Components Of Bone Marrow Niche Research Articles

Human umbilical cord blood-derived mesenchymal stem cells restored hematopoiesis by improving radiation induced bone marrow niche remodeling in rats

BackgroundFunctional hematopoiesis is governed by the bone marrow (BM) niche, which is compromised by radiotherapy, leading to radiation induced BM failure. The aim of this study was to demonstrate the radiation induced pathological remodeling of the niche and the efficacy of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in restoring hematopoiesis via improvement of the niche. MethodsThirty male Wistar rats were equally assigned to three groups: control (CON), irradiated (IR), and IR+hUCB‐MSCs. Biochemical, histopathological and immunohistochemical analyses were performed to detect collagen type III and IV, Aquaporin 1+ sinusoidal endothelial cells and immature hematopoietic cells, CD11c+ dendritic cells, Iba1+ macrophages, CD9+ megakaryocytes, Sca-1+, cKit+, CD133 and N-cadherin+ hematopoietic stem and progenitor cells, CD20+, Gr1+ mature hematopoietic cells, in addition to ki67+ proliferation, Bcl-2+ anti-apoptotic, caspase-3+ apoptotic, TNF-α+ inflammatory cells. Histoplanimetry data were statistically analyzed using the one-way analysis of variance followed by the post hoc Duncan’s test. Moreover, Pearson’s correlation was used to assess the correlation between various parameters. ResultsIn comparison to the IR group, the IR+hUCB-MSCs group showed restored cell populations and extracellular collagen components of the BM niche with significant increase in hematopoietic stem, progenitor, mature and proliferating cells, and a considerable decrease in apoptotic and inflammatory cells. Furthermore, highly significant correlations between BM niche and blood biochemical, histopathological, and immunohistochemical parameters were observed. ConclusionhUCB-MSCs restored functional hematopoiesis through amelioration of the BM niche components via reduction of oxidative stress, DNA damage, inflammation, and apoptosis with upregulation of cellular proliferation.

Critical role of Lama4 for hematopoiesis regeneration and acute myeloid leukemia progression

AbstractImpairment of normal hematopoiesis and leukemia progression are 2 well-linked processes during leukemia development and are controlled by the bone marrow (BM) niche. Extracellular matrix proteins, including laminin, are important BM niche components. However, their role in hematopoiesis regeneration and leukemia is unknown. Laminin α4 (Lama4), a major receptor-binding chain of several laminins, is altered in BM niches in mice with acute myeloid leukemia (AML). So far, the impact of Lama4 on leukemia progression remains unknown. We here report that Lama4 deletion in mice resulted in impaired hematopoiesis regeneration following irradiation-induced stress, which is accompanied by altered BM niche composition and inflammation. Importantly, in a transplantation-induced MLL-AF9 AML mouse model, we demonstrate accelerated AML progression and relapse in Lama4−/− mice. Upon AML exposure, Lama4−/− mesenchymal stem cells (MSCs) exhibited dramatic molecular alterations, including upregulation of inflammatory cytokines that favor AML growth. Lama4−/− MSCs displayed increased antioxidant activities and promoted AML stem cell proliferation and chemoresistance to cytarabine, which was accompanied by increased mitochondrial transfer from the MSCs to AML cells and reduced reactive oxygen species in AML cells in vitro. Similarly, we detected lower levels of reactive oxygen species in AML cells from Lama4−/− mice post–cytarabine treatment. Notably, LAMA4 inhibition or knockdown in human MSCs promoted human AML cell proliferation and chemoprotection. Together, our study for the first time demonstrates the critical role of Lama4 in impeding AML progression and chemoresistance. Targeting Lama4 signaling pathways may offer potential new therapeutic options for AML.

The bone marrow niche components are adversely affected in sepsis

Multiple organ dysfunction is an important cause of death in patients with sepsis. Currently, few studies have focused on the impact of sepsis on bone marrow (BM), especially on the cell components of BM niche. In this study, we performed mouse sepsis models by intraperitoneal injection of LPS and cecal ligation and puncture (CLP). The changes of niche major components in the mouse BM among vascular structures, mesenchymal stem cells and Treg cells were observed and analyzed. The results showed that pathological changes in BM was earlier and more prominent than in other organs, and various cell components of the BM niche changed significantly, of which vascular endothelial cells increased transiently with vascular remodeling and the regulatory T cells decreased over a long period of time. These results indicated that the components of the BM niche underwent series of adaptive changes in sepsis.

Osteogenic/Adipogenic Imbalance in Bone Marrow Nestin+ mesenchymal Stem Cells in Patients with Chronic Graft-Versus-Host Disease

Background: Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The roles of bone marrow niche in cGVHD pathogenesis have gained increasing attention in recent years. Nestin+ mesenchymal stem cells (MSCs) is an important component of bone marrow niche. However, the potential implication of Nestin+ MSCs in the pathophysiology of cGVHD has not been completely clarified. Methods: A total of 68 patients with hematologic malignancies who underwent allo-HSCT at Nanfang Hospital between April 2016 and October 2018 were enrolled in this experimental study. We analyzed expanded Nestin+ MSCs from patients with cGVHD and compared them with patients without cGVHD. The diagnosis and grade of cGVHD was made at the time of sample collection according to National Institutes of Health criteria (Howard M.S. et. al. BBMT 2015). The results were compared using one-way ANOVA and unpaired two-tailed Student t-test. Statistical significance was defined as P value of <.05. Result: The Nestin+ MSCs from both groups showed similar morphology, immunophenotype, proliferation, and apoptosis. However, the adipogenic differentiation capacity of Nestin+ MSCs in patients with cGVHD was significantly reduced compared with patients without cGVHD (relative expression of PPARγ 2.22±0.27, and 6.82±0.87, respectively, P<0.05). The osteogenic differentiation capacity was significantly increased in patients with cGVHD (relative expression of RUNX2 3.84±0.38, and 1.95±0.52, respectively, P<0.05)(Figure 1A,B). These abnormal differentiations were more significant in patients with moderate/severe cGVHD. Furthermore, β-catenin phosphorylation decreased and nuclear β-catenin increased in the Nestin+ MSCs of cGVHD patients(Figure 1 C). Conclusion: These results demonstrate that Nestin+ MSCs from cGVHD patients had abnormal differentiation characterized by decreased adipogenic differentiation capacity and enhanced osteogenic capacity. The reduction of phosphorylation of β-catenin play an important role in these abnormal differentiations. Disclosures No relevant conflicts of interest to declare.

Upregulation of Pro-inflammatory Cytokine Genes by Parvovirus B19 in Human Bone Marrow Mesenchymal Stem Cells.

Chronic inflammation plays a prominent role in cancer initiation and development. On the other hand, the Inflammation can be established by a number of factors such as viral infections. Parvovirus B19 (B19V) is a pathogen with widespread infection, which infects bone marrow erythroid progenitor cells. It has been shown that B19V can also enter human bone marrow mesenchymal stem cells (BM-MSCs). In this study, we hypothesized that BM-MSCs as the main cellular component of bone marrow niche may be induced to secret pro-inflammatory cytokines after B19V infection. BM-MSCs were cultured up to passage 3. The cells were then subjected to nucleofection to transfer a plasmid containing B19V genome. After 36h, total RNA was extracted and the expression levels of IL-1β, IL-6, TNF-α and NF-κB genes were examined using qRT-PCR. Data analysis showed the significant increase in expression levels of all studied genes in the B19V-transfected cells (P < 0.05). Although further researches are required, our findings for the first time suggest the importance of B19V infection to establish an inflammatory microenvironment in the bone marrow and its involvement in inflammation-related diseases. Finally, based on our results, molecular assay to diagnose B19V infection of BM-MSCs prior to stem cell therapy is strongly recommended.

Role of Vascular Niche of Bone Marrow in the Development of MLL-AF9 Induced Acute Myclaid Leukemia

To investigate the role of bone marrow vascular niche in the development of MLL-AF9 acute myeloid leukemia (AML). Transplantation experiments were performed to establish non-radiated MLL-AF9 AML model; the half-bone immunofluorescence staining and tile-scan imaging of two-photon confocal microscopy were used to obtain the data of 3 main bone marrow niche cells; flow cytometry analysis was performed to characterize leukemia cells in different anatomical sites. In the early stage of MLL-AF9 AML, the proportion of leukemia cells in the metaphysis of the femur was significantly higher than that in the diaphysis. The detection of apoptosis and proliferation rate of leukemia cells showed that the percentage of leukemia cells in metaphysis significantly decreased, and the proliferation (S/G2/M phase) was also significantly more active. These different features of leukemia cells may relate with different bone marrow microenvironment. The image data of 3 major components of bone marrow niche (endothelial cells, endosteum, megakaryocytes) showed different distribution of blood vessels in metaphysis and diaphysis. Furtherly comparing the spatial distance between leukemia cells and endothelial cells, endosteum, megakaryocytes indicated that leukemia cells are closer to the blood vessels, suggesting the important role of blood vessels in the development of leukemia. Glucose uptake assays and intracellular ROS detection showed that the supportive role of blood vessels for leukemia cells did not related with nutrient metabolism pathway. The vascular niche plays an important role in the development of leukemia, and does not relate with the transport of nutrients and the elimination of metabolic waste, instead, which may relate with perivascular cytokines or other vascular functions.

Microvesicles of osteoblasts modulate bone marrow mesenchymal stem cell-induced apoptosis to curcumin in myeloid leukemia cells.

Microvesicles (MVs) derived from bone marrow niche components have an important role in genetic reprogramming and subsequent drugs induce apoptosis in leukemic cells. Here, we have found that undertreatment of curcumin or daunorubicin, the cross-talk through MVs of KG-1-bone marrow mesenchymal stem cells (BMSCs), significantly downregulates the expression of the survival geneosteopontin (OPN), CXCL-12, IL-6 (interleukin-6), STAT-3, and VCAM-1 (vascular cell adhesion molecule 1) in treated-KG-1 cells as well as exclusively upregulates CXCL-12 in BMSCs. Drug treated-cell populations' MVs of both single cultured osteoblasts (OBs) and cocultured KG-1 + BMSCs + OBs similarly upregulate survival mediators' OPN, CXCL-12, IL-6, STAT-3, and VCAM-1 in treated-KG-1 cells. Likewise, isolated MVs from KG-1 cells or communication between KG-1, BMSCs, and OBs treated by drugs increase the expression of genes OPN, CXCL-12, IL-6, STAT3, and VCAM-1 by OBs. MVs derived from KG-1 + BMSCs + OBs reducedrug-induced apoptosisin KG-1 cells. Thissuggests MVs-mediated information transfer is a procedure whereby OBs could overcome BMSCs-induced apoptosis in drug-treated-KG-1 cells.

A computational model of feedback-mediated hematopoietic stem cell differentiation in vitro.

Hematopoietic stem cells (HSCs) play an important physiological role as regulators of all blood and immune cell populations, and are of clinical importance for bone marrow transplants. Regulating HSC biology in vitro for clinical applications requires improved understanding of biological inducers of HSC lineage specification. A significant challenge for controlled HSC expansion and differentiation is the complex network of molecular crosstalk between multiple bone marrow niche components influencing HSC biology. We describe a biology-driven computational approach to model cell kinetics in vitro to gain new insight regarding culture conditions and intercellular signaling networks. We further investigate the balance between self-renewal and differentiation that drives early and late hematopoietic progenitor populations. We demonstrate that changing the feedback driven by cell-secreted biomolecules alters lineage specification in early progenitor populations. Using a first order deterministic model, we are able to predict the impact of media change frequency on cell kinetics, as well as distinctions between primitive long-term HSCs and differentiated myeloid progenitors. Integrating the computational model and sensitivity analyses we identify critical culture parameters for regulating HSC proliferation and myeloid lineage specification. Our analysis suggests that accurately modeling the kinetics of hematopoietic sub-populations in vitro requires direct contributions from early progenitor differentiation along with the more traditionally considered intermediary oligopotent progenitors. While consistent with recent in vivo results, this work suggests the need to revise our perspective on HSC lineage engineering in vitro for expansion of discrete hematopoietic populations.

Engineered humanized bone organs maintain human hematopoiesis in vivo

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) environment, the so-called HSC niche, that provides pivotal factors for their maintenance. Although the cellular and molecular components of the mouse BM HSC niche have been extensively studied using genetically modified animals, relatively little is known about the counterpart human BM niche components. We previously illustrated, with a developmental tissue engineering approach, that human adult BM-derived mesenchymal stromal cells (MSCs) can develop into human bone organs (so-called ossicles) through endochondral ossification in vivo and that these human ossicles are able to maintain functional mouse HSCs. We here report that human ossicles in immunodeficient mice maintain human immature and mature hematopoiesis in vivo. Moreover, a higher percentage of human stem and progenitor cells are kept in quiescence in human ossicles as compared with mouse BM. These findings indicate that the human MSC-derived ossicles function as a hematopoietic niche and may potentially serve as a re-engineerable platform to study normal and diseased human hematopoiesis in a physiologically optimized environment.

G-CSF-induced sympathetic tone provokes fever and primes antimobilizing functions of neutrophils via PGE2

AbstractGranulocyte colony-stimulating factor (G-CSF) is widely used for peripheral blood stem/progenitor mobilization. G-CSF causes low-grade fever that is ameliorated by nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting the activation of arachidonic acid (AA) cascade. How G-CSF regulated this reaction was assessed. G-CSF treatment in mice resulted in fever, which was canceled in prostaglandin E synthase (mPGES-1)-deficient mice. Mobilization efficiency was twice as high in chimeric mice lacking mPGES-1, specifically in hematopoietic cells, suggesting that prostaglandin E2 (PGE2) from hematopoietic cells modulated the bone marrow (BM) microenvironment. Neutrophils from steady-state BM constitutively expressed mPGES-1 and significantly enhanced PGE2 production in vitro by β-adrenergic stimulation, but not by G-CSF, which was inhibited by an NSAID. Although neutrophils expressed all β-adrenergic receptors, only β3-agonist induced this phenomenon. Liquid chromatography–tandem mass spectrometry traced β-agonist-induced PGE2 synthesis from exogenous deuterium-labeled AA. Spontaneous PGE2 production was highly efficient in Gr-1high neutrophils among BM cells from G-CSF-treated mice. In addition to these in vitro data, the in vivo depletion of Gr-1high neutrophils disrupted G-CSF-induced fever. Furthermore, sympathetic denervation eliminated both neutrophil priming for PGE2 production and fever during G-CSF treatment. Thus, sympathetic tone-primed BM neutrophils were identified as one of the major PGE2 producers. PGE2 upregulated osteopontin, specifically in preosteoblasts, to retain progenitors in the BM via EP4 receptor. Thus, the sympathetic nervous system regulated neutrophils as an indispensable PGE2 source to modulate BM microenvironment and body temperature. This study provided a novel mechanistic insight into the communication of the nervous system, BM niche components, and hematopoietic cells.

Mesenchymal Stromal Cells, Instigator or Suppressor for the Development of MLL-AF9 Induced Acute Myeloid Leukemia?

It has been reported that bone marrow (BM) hematopoietic niche is critical for the maintenance of normal hematopoiesis and the development of hematopoietic disorders. However, little is known about how the different BM niche components contribute to the initiation and progression of acute myeloid leukemia (AML). To explore the role of BM stromal cells in the development of AML, we here characterized the BM stromal cells in AML mouse model induced by transplantation of mouse BM cellstransduced with MLL-AF9 oncogene. We subdivided the BM stromal cells (CD45-TER119-) into mesenchymal stem cells (MSCs, CD31-CD44-CD51+SCA1+), mesenchymal progenitor cells (MPCs, CD31-CD44-CD51+SCA1-), and endothelial cells (CD31+) in the mice after development of AML by multicolor fluorescent activated cell sorting (FACS) (Qian et al., Mol Cell Biol, 2013). We found significantly increased frequencies of BM MSCs (p < 0.0001), MPCs (p < 0.0001), and endothelial cells (p < 0.0001) in the AML mice, indicating an AML-induced alteration of BM stromal cell composition. The increased frequency of MSCs in AML mouse BM was confirmed by the increased number of colony foming unit-fibroblasts in the AML mice (p < 0.0001). Furthermore, in vitro multi-lineage differentiation assay revealed increased adipogenic and osteogenic differentiation capacities of the BM MSCs in the AML mice. Consistent with the increased osteogenic differentiation capacity of AML MSCs, Runx2 mRNA expression was increased in freshly sorted AML MSCs compared to that in the control mice. In addition, quantitative real time PCR on freshly sorted BM MPCs showed a dramatic downregulation of the genes important in hematopoietic stem cell regulation, including angiopoietin like 1 (Angptl1), C-X-C motif chemokine (Cxcl12), and kit ligand (Kitl) in the AML mice. The altered gene expression might contribute to the AML progression.To specifically determine the contribution of MSCs to AML development, we analyzed the MSC population expressing Early B cell factor 2 (Ebf2) (Qian et al., Mol Cell Biol, 2013) by establishing the AML in Ebf2-Egfp transgenic recipient mice. We detected an increased frequency of Ebf2+ cells in the recipient BM after development of AML (p = 0.0018). In addition, by using the triple transgenic Tg(Ebf2-Egfp x Ebf2-CreER x Rosa26-loxpStoploxp-Tomato) mouse model, where Ebf2+ MSCs and their progenies can be traced by tomato fluorescent protein after tamoxifen treatment, we found that the MSCs and MPCs generated from the Ebf2+ MSCs were also increased in the AML mouse BM (p < 0.05).To further investigate the functional contribution of Ebf2+ MSCs to the AML progression, we took advantage of the transgenic Tg(Ebf2-CreER x Rosa26-loxpStoploxp-DTA) mouse model allowing specific deletion of the Ebf2-expressing MSCs. Strikingly, we observed an accelerated initiation of AML, illustrated by the shorter survival of the MSC depleted recipients (Figure 1), compared to the non-depleted recipients, suggesting that Ebf2+ MSCs could suppress AML progression.Taken together, our data show that BM stromal cell composition and function are altered in the mice with AML and the BM MSCs may act to suppress the progression of the MLL-AF9 induced AML. These findings provide new evidence for the niche contribution to the development of AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Increased phosphorylation of eIF2α in chronic myeloid leukemia cells stimulates secretion of matrix modifying enzymes.

Recent studies underscore the role of the microenvironment in therapy resistance of chronic myeloid leukemia (CML) cells and leukemia progression. We previously showed that sustained mild activation of endoplasmic reticulum (ER) stress in CML cells supports their survival and resistance to chemotherapy. We now demonstrate, using dominant negative non-phosphorylable mutant of eukaryotic initiation factor 2 α subunit (eIF2α), that phosphorylation of eIF2α (eIF2α-P), which is a hallmark of ER stress in CML cells, substantially enhances their invasive potential and modifies their ability to secrete extracellular components, including the matrix-modifying enzymes cathepsins and matrix metalloproteinases. These changes are dependent on the induction of activating transcription factor-4 (ATF4) and facilitate extracellular matrix degradation by CML cells. Conditioned media from CML cells with constitutive activation of the eIF2α-P/ATF4 pathway induces invasiveness of bone marrow stromal fibroblasts, suggesting that eIF2α-P may be important for extracellular matrix remodeling and thus leukemia cells-stroma interactions. Our data show that activation of stress response in CML cells may contribute to the disruption of bone marrow niche components by cancer cells and in this way support CML progression.

Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells.

Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Using next-generation sequencing we show that human osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of human umbilical cord blood-derived CD34+ HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34+ cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and successfully engraft in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders.

Prostaglandin E2 Signaling through EP4 Receptor Promotes Hematopoietic Stem Cell Niche Regeneration and Enhances Hematopoietic Recovery

High-dose radiation treatment for hematological malignancies such as leukemia, lymphoma or multiple myeloma induces damage to the bone marrow microenvironment that limits hematopoietic regeneration. We previously demonstrated that single administration of dimethyl prostaglandin E2 (dmPGE2) at 24 hrs post lethal irradiation (796 cGys; LD70/30) in mice increases survival and accelerates hematopoietic recovery. Since bone marrow niches regulate hematopoiesis, we investigated whether the effects of PGE2 administration on hematopoietic recovery and survival were dependent on PGE2 signaling in bone marrow stromal cells. Total body irradiation (TBI) severely disrupts bone marrow niche components including MSC, osteoblasts and endothelial cells. Multivariate flow cytometry revealed that exposure of mice to 650 cGy TBI reduced the total number of bone marrow MSC defined as CD45- Ter119- CD31- PDGFR+ CD51+ MSC by 8 fold and reduced functional fibroblast colony formation (CFU-F) by 8.5 fold. Osteoblast (OB) and endothelial cell (EC) counts were reduced by 6.5 fold and 6 fold respectively. Treatment of mice with dmPGE2 at 24 hours post-irradiation substantially rescued MSC and EC, which were 3.5 and 2.2-fold higher compared to un-treated irradiated mice. Histological and flow cytometric analysis indicated that total OB, OB precursors and mesenchymal progenitor cells (MPC) in bone marrow were also enhanced by PGE2 administration. Interestingly, dmPGE2 failed to rescue MSC and OB from aged (24-27 mo/old) mice compared to young (4-6 mo/old) mice. Since PGE2 signals through four receptors (EP1-4), each with unique signaling pathways, we hypothesized that the hematopoietic niche regeneration was due to activation of PGE2 signaling via one or more EP receptors. Treatment of mice with the EP4 receptor agonist L-902,688 following irradiation enhanced the recovery of bone marrow MSC by 3.1 fold and EC by 2.0 fold compared to untreated irradiated control mice. EP1, EP2 and EP3 receptor agonists failed to enhance hematopoietic niche regeneration in irradiated mice. To further investigate the role of EP4 signaling in bone marrow niche reconstruction and hematopoietic recovery, we transplanted BM cells from wild-type mice into syngeneic wild-type or conditional inducible EP4-/- recipients after lethal irradiation, and analyzed stromal cells recovery and hematopoietic reconstitution. At 15 days post-transplantation, chimeric mice with EP4-/- stroma displayed attenuated niche recovery and hematopoietic reconstitution compared to mice with wild-type stroma. PGE2 or EP4 agonist administration increased the expression of the endogenous anti-apoptotic protein Survivin, and enhanced survival of MSC and EC. In conclusion, our study suggests that PGE2 signaling through the EP4 receptor increases hemotopoietic recovery after TBI by enhancing the survival and expansion of MSC and endothelial cells. Furthermore, our data suggest the modulation/regulation PGE2 signaling in hematopoietic niche components could be beneficial to enhance HSC recovery following clinical hematopoietic transplantation. Disclosures No relevant conflicts of interest to declare.

Abstract 983: Gas6/Axl mediation of prostate cancer cell dormancy requires cooperation with additional bone marrow niche components

Abstract Early metastatic events are often responsible for the recurrence and lethality of prostate cancer. The most frequent site of metastasis is the bone marrow, where small populations of disseminated tumor cells (DTCs) preferentially take residence in the hematopoietic stem cell (HSC) niche. These micro-metastases may survive in a benign, dormant state for years before they become “reawakened” to form full metastases, and are thus difficult to detect and treat. It is unknown what induces and maintains DTC dormancy, and it remains a key question with regard to metastatic prostate cancer treatment and prevention strategies. An essential component of the HSC niche is the osteoblast population to which DTCs bind for support and uptake of secreted factors. We have previously shown that dormant DTCs in the HSC niche display high levels of the receptor tyrosine kinase Axl, and are bound to osteoblasts that secrete its ligand, Gas6. The requirement of Gas6/Axl in DTC dormancy is striking in vivo, but in vitro is not replicable. Addition of Gas6 to prostate cancer cells expressing Axl does not have a significant inhibitory effect on cell proliferation, suggesting the cooperation with other niche factors absent in culture. As the elements of the DTC niche are still being actively explored, this study further supports the complex nature of the mechanisms governing DTC dormancy and preferential survival in the bone marrow niche. Selective targeting of these elements may allow for long term maintenance of the benign, dormant state of DTCs, or coerced reawakening to a more chemotherapy-sensitive state. Citation Format: Haley Axelrod, Kenneth J. Pienta. Gas6/Axl mediation of prostate cancer cell dormancy requires cooperation with additional bone marrow niche components. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 983. doi:10.1158/1538-7445.AM2015-983

Cathecholamines Differently Regulate Human AML and Normal Hematopoietic Progenitor Cell Motility Via miR126 and RGS16

Abstract Introduction Motility, proliferation, bone marrow (BM) retention and egress to the circulation of hematopoietic stem and progenitor cell (HSPCs) are key elements in normal hematopoiesis and also in the pathogenesis of acute myeloid leukemia (AML). HSPCs are tightly regulated in the BM niche by various molecules. Among them, CXCL12 and its major receptor CXCR4, expressed by both HSPCs and BM niche components, govern HSPC retention in the BM. Administration of CXCR4 antagonists lead to mobilization of both normal HSPCs and leukemia-inducing stem cells (LICs) from the BM to the circulation. We have previously found that normal human HSPCs functionally express b2-adrenergic receptors (b2-AR) that are up-regulated by myeloid cytokines such as G-CSF. The catecholaminergic neurotransmitters epinephrine and norepinephrine (NE) activate both Wnt and GSK3β signaling pathways via b2-AR, leading to enhanced HSPC proliferation, motility and BM repopulation (Spiegel et al, Nat Immunol 2007; Lapid et al, JCI 2013). These findings indicate HSPC regulation by dynamic interactions of the sympathetic nervous and hematopoietic systems. However, the role of catecholamines in regulation of AML remains elusive. Results We found that several human AML cell lines from different FAB subtypes express b2-AR. NE, a b2-AR activating ligand, increased b2-AR expression and the CXCL12-induced migration of AML primary patients’ cells and cell lines. In addition, NE treatment significantly enhanced CXCL12induced actin polymerization, which drives most of the cellular movements. Looking for a downstream effector of b2-AR, we focused on RGS16, a G-protein signaling regulator, which negatively regulates CXCL12/CXCR4 axis (Berthebaud et al., Blood 2005). Concurrently with the increase in cell migration, NE decreased RGS16 expression (both in protein and mRNA level) in monocytic AML cells. However, no effect on either cell migration or RGS16 expression was observed in non-monocytic AML cells. These data provided evidence for the involvement of catecholamines in the regulation of AML cell migration and showed a correlation between AML FAB subtypes, cell motility and RGS16 expression. One of the regulators of RGS16 levels is miR126, which is highly expressed in AML and normal HSPCs and plays an important role in mobilization and proliferation of normal HSPCs. Indeed, in search for the mechanisms underlying the above observed differences, we found that the enhancing effect of NE on CXCL12-induced migration of monocytic AML cells was accompanied by up-regulation of miR126 expression concurrently with down-regulation of RGS16 expression, whereas in non-monocytic AML cells we observed the opposite effects, suggesting that NE differently regulates AML cells belonging to different FAB subtypes. Upon studying human normal HSPCs, we found that in steady state, normal cells express low levels of β2-AR and NE did not affect either RGS16 expression or CXCL12-induced migration of both mononuclear and CD34+ cells derived from human cord blood and BM. Conclusions Our results demonstrate that while normal and AML cells share common mechanisms that govern their motility, there are unrevealed yet mechanisms, apparently cell-type dependent, which uniquely lead to opposite effects in normal HSPCs, monocytic and non-monocytic AML cells. Altogether, these findings suggest that targeting of miR126 and RGS16 pathways by specific agonists and antagonists may serve as a new approach for selective eradication of LICs. Disclosures: No relevant conflicts of interest to declare.

Defined Bone Marrow Niche Components Mediate the In Vitro Resistance of Acute Myeloid Leukaemic Stem and Progenitor Cells (LSPC) to Cytosine Arabinoside.

The majority of Acute Myeloid Leukaemia (AML) patients respond to remission-induction chemotherapy, but many patients will suffer disease relapse following induction therapy due to the presence of minimum residual disease (MRD) cells in the bone marrow. Relapse is underpinned by outgrowth of leukaemic stem and progenitor cells (LSPC). We aimed to establish a methodology to distinguish normal from leukaemic SPC, to optimise maintenance of the LSPC phenotype in 48 hour culture and to quantify viable LSPC following culture with and without drugs. The CD34+ CD38− CD123+ high phenotype was used to distinguish LSPC. CD123 fluorescence intensity was measured using fluorescence standards. Miltenyi CD 133 and CD34 coated beads were compared as enrichment strategies. The concentration of LSPC at the start of 48h culture ranged from 0.2 – 16 × 104/ml (median 3.4 × 104/ml). We compared various culture conditions aimed at maintaining these cells in culture without differentiation (i.e. without loss of phenotype), including serum, immobilized fibronectin, SCF, IL-3, IL-6, GM-CSF and angiopoietin-1. We used two flow cytometric assays in parallel for analysis of LSPC survival: in the first assay, viable bulk cells were enumerated using the dye 7-AAD along with an internal standard for cell counting. In the second assay, cells were labelled with CD34FITC, CD123PE, 7-AAD and CD38APC in order to quantify LSPC as a percentage of viable cells. We found that serum-free culture medium, fibronectin-coated wells and a cocktail of IL-3, IL-6, SCF, and angiopoietin-1 was the most successful at maintaining the concentration of CD34+ CD38− CD123+ cells in culture for 48h (median 0% change), although there was considerable variation between samples. We then examined the effect of these niche conditions on the sensitivity of LSPC to cytosine arabinoside (Ara-C). Our data indicates that the survival of LSPC treated with 500 ng/ml Ara-C was 50 per cent of untreated cell survival in the presence of fibronectin and cytokine support (n=9), but 33% per cent of untreated cells survived without this support. Moreover, under these conditions LSPC were less sensitive to Ara-C (median 50% survival, n=10) than unselected cells (median 26% survival). We have defined a system for assessing the in vitro chemosensitivity of LSPC from AML samples to anti-leukaemic agents and have validated the assay by showing that phenotypically-identified LSPC as well as niche components fibronectin and cytokines mediate in vitro resistance to cytosine arabinoside.