adipocytes, and reticular cells) are considered as key cellular components of the bone marrow haemopoietic niche. MSCs may interfere with haemopoietic as well as immune regulation. Evidence suggests that bone marrow MSCs may be involved in immune-mediated BMFS underlying pathophysiology, harboring either native abnormalities and/or secondary defects, caused by exposure to activated marrow components. This review summarizes previous as well as more recent information related to the biologic/functional characteristics of bone marrow MSCs in myelodysplastic syndromes, acquired aplastic anemia, and chronic idiopathic neutropenia.

Aplastic anemia associated to systemic lupus erythematosus in an AIDS patient: a case report

Aplastic anemia is a bone marrow failure syndrome characterized by peripheral cytopenias and hypocellular bone marrow. Although aplastic anemia is idiopathic in most cases, rheumatic diseases such as systemic lupus erythematosus are recognized as causes of aplastic anemia, with their possible etiological mechanisms being T and B lymphocyte dysfunction and pro-inflammatory cytokines and autoantibody production directed against bone marrow components. In the course of the human immunodeficiency virus infection/acquired immunodeficiency syndrome, the identification of autoantibodies and the occurrence of rheumatic events, in addition to the natural course of systemic lupus erythematosus which is modified by immune changes that are characteristic of human immunodeficiency virus infection/acquired immunodeficiency syndrome, make the diagnosis of systemic lupus erythematosus challenging. This study reports the case of a woman with acquired immunodeficiency syndrome treated with a highly active antiretroviral therapy, who had prolonged cytopenias and hypocellular bone marrow consistent with aplastic anemia. The clinical picture, high autoantibodies titers, and sustained remission of the patient's hematological status through immunosuppression supported the diagnosis of systemic lupus erythematosus-associated aplastic anemia. This is the first report of aplastic anemia concurrent with systemic lupus erythematosus and acquired immunodeficiency syndrome, providing additional evidence that immune dysfunction is a key part of the pathophysiological mechanism of aplastic anemia.

Suppression of Mir-93 May Regulate Anti-Oxidant Metabolism in Mesenchymal Stromal Cells Derived From Acute Myeloid Leukemia Patients.

Abstract 2354Though still in its infancy, increasing interest is focused on the role of metabolism in cancers and leukemias. At present, most of the studies addressing this question have centered on the malignant cells themselves and not on the cells that comprise the tumor microenvironment. The leukemia microenvironment in the bone marrow has been found to provide survival advantage to AML stem cells, and to protect against chemotherapy. The underlying mechanisms, however, are not well understood. Furthermore, it is not known how the leukemic microenvironment in AML patients differs from the hematopoietic microenvironment of healthy individuals. A better understanding of leukemia will be achieved when we determine if such differences exist and importantly, how these differences could impact the microenvironment’s ability to render cells resistant to therapy and possibly also support leukemogenesis. Mesenchymal stromal cells (MSC) are a key component of the bone marrow and play a key regulatory role in the leukemic niche. We have previously shown that co-cultured MSC can alter metabolism of leukemic cell lines by a mechanism involving mitochondrial uncoupling induced by the activation of Uncoupling Protein 2, to promote the Warburg Effect (Samudio et al Cancer Research, 2008; 68: 5198–5205). However, the metabolic nature of MSC from the leukemic microenvironment has not been investigated. In the present study, analysis of 352 known metabolites was performed on MSC from normal healthy donors (N = 15) and AML-derived MSC (N = 14) using Liquid Chromatography/Mass Spectrometry (LC/MS) and Gas Chromatography/Mass Spectrometry (GC/MS) (Metabolon, Durham, NC). Results indicated that metabolites involved in signaling and anti-oxidant rather than in energy regulation showed significant differences. AML derived MSC had significantly elevated levels of both reduced and oxidized glutathione compared to healthy donor MSC. Homocysteine, 5-methyltetrahydrofolate, and opthalmate levels were higher in AML MSC which is consistent with increased glutathione synthesis. The antioxidants α- and γ-tocopherol (components of vitamin E) were also significantly increased in AML MSC. These changes in key components of cellular redox homeostasis may reflect a cellular response that leads to the upregulation of glutathione synthesis and tocopherol uptake to protect the MSC cells from a more oxidative environment. In addition to metabolic analysis, we conducted miR profiling on MSC from normal healthy donors (N = 15) and AML derived MSC (N = 28). Eighteen miRs showed significant differences in expression between the 2 groups. AML derived MSC were found to express 2.3 fold less miR-93 compared to MSC from healthy donors. Previous analysis of miRs in AML cells by our groups indicated that miR-93 levels were lower in AML blast cells compared to normal counterparts (Garzon et al Blood, 2008; 111: 3183–3189). Mir-93 is a member of the miR-106b-25 cluster that comprises a group of three miRNAs on human chromosome 7q22. A recent report by Li and colleagues (Mechanisms of Ageing and Development, 2011; 132: 75–85) demonstrated in a mouse model that the aging liver is subject to damage by oxidative stress due to increased levels of miR-93. MiR-93 targets microsomal glutathione S-transferase 1 (MGST1) and the potential increase in MGST1 and the higher abundance of gluthathione support a model in which leukemia MSC could have increased xenobiotic (chemotherapy) detoxifying capacity. Future studies will include analysis of MGST1 protein expression to determine if this enzyme is indeed elevated in AML-derived MSC. In conclusion, AML-derived MSC exhibit lower expression of miR-93 that could support production of anti-oxidant metabolites to protect the cells from oxidative stress damage in the leukemic microenvironment. Disclosures:No relevant conflicts of interest to declare.

A Plant Triterpenoid Avicin D Stimulates Adipocytic Differentiation of Bone Marrow Stromal Cells and Promotes Their Pro-Survival Effects On Acute Monoblastic Leukemia Cells

Abstract 4315A natural triterpene Avicin D induces apoptosis in various tumor cells and regulates cellular metabolism via Glucocorticoid receptor (GR) signalling, known to be involved in adipocyte differentiation (Tang, Annu Rev Biochem. 2012;81:715). Since adipocytes represent an essential component of the aging bone marrow (BM) microenvironment (Tabe, Blood 2004;103:1815), and promote monocytic differentiation and survival of monoblastic leukemia cells (Tabe, ASH, 2011), we examined the potential of Avicin D to modulate BM adipocytes and its effects on the survival of the leukemic cells.In mature adipocytes derived from BM mesenchymal stem cells (MSCs), Avicin D (1ƒE′M, 48 hrs) significantly increased the number of lipid vesicles without apoptosis induction or significant cell cycle arrest. This was associated with upregulation of adipose-specific genes, PPARG and fatty acid binding protein 4 (FABP4), and inflammation-related haptoglobin (HP) gene (p<0.005, Figure 1). Avicin D further enhanced leptin release from adipocytes and inflammatory cytokines production, IL-6 and IL-8, from both MSCs and adipocytes (Figure 2). Of note, Avicin D increased pro-survival Bcl-2 mRNA expression in adipocytes (fold increase compared to MSCs: MSCs+Avicin D 1.0±0, adipocytes 1.8±0.1, adipocytes+Avicin D 4.4±0.6, p=0.02).We next investigated the effects of MSCs and adipocytes pre-treated with 1mM Avicn D for 24 hours on co-cultured monoblastic leukemia cell line U937. Avicin D (1ƒE′M) moderately (26±5 %) inhibited cell proliferation of U937 cells cultured alone as detected by MTT assay. Serum-starvation induced cell death in U937 cells was inhibited by MCS or adipocyte co-culture, and further blocked by Avicin D-pretreated adipocytes, which was accompanied with cell cycle arrest (sub-G1 fraction; control U937 36.1±7.1%, co-culture with MSC 17.9±4.1%, MSC+Avicin D 20.9±3.9%, adipocyte 16.9±2.9, adipocyte+Avicin D 11.5±0.8%, p=0.03, G0/G1 phase; control 39.1±5.9%, with MSC 44.3±1.0%, MSC+Avicin D 40.3±1.3%, adipocyte 40.6±1.3, adipocyte+Avicin D 50.4±2.3%, p=0.03). Co-culture of U937 cells with Avicin D-pretreated adipocytes induced upregulation of PPARG and CD36, markers of monocytes/macrophages maturation in U937 cells (fold increase compared to control U937: PPARG: co-culture with adipocytes 2.1±0.4, adipocytes+Avicin D 13.8±6.2; CD36: adipocytes 2.2±0.4, adipocytes+Avicin D 4.7±2.9). Co-culture with Avicin D-pretreated adipocytes also caused 3.1 ±0.4 fold increase of BCL2 mRNA in U937 cells compared to the one of co-cultured with control adipocytes (p=0.02). The purity of U937 cells separated from adipocytes was confirmed by lack of CD90 mRNA expression by PCR. Utilization of the proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ) with two-dimensional-liquid chromatography-tandem mass spectrometry allowed the identification of 1,634 proteins. We found changes in 14 proteins in U937 cells co-cultured with Avicin D-pretreated adipocytes compared to untreated adipocytes. Among 13 down-regulated proteins, three are involved in pyruvate metabolism (Glyoxalase I; p=0.01, Lactate dehydrogenase B; p=0.02, Pyruvate kinase; p=0.03), and four proteins participate in cell cycle progression (DNA replication licensing factor MCM4, MCM5; p=0.02, p=0.03, DNA-dependent protein kinase; p=0.02, Interleukin enhancer-binding factor 3; p=0.005). Voltage-dependent anion-selective channel protein 2, which is involved in the mitochondrial apoptotic pathway via regulation of Bcl-2, and an enzymatic antioxidant Superoxide dismutase were up-regulated (p=0.04, p=0.03). These data indicate that U937 cells co-cultured with Avicin D pretreated adipocytes were induced to undergo cell cycle arrest, escape apoptosis with down-regulation of glucose metabolism.Altogether, these results suggest that Avicin D induces adipocytic differentiation of BM-derived stromal cells, promotes production of leptin and inflammatory cytokines, which in turn supports survival of monocytic leukemia cells. These findings suggest a contributory role of the aging “pro-inflammatory” bone marrow microenvironment in the reduced efficacy of cytotoxic chemotherapy which is prevalent in elderly AML patients. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Exosomes Mediate Communication Between the Microenvironment and Leukemic Cells in Acute Myeloid Leukemia

Abstract 3469It is becoming clear that the microenvironment plays a critical role in tumorigenenesis and drug resistance in cancers including Acute Myeloid Leukemia (AML). Mesenchymal stromal cells (MSC) are a key component of the bone marrow (BM) niche where AML cells reside. Our group and others have reported that MSC are critical mediators of leukemic cell engraftment and survival. Much of the emphasis on studies of the role of MSC in the microenvironment has focused on chemokines and cytokine effects especially those involving the SDF-1/CXCR4 axis. While paracrine mechanisms of MSC regulation of leukemic cells are important, in this report we suggest a novel role for MSC in altering AML cells by exosome-mediated transfer of genetic material. It is only within the last 10 years that microvesicles such as exosomes have been implicated in mammalian cell-to-cell communication. In blood biology, this is perhaps best evidenced in the regulation of antigen presenting cells by exosome containing miRs from T cells (Mittelbrunn et al Nat Commun. 2011; 2:282). At present, the role for exosomes in leukemia biology is unknown. Exosomes are derived from late endosomal processing, are enriched in ceramides, and contain proteins that are critical in membrane trafficking such as the tetraspanin CD63. To determine if MSC could transfer exosomes to AML cells, we introduced GFP-tagged CD63 into MSC. Live confocal imaging of MSC revealed that MSC can indeed secrete exosomes. The secreted exosomes are often observed in clusters, likely due to the high ceramide content of the particles. GFP-CD63 containing exosomes from MSC were purified using the Exoquick kit from System Biosciences (Mountain View, CA) and introduced to OCI-AML3 and THP-1 cells. Confocal imaging revealed exosomal uptake by only few leukemia cells; however, cells that contained the microvesicles appeared to contain clusters of these particles. Co-culture of GFP-CD63+ containing MSC with AML derived OCI-AML3 cells or KG-1 cells for 48 hours resulted in transfer of exosomes to leukemic cells as determined by analytical flow cytometry. Roughly 1.5 % of OCI-AML3 and 0.8 % of KG1 cells were found to have incorporated GFP-CD63. Though exosome transfer from MSC to AML cells appears to occur at low frequency, at least under the conditions employed here, the possibility that even a small number of leukemic cells could be altered by this mechanism could have significant ramifications. Introduction of miRs or other non-coding RNAs could profoundly modify the leukemic cells. If the primary site of residence of leukemic stem cells (LSC) is in the BM niche, then MSC could alter LSC thus creating a heterogeneous population of leukemic cells. The question arises how the BM microenvironment of a healthy individual might differ from that of an AML patient. We conducted miR profiling on MSC from normal healthy donors (N = 15) and AML derived MSC (N = 28). Eighteen miRs showed significant differences in expression between the two groups: AML derived MSC were found to express > 2 fold more miR-450b-5p, mir-382, and miR-539. On the other hand, healthy donor MSC express > 2 fold higher levels of mir-19a, miR-93, and miR-542-5p compared to MSC from AML patients. While it is not known how the exosomes of AML derived MSC differ from those of healthy donors, the difference in miR profiles between the two groups suggests that the miR content of their exosomes could vary and that exosomes from each group could have different effects. Determination of the miR content in the exosomes from each group will shed light on possible mechanisms and analysis is underway. Finally, it is also possible that MSC could use exosomes to alter the microenvironment itself. Preliminary data obtained by confocal microscopy revealed transfer of CD63+ exosomes from GFP-CD63+ MSC to MSC expressing red fluorescent protein (RFP) during co-culture conditions. At this point, the mechanism of transfer is not clear as it is possible that transfer occurs via cell-to cell contact between MSCs and/or incorporation of secreted exosomes by recipient MSC. Still, this data suggests that MSC can exchange genetic materials and that this novel mechanism could act as a plausible means of modifying the microenvironment. In conclusion, exosomal transfer represents a new mechanism in cell-to-cell communication and is active in the leukemia microenvironment with transfer from normal MSC to AML cells or other component cells of the leukemic bone marrow niche. Disclosures:No relevant conflicts of interest to declare.

The Bone Marrow Component of Vascularized Composite Allografts Combined with Costimulatory Blockade Promotes Donor-Derived Hematopoietic Stem Cell Engraftment, Chimerism and Tolerance

The Bone Marrow Component of Vascularized Composite Allografts Combined with Costimulatory Blockade Promotes Donor-Derived Hematopoietic Stem Cell Engraftment, Chimerism and Tolerance

The Bone Marrow Component of Vascularized Composite Allografts Combined with Costimulatory Blockade Promotes Donor-Derived Hematopoietic Stem Cell Engraftment, Chimerism and Tolerance

The Bone Marrow Component of Vascularized Composite Allografts Combined with Costimulatory Blockade Promotes Donor-Derived Hematopoietic Stem Cell Engraftment, Chimerism and Tolerance