Bone Marrow Resident Cells Research Articles

Abstract 013: Genetic Ablation of Mas Receptor Impairs Mobilization of Bone Marrow Progenitor Cells

Angiotensin (Ang)-(1-7)/Mas receptor (MasR) pathway accelerates vascular repair in ischemic conditions partly by stimulating the mobilization of vascular reparative bone marrow progenitor cells (BMPCs) into blood circulation. This study tested if the endogenous MasR expression is required for the mobilization of BMPCs in response to ischemic injury. Hind limb ischemia (HLI) was induced in wild type (WT) or MasR knock out mice (MasR-KO) (in C57Bl/6J background). BMPCs in the blood circulation were quantitated by flow cytometric enumeration of Lineage - , Sca-1 + and cKit + (LSK) cells in peripheral blood or by colony forming unit (CFU) assay. Subcutaneous osmotic pumps were used for continuous infusion of Ang-(1-7) at the rate of 1 μg/kg/min for four weeks. In vitro migration of LSK cells in response to hypoxia-regulated factors, stromal-derived factor (SDF) or by vascular endothelial growth factor (VEGF) were determined. In WT mice, HLI stimulated mobilization of LSK cells that reached maximum by day 2 (110±11 cells/mL blood, n=6). Ang-(1-7)-treatment potentiated the peak mobilization (206±24 cells/mL blood, n=8, P<0.01 compared to the untreated). MasR-KO mice have reduced number of circulating LSKs (12±3 vs 43±9 per mL blood in WT, P<0.01, n=5) (CFUs/mL blood 28±5 vs 54±8 in WT, P<0.05, n=5). In MasR-KO mice, HLI did not induce mobilization, and blood flow recovery post-HLI was lower compared to WT (52±4% vs 89±6% in WT, P<0.001, n=5), both of which were not improved by treatment with Ang-(1-7). Number of bone marrow-resident LSK cells was higher in MasR-KO mice compared to WT. Migration induced by SDF (84±6% vs 160±8% in WT, P<0.001, n=5) or VEGF (97±4% vs 146±5% in WT, P<0.001, n=4) was decreased in MasR-KO. These results suggest that MasR deficiency causes impaired mobilization of BMPCs likely by decreasing their sensitivity to hypoxia-regulated factors. Therefore endogenous MasR expression is essential for ischemia-dependent mobilization of BMPCs.

Characterization of a novel bone marrow resident cell population with the potential to regulate early hematopoiesis and skeletogenesis.

Abstract Hematopoiesis and skeletogenesis are tightly regulated with participation of cells from both systems. The main hematopoietic component is the osteoclast, a cell crucial for bone remodeling. However, beyond bone homeostasis, other hematopoietic cells may exist as regulators of hematopoiesis within the bone marrow (BM) microenvironment. Histological analyses of mice expressing cre under the control of Dental Matrix Protein-1 (Dmp-1) promoter and crossed with the reporter strain Ai9, showed a fluorescent (tomato+) dendritic cell–like population interspersed within the BM space. These tomato+ cells were isolated, and showed efficient expression of RANK-L and IL-7, suggesting that they could regulate hematopoiesis. These cells also express surface markers of myeloid lineage including: CD11b, F4.80, CD169, and Ly-6C. In vitro hematopoietic progression assays failed on generating these cells from isolated hematopoietic stem cells. However, they can be generated and expanded ex vivo from CD11b+ tomato negative cells. Furthermore, using a tamoxifen inducible Dmp-1/tomato mouse, hematopoietic tomato+ cells appeared 2 days after tamoxifen treatment. In a transgenic mouse bearing the tamoxifen inducible Dmp-1/tomato plus a transgene allowing the ablation of bone formation after injecting ganciclovir; bone ablation for 16 days followed by tamoxifen two days prior to analysis, showed a compromise in the generation of tomato+ hematopoietic cells indicating that their generation is linked to bone formation. Finally, these cells exist in the BM of late gestation embryos and neonates suggesting a possible fetal origin. Currently we are addressing their phagocytic function and their ability to influence hematopoietic progression.

Age-related changes in the transcriptome of antibody-secreting cells.

We analyzed age-related defects in B cell populations from young and aged mice. Microarray analysis of bone marrow resident antibody secreting cells (ASCs) showed significant changes upon aging, affecting multiple genes, pathways and functions including those that play a role in immune regulation, humoral immune responses, chromatin structure and assembly, cell metabolism and the endoplasmic reticulum (ER) stress response. Further analysis showed upon aging defects in energy production through glucose catabolism with reduced oxidative phosphorylation. In addition aged B cells had increased levels of reactive oxygen-species (ROS), which was linked to enhanced expression of the co-inhibitor programmed cell death (PD)-1.

Activation of the polycomb repressive complex pathway in the bone marrow resident cells of diffuse large B-cell lymphoma patients

The present study investigated the activation of polycomb repressive complex 2 (PRC2) pathway proteins in the resident cells within the bone marrow hematopoietic microenvironment of diffuse large B-cell lymphoma (DLBCL) patients. PRC2 proteins (enhancer of zeste homolog 2, suppressor of zeste 12 homolog, and embryonic ectoderm development), histone methylation mark (H3K27me3), and c-MYC activation were evaluated in pretreatment bone marrow from 208 DLBLC patients. Positive expression of the PRC2, H3K27me3, and c-MYC in the bone marrow resident cells was more frequent in cases with bone marrow involvement of tumor. The expression among PRC2, H3K27me3 mark, and c-MYC was closely correlated. Positive PRC2 expression in bone marrow resident cells was significantly associated with inferior progression-free survival (PFS) and overall survival (OS) and determined to be an independent prognostic factor of inferior PFS and OS. In conclusion, the PRC pathway was frequently activated in bone marrow resident cells of DLBCL patients, and PRC activation was tumor-related and associated with poor clinical outcomes.

Regulation of Hematopoiesis By the Coagulation Receptor Thrombomodulin

BACKGROUND: Pharmacologic supplementation of protein C pathway function by infusion of recombinant Thbd or activated protein C supports recovery of hematopoietic function from lethal radiation injury in mice [Geiger et al., Nature Medicine, 2012]. Partial Thbd deficiency in hematopoietic stem and progenitor cells (HSPC) or bone marrow endothelium results in augmented sensitivity towards radiation injury [Geiger et al., Nature Medicine, 2012]. The underlying cellular and molecular mechanisms of Thbd function in hematopoiesis are not yet characterized. The objective of the current study was to determine the expression pattern and functional role of Thbd in HSPC.RESULTS: Flow cytometric analysis was employed to detect Thbd expression in defined subsets of murine HSPC. Thbd was co-expressed with the endothelial protein C receptor (Procr/EPCR) in the majority of bona fide stem cells with long-term-repopulating capacity (LT-HSC), and was also expressed on EPCR-negative stem cells with short-term repopulating capacity (ST-HSC), multipotent progenitors (MPP), common lymphoid progenitors (CLP), common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP). In contrast, only a subset of megakaryocyte-erythrocyte progenitors (MEP) expressed low levels of Thbd. In the bone marrow, Thbd was also expressed by B cells in early stages of maturation (from progenitor B cell stage till immature stage). In this lineage, the fraction of Thbd-positive cells was inversely correlated with the stage of B cell maturation. In addition, Thbd was detected in three distinct subsets of bone marrow-resident myeloid cells (CD11b+CD115+, CD11b+CD11c+ and CD11b-CD115+). Thbd expression outside the bone marrow was limited to a small fraction of hematopoietic cells (2-5% in the peripheral blood and spleen). These cells included myeloid cells (macrophage/monocyte and dendritic cells). Approximately 1-2% of all B cells in the peripheral blood and the spleen expressed Thbd, possibly reflecting recent bone marrow emigrants. Thbd expression was largely absent from splenic follicular and marginal zone B cells.Adult mice with complete, ubiquitous ablation of Thbd gene function (Meox2Cre-ThbdloxP -mice; "Thbd-null") were generated to analyze the functional role of Thbd in hematopoiesis. Thbd-null mice exhibited low birth weight, but only a mild prothrombotic diathesis, reflected in occasional peripheral vascular occlusion limited to the tail vein. Flow cytometric analyses revealed increased frequency of LT- and ST-HSC, a trend towards reduced CLP frequency, but normal relative abundance of MPP, CMP, GMP, and MEP. Thbd deficiency was also associated with a significant increase in the absolute number of LT-HSC and a reduction in the absolute number of CLP in the bone marrow. No such derangements were observed in mice lacking EPCR. In functional assays, bone marrow from wildtype and mutant mice yielded comparable numbers of CFU-GM. In contrast, the number of CFU-GM was increased in the spleen and peripheral blood of Thbd-null animals. The absolute number and frequency of all B cell precursors, as well as mature B cells in the bone marrow was reduced by ~50%. In the spleen, the absolute number of B cells was increased, whereas other hematopoietic populations in peripheral organs of Thbd-null mice were identical to that of wildtype controls.CONCLUSION: Thbd is expressed in the majority of hematopoietic progenitor cells in the bone marrow, including LT- and ST-HSC, and B cell precursors. Despite abundant Thbd expression in bone marrow resident cells and a modest prothrombotic phenotype, complete Thbd deficiency had only mild effects on steady-state hematopoiesis. Hematopoietic derangements were limited to the B cell compartment, and an Increased presence of CFU-GM in the spleen and peripheral blood of Thbd-null mice, possibly reflecting stimaulation of extramedullary hematopoiesis and/or altered bone marrow retention of precursors. DisclosuresNo relevant conflicts of interest to declare.

Targeting bone marrow lymphoid niches in acute lymphoblastic leukemia

In acute lymphoblastic leukemia (ALL) the bone marrow microenvironment provides growth and survival signals that may confer resistance to chemotherapy. Granulocyte colony-stimulating factor (G-CSF) potently inhibits lymphopoiesis by targeting stromal cells that comprise the lymphoid niche in the bone marrow. To determine whether lymphoid niche disruption by G-CSF sensitizes ALL cells to chemotherapy, we conducted a pilot study of G-CSF in combination with chemotherapy in patients with relapsed or refractory ALL. Thirteen patients were treated on study; three patients achieved a complete remission (CR/CRi) for an overall response rate of 23%. In the healthy volunteers, G-CSF treatment disrupted the lymphoid niche, as evidenced by reduced expression of CXCL12, interleukin-7, and osteocalcin. However, in most patients with relapsed/refractory ALL expression of these genes was markedly suppressed at baseline. Thus, although G-CSF treatment was associated with ALL cell mobilization into the blood, and increased apoptosis of bone marrow resident ALL cells, alterations in the bone marrow microenvironment were modest and highly variable. These data suggest that disruption of lymphoid niches by G-CSF to sensitize ALL cells to chemotherapy may be best accomplished in the consolidation where the bone marrow microenvironment is more likely to be normal.

Emigration of tumor specific regulatory T cells from the bone marrow is triggered by S1P1 and correlates with their accumulation in breast tumors (TUM9P.1001)

Abstract High regulatory T cell (Treg) infiltration in breast tumors is associated with reduced survival. However, the source of tumor infiltrating Treg and signals underlying their migration from lymphoid organs to the tumor tissue remain elusive. We here demonstrate that pronounced Treg infiltration in human breast tumors correlates with a selective reduction of tumor antigen specifc Treg from the bone marrow. Using MHC-II tumor peptide tetramers we furthermore show that tumor specific bone marrow Treg selectively express Sphingosine-1-phosphate receptor 1 (S1P1), the receptor that mediates cell egress. S1P1 was upregulated in Treg upon TCR stimulation mediated by bone marrow resident antigen presenting cells and triggered selective Treg but not conventional T cell migration in response to S1P gradients between bone marrow and blood which we found to be significantly increased in breast cancer patients. Taken together, our data suggests a crucial role for bone marrow antigen presenting cells in regulating the surface expression of S1P1 on tumor specific Treg in the bone marrow which may represent an important source of tumor infiltrating Treg.

Relationship between perioperative cardiovascular risk factors and bone marrow cells from patients undergoing coronary artery bypass grafting surgery.

Cell therapy through the implantation of autologous bone marrow cells has long been used in clinical trials for the treatment of ischemic heart diseases. However, as the outcomes of cell implantation vary among patients, risk factors that might influence the level and function of bone marrow progenitor cells should be determined, to identify patients who would benefit the most from this treatment. We collected clinical and laboratory data from 44 patients scheduled to undergo sternotomy for coronary artery bypass grafting (CABG). Bone marrow was aspirated from the sternum during the operation, and bone marrow mononuclear cells (BMMNCs) were isolated through density centrifugation. A negative correlation was observed between the number of BMMNCs and age (N = 44, r = -0.788, P = 0.001). The level of CD34+ cells in BMMNCs was 0.94 ± 0.39%, CD133+ cells 0.46 ± 0.28%, and CD34+CD133+ cells 0.53 ± 0.26%. The levels of CD34+ and CD133+ cells in diabetic patients were significantly lower than those in nondiabetic patients. Female gender, advanced age, and poor heart function were related with reduced progenitor cell clonogenic function. A positive correlation was observed between the level of CD34+ cells and BMMNC migration ability. Aging and diabetes were the major risk factors that influence the level and function of bone marrow resident progenitor cells in patients with coronary heart disease undergoing CABG. Further study is needed to determine whether these two factors can influence the outcome of bone marrow cell therapy for ischemic heart disease.

Myeloma Cell-Derived Runx2 Promotes Myeloma Progression and Bone-Homing

Background. Multiple myeloma (MM), an incurable plasma-cell malignancy, preferentially grows in bone marrow and frequently metastasizes to new bone sites. The molecular network responsible for progression and bone-metastasis of MM remains largely unknown. Recent work from our laboratory suggests that the Runx2 transcription factor, well-known for its essential role in osteoblast differentiation and bone development, is also required for coordinating the network of molecular signals in MM cells that drive MM progression and bone metastasis. Substantial evidence exists about expression of Runx2 in various cancer cells, including myeloma cells. Moreover, Runx2 is shown to promote tumor-to-bone metastasis, osteolytic bone disease, and tumor progression in solid tumors. However, the role of Runx2 in MM progression and bone metastasis remains unknown. In the present study, we determined the mechanism(s) governing the action of Runx2 in MM cell, especially during tumor growth and bone-homing.Methods. Molecular, biochemical, and in vivo approaches were used to assess the role of Runx2 in growth and homing of MM to bone tissue. Initially, bone marrow biopsies from 35 MM patients and 14 healthy bone marrow donors were examined for the expression of Runx2. For a mechanistic understanding of the role of Runx2 in MM, we over-expressed and knocked-down Runx2 expression in both murine 5TGM1 and human MM1S myeloma cells. The following methods and strategies were used to examine the Runx2 over-expression (Runx2 k/in), Runx2 knockdown (Runx2 k/d) and control MM cell lines in both ex vivo and in vivo models. (1) Assessment of tumor growth and homing in the 5TGM1 syngenic model of murine myeloma (for 5TGM1 cells) and SCID mice (for MM1S cells). (2) RNA-Sequencing, Real-time PCR and Western blot analyses to determine downstream genes/molecules regulated by Runx2 in MM cells. (3) Cytokine array and ELISA of the conditioned media to identify Runx2 regulated soluble factors secreted by MM cells.Results. Immunostaining of bone marrow biopsies indicated that Runx2 expression was significantly increased in both cytoplasm and nuclei of MM cells compared to normal plasma cells (p< 0.001). In vivo studies using three different animal models (i.v., subcutaneous and intra-tibial injection) demonstrated that the overexpression of Runx2 in MM cells promoted tumor growth and homing to bone whereas knockdown of Runx2 inhibited tumor progression and bone-homing. Interestingly, Runx2 over-expression in MM cells leads to the increased expression of active β-catenin and phospho-AKT, indicating that Runx2 promotes tumor growth through Wnt/β-catenin and AKT signaling pathway. This regulatory mechanism was further confirmed in 5TGM1 Runx2 k/d cells, in which both the expression of active β-catenin and phospho-AKT was significantly decreased. RNA-sequencing and real-time PCR revealed that, over-expression of Runx2 induced the expression of multiple marker genes of osteoblast (Osteocalcin, osteopontin), the osteocytes (DMP1) as well as the osteoclast (cathepsin K, RANK). Expression of these marker genes was significantly down-regulated in Runx2 k/d 5TGM1 cells. These results imply that Runx2 promotes tumor bone-homing by providing MM cells with the ability to “mimic” bone marrow resident cells and therefore escape immune surveillance in the host bone marrow. Finally, we provided evidence that MM cell-derived Runx2 promote secretion of multiple soluble factors linked with increased metastasis such as DKK1, MMP9, SDF-1, and osteopontin by MM cells.Conclusions. MM cell-derived Runx2 is a central regulator of MM progression. Runx2 expression promotes MM growth and bone-metastasis via upregulation of (i) Wnt/β-catenin and AKT signaling, (ii) the expression of multiple osteogenic genes, and (iii) the secretion of metastatic soluble factors, in MM cells. This activation could activate a pre-metastatic niche in bone thereby providing MM cells with the ability to avoid immune surveillance in the bone marrow, and supporting survival and growth of tumor cells that arrive in bone. These discoveries provide insights into MM pathology, help to predict the prognosis of MM patients, and identify novel therapeutic targets for MM. DisclosuresNo relevant conflicts of interest to declare.

Differential induction of inflammatory cytokines and reactive oxygen species in murine peritoneal macrophages and resident fresh bone marrow cells by acute staphylococcus aureus infection: contribution of toll-like receptor 2 (TLR2).

Among the known Toll-like receptors (TLRs), Toll-like receptor 2 (TLR2) is a key sensor for detecting Staphylococcus aureus invasion. But the function of TLR2 during S. aureus infection in different cell populations is unclear. Two different cell subtypes were chosen to study the interaction of S. aureus with TLR2 because macrophages are extremely different from one compartment to another and their capacity to respond to live bacteria or bacterial products differs from one site to another. The contribution of TLR2 to the host innate response against acute live S. aureus infection and heat-killed S. aureus (HKSA) using anti-TLR2 antibody in murine peritoneal macrophages and resident fresh bone marrow cells has been investigated here. TLR2 blocking before infection induces the release of interleukin (IL)-10 by macrophages thereby inhibiting excessive production of oxidants by activating antioxidant enzymes. TLR2-blocked peritoneal macrophages showed impaired release of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and IL-6 in response to both live and heat-killed S. aureus infection except bone marrow cells. TLR2-mediated free radical production and killing of S. aureus were modulated by TLR2 blocking in peritoneal macrophages and resident bone marrow cells. This study supported that S. aureus persists in resident bone marrow cells in a state of quiescence.

Proteomic profile of pre - B2 lymphoblasts from children with acute lymphoblastic leukemia (ALL) in relation with the translocation (12; 21).

BackgroundUntil now, the major prognostic factors for pediatric acute lymphoblastic leukemia (ALL), age, white blood cell count and chromosomal alterations are initially taken into account for the risk stratification of patients. In the light of protein marker studies to classify subtypes of Acute Myeloblastic Leukemia efficiently, we have compared the lymphoblastes proteome in Childhood ALL in accordance with the presence of t(12;21), indicator of good prognosis, usually.MethodsProtein expression in pre-B2 lymphoblastic cells, collected from residual bone marrow cells after diagnostic procedures, was analyzed using two dimensional gel electrophoresis protocol. Protein spots whose average normalized volumes were statistically different in the two patients groups (n = 13; student t test p < 0.01), were excised. Tryptic peptides were then analyzed using a nano-LC1200 system coupled to a 6340 Ion Trap mass spectrometer equipped with a HPLC-chip cube interface. The tandem mass spectrometry peak lists extracted using the DataAnalysis program, were compared with the protein database Mascot Daemon.ResultsWe focused on twelve spots corresponding to sixteen identified candidate proteins among the 26 found differentially expressed (p ≤ 0.05) regarding the presence of t(12;21). Among over expressed proteins, two proteins were implicated in cellular growth arrest (i.e. calponine 2, p ≤ 0.001 and phosphatidylinositol transfer protein beta, p ≤ 0.001) in accordance with good prognosis, while two other proteins favored cell cycle proliferation (i.e. methionine adenosyl transferase 2β, p ≤ 0.005 and heterogeneous nuclear ribonucleo-proteins A2 p ≤ 0.01) and could therefore be good marker candidates of aggressiveness. Level of expression of proteasome subunit beta type-2 (p ≤ 0.01) and protein casein kinase 2α (p ≤ 0.01) which both favored apoptosis, deubiquitinating enzyme OTUB1 (p ≤ 0.05) and MLL septin-like fusion protein MSF-B, septin 9 i4 (p ≤ 0.01) were in accord with a good prognosis related to t(12;21) lymphoblasts.ConclusionBy drawing up the protein map of leukemic cells, these new data identified marker candidates of leukemic aggressiveness and new t(12;21) patients subgroups. These preliminary results will be in the near future confirmed by using a larger sample of pre-B2 childhood ALLs from national lymphoblastic cell collections.

Effective Inhibition Of Tumor Microenvironment Interactions In CLL Patients Treated With The BTK Inhibitor Ibrutinib Results In Sustained Inhibition Of Tumor Proliferation and Survival Pathways

▪Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental factors for proliferation and survival. In particular, tissue resident CLL cells show an increase in both B-cell receptor (BCR) and NF-κB signaling; pathways known to regulate survival, proliferation and migration of CLL cells. One key signaling molecule in this pathway is Bruton's tyrosine kinase (BTK) that is activated directly downstream of the BCR and known to be up-regulated in CLL cells (Herman et al., Blood 2011). Ibrutinib, a covalent BTK inhibitor currently in clinical trials for CLL, has been shown to induce apoptosis and inhibit proliferation and tumor burden both in vitro and in mouse models of CLL (Herman et al., Blood 2011; Ponader et al., Blood 2012, Herman et al., Leukemia 2013). Recently, in a multicenter study, ibrutinib has been shown to induce objective clinical responses and reduce lymphadenopathy in the majority of patients, regardless of the presence of adverse prognostic markers (Byrd et al., NEJM 2013).It has been shown that CLL cells in the lymph node and bone marrow microenvironments demonstrate higher levels of BCR and NF-κB signaling as well as increased cell activation and proliferation (Herishanu et al., Blood 2011). We therefore sought to determine the in vivo effect of ibrutinib on tumor cell activation and proliferation in these microenvironmental niches. We have previously demonstrated that ibrutinib inhibits BCR and NF-κB in the lymph node microenvironment within the first 24 hours after initiation of therapy (Herman et al., ASH 2012). We here expand our previous results by evaluating the long term effects of ibrutinib in the tissue compartment. Because repeat sampling of lymph node tissue on therapy is not practical, we assessed changes in the bone marrow compartment. We obtained bone marrow aspirates pre-treatment and after two cycles of ibrutinib (Day 56). We first evaluated the BCR gene signature using a previously validated set of BCR regulated genes (Herishanu et al., 2011). We found that BCR signaling was significantly inhibited in CLL cells sampled from the bone marrow in 8/8 patients evaluated (P = 0.01). Similarly we also found inhibition of the NF-κB gene signature in all patients evaluated (P = 0.01). In fact, every patient evaluated demonstrated a reduction in both signatures, but there was substantial variation among patients in the extent to which these pathways were inhibited. This variability did not appear to correlate with clinical and prognostic factors, such as IGHV mutation status, deletion of chromosome 17p, or prior treatment status. However, the degree of inhibition of NF-κB signaling was strongly correlated with the degree of inhibition of BCR signaling (r = 0.96, P < 0.001), suggesting that the BCR (and/or a second equally BTK dependent pathway) plays a major role in activating NF-κB also in bone marrow resident CLL cells. To confirm our results we next evaluated the phosphorylation of proteins activated downstream of the BCR. We found that CLL cells showed a significant reduction in both PLCγ2 and ERK phosphorylation (mean reduction 52.9% and 71.2%, respectively; P < 0.01). Next, using the proliferation marker Ki67 we found a significant reduction in tumor proliferation in the bone marrow on ibrutinib (from a median of 6.6% KI67+ CLL cells pre-treatment to 1.1% on Day 56, P = 0.003). Lastly, we also found a significant reduction in the cell surface expression of the activation markers CD69 and CD86 (mean reduction of 57% (P = 0.001) and 82% (P = 0.03), respectively).In conclusion, our data demonstrate that ibrutinib effectively inhibits BCR, NF-κB, and ERK signaling. This occurs very quickly as demonstrated in the lymph node and is sustained on treatment as shown in the bone marrow. The strong and sustained reduction in proliferation and activation of CLL cells in the tissue microenvironment suggests that BTK is indeed a central hub mediating the nourishing and protective effects of the tumor microenvironment.This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for donating blood and tissue samples to make this research possible. We acknowledge Pharmacyclics for providing study drug. Disclosures:Off Label Use: Ibrutinib in chronic lymphocytic leukemia.

Cycling Marrow Stem Cells Are Lost with Purification.

Abstract 2308Hematopoietic stem cell biologists have amassed a tremendous depth of knowledge about the biology of the marrow stem cell over the past few decades, facilitating invaluable basic scientific and translational advances in the field. Most of the studies to date have focused on highly purified populations of marrow cells, with emphasis placed on the need to isolate increasingly restricted subsets of marrow cells within the larger population of resident bone marrow cells in order to get an accurate picture of the true stem cell phenotype. Such studies have led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to homogeneity. However, work from our laboratory, focusing on the stem cell potential in un-separated whole bone marrow (WBM), supports an alternate view of marrow stem cell biology in which a large population of marrow stem cells are actively cycling, continually changing phenotype with cell cycle transit, and therefore, cannot be purified to homogeneity. Our studies separating WBM into cell cycle-specific fractions using Hoechst 33342/Pyronin Y or exposing WBM to tritiated thymidine suicide followed by competitive engraftment into lethally irradiated mice revealed that over 50% of the long-term multi-lineage engraftment potential in un-separated marrow was due to cells in S/G2/M. This is in stark contrast to studies showing that highly purified stem cell populations such as LT-HSC (Lineage–c-kit+sca-1+flk2−) engraft predominantly when in G0. Additionally, by performing standard isolation of a highly purified population of stem cells, SLAM cells (Lineage–c-kit+sca-1+flk2−CD150+CD41−CD48−), and testing the engraftment potential of different cellular fractions created and routinely discarded during this purification process, we found that 90% of the potential engraftment capacity in WBM was lost during conventional SLAM cell purification. Incubation of the Lineage-positive and Lineage-negative fractions with tritiated thymidine, a DNA analogue which selectively kills cells traversing S-phase, led to dramatic reductions in long-term multi-lineage engraftment potential found within both cellular fractions (over 95% and 85% reduction, respectively). This indicates that the discarded population of stem cells during antibody-based stem cell purification is composed largely of cycling cells. In sum, these data strongly support that 1) whole bone marrow contains actively cycling stem cells capable of long-term multi-lineage engraftment, 2) these actively cycling marrow stem cells are lost during the standard stem cell purification strategies, and 3) the protean phenotype of actively cycling cells as they transit through cell cycle will render cycling marrow stem cells difficult to purify to homogeneity. Given the loss of a large pool of actively cycling HSC during standard stem cell isolation techniques, these data underscore the need to re-evaluate the total hematopoietic stem cell pool on a population level in addition to a clonal level in order to provide a more comprehensive study of HSC biology. Disclosures:No relevant conflicts of interest to declare.

Abstract 4264: NFκB activation in mesenchymal stromal cells induced by leukemia-stroma interaction plays a central role in stroma-mediated chemo-resistance of leukemic cells

Abstract Within the bone marrow (BM) microenvironment, BM mesenchymal stromal cells (BM-MSC) produce cytokines and chemokines and initiate cellular adhesion-mediated signals that tightly regulate normal and malignant hematopoietic cell development. This rich environment serves as a sanctuary for malignant hematopoietic cells, offering protection from chemotherapeutic agents and promoting residual disease with greater potential for developing acquired drug resistance. In this study we utilized gene expression profiling (GEP) as a genome-wide exploratory approach to characterize changes caused by the interaction between leukemic cells and BM-MSC, and how these changes can contribute to the observed microenvironment-mediated chemoresistance. Co-culture of REH, a pre-B-ALL cell line, with normal-donor BM-MSCs for 48 hours upregulated a variety of genes encoding cytokines and chemokines in BM-MSCs such as CXCR6, CCL5, CCL2, IL32, IL34, IL8, IL6, CXCL12, and the VLA4 ligand VCAM-1. These microarray-based GEP findings were confirmed by qRT-PCR. Gene Set Enrichment Analysis (GSEA) implicated activation of NFκB in BM-MSCs as a potential cause of these changes. Similar upregulation of NFκB target genes was found in MSC co-cultured with the AML cell line OCI-AML3, suggesting that NFκB activation is a common consequence of leukemia-stroma interation. Given known paracrine functions of NFκB-regulated target genes such as IL-8, we hypothesized that stromal NFκB activation contributes to stroma-mediated chemo-resistance of leukemic cells. Stable transduction of BM-MSC to overexpress a super-repressor form of IαBβ (IαBβ-SR), to block canonical-pathway NFκB activation in the stromal compartment, significantly reduced the stroma-mediated chemo-resistance to Vincristine (VCR) of REH cells. NFκB inhibition using a specific IKKα inhibitor (MLN120B), affecting both BM-MSC and tumor cells, highly increased the apoptotic effects in leukemia-stroma co-cultures of VCR in REH cells and NALM6, another pre-B-ALL cell line. Similar results were obtained with CDDO-Me, another NFκB inhibitor. Altogether these results indicate that the interaction of leukemic cells with BM stromal cells activates NFκB in the stromal compartment, with subsequent transcriptional upregulation of a number of cytokines and chemokines ultimately favoring the survival of leukemic cells. We propose that targeting NFκB may ameliorate stroma-mediated chemo-resistance and help in eliminating bone-marrow resident leukemic cells. 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 4264. doi:1538-7445.AM2012-4264

P006 Regulation of inflammatory bowel diseases by myeloid-derived suppressor cells

Background: Restoration of immune homeostasis and selftolerance represent the ultimate goal of autoimmune diseases. Although a variety of therapeutic targets are employed, a large number of patients with autoimmune syndromes fail to either respond to current therapy or to achieve long-lasting remission after its cessation. Myeloid-derived suppressor cells (MDSCs) encompass a newly described population of cells that potently suppress immune responses; however their role in autoimmune inflammatory diseases is poorly understood. Methods: Using flow cytometry, we assessed the presence of human MDSCs, denoted as CD14-HLA-DR-/lowCD15+CD33+cells, in peripheral blood from patients with active Crohn’s disease (CD) and ulcerative colitis. Further, we monitored the expansion of CD11b+Gr1+ MDSCs in peripheral lymphoid compartments of mice with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Following, we evaluated the disease progress after in vivo depletion of MDSCs, by using anti-Gr1 mAb (RB6 8C5). Results: We observed that CD15+CD33+ MDSCs are significantly enriched in the periphery of subjects with active CD compared to healthy controls (4.336±0.8747 vs 14.34±2.462, ***p < 0.0001). In addition, Gr1+CD11bhi MDSCs are largely accumulated in the spleen of mice with experimental colitis, during the effector phase of the disease, while they contracted upon disease resolution (5.328±0.7109 vs 1.170±0.2740, **p < 0.0055). Importantly, anti-Gr1 mAb mediated depletion of MDSCs aggravated the colitic phenotype. These findings suggest that MDSCs might play an important role in the regulation of TNBS-induced colitis. Conclusions: Our data demonstrate a significant enrichment of MDSCs in the periphery of CD patients, as well as the peripheral lymphoid compartments of colitic mice. Since MDSCs are bone marrow (BM) resident cells, ongoing work would address the potential role of BM-derived MDSCs in inhibiting established disease and elucidate the mechanisms used by MDSCs for the suppression of inflammatory bowel diseases.

Bone Marrow-Infiltrating Human Neuroblastoma Cells Express High Levels of Calprotectin and HLA-G Proteins

Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Sixty-six Italian children over 18 months of age, diagnosed with stage 4 NB, were included in the study. Metastatic NB cells were freshly isolated from patients' BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients.

HTLV-1 affects the adipogenic differentiation of human mesenchymal stem cells

Background Mesenchymal stem cells (MSCs) are multipotent cells mainly found in the bone marrow (BM) and are considered to support human hematopoiesis. During HTLV-1 infection, T cells that are infected may migrate to the BM, and establish a progressive viral invasion. The persistence of these cells in the BM may affect the function of the resident BM cells and the biological properties of this microenvironment. However, the exact mechanisms of this phenomenon and the relative contribution of HTLV-1 to BM cell dysfunction remain to be elucidated. In this study, we evaluated the ability of HTLV-1 to disturb MSCs normal functions. Methods Human MSCs were cultured on the presence of MT-2 cell line. MSCs markers and adipogenic differentiation potential was evaluated. Results We observed that MSCs that had contact with HTLV-1 were able to differentiate into adipocytes. However, gene expression of PPARG, cell morphology and intracellular lipid formation analyses revealed that adipogenic differentiation of MSCs that were exposed to HTLV-1 was decreased compared to MSCs not exposed (p<0.03). In addition, analyses by flow cytometry showed a reduction of the expression of MSCs markers (CD271 and HLADR) in MSCs that had contact with HTLV-1. Conclusion HTLV-1 caused significant changes in the characteristics and functions of MSCs. This finding suggests the importance of HTLV infection on MSCs, and this could contribute to the understanding the viral infection mechanisms and pathogenesis.

138 Cell-specific role of NOX2 NADPH oxidase in development of angiotensin ii-induced cardiac fibrosis in vivo

<h3>Introduction</h3> Mice globally deficient in Nox2 are protected against cardiac fibrosis in response to chronic AngII infusion even though the degree of hypertrophy was unaltered. The selective effect of Nox2 on fibrosis may reflect its activation in a non-cardiomyocyte cell type. We hypothesised that Nox2, which is expressed in endothelial cells and inflammatory cells, may be important for cardiac fibrosis in these cell types. <h3>Methods</h3> To investigate the role of Nox2 in inflammatory cells, we generated chimeric mice by irradiation (10Gy, 15 min) to deplete resident bone marrow cells, followed by bone marrow (BM) transplantation, using the following permutations: wild-type (WT) recipient with either KO or WT BM, and KO recipients with WT BM. To assess the role of endothelial Nox2, we used transgenic mice with endothelial-specific overexpression of Nox2 (TG) utilising the tie2 promoter construct. <h3>Result</h3> AngII (1.1 mg/kg/day, 14-day) infusion caused similar increase in systolic hypertension and cardiac hypertrophy in all 3 chimeric groups. However, cardiac fibrosis assessed by Sirius red staining was significantly lower in KO mice receiving WT BM (0.5±0.1%) compared to the WT:WT group (2.7±0.7%) or in WT receiving KO BM (2.3±0.6%). These data suggested that resident Nox2-expressing cells are responsible for the protective effect observed in global Nox2 KO mice. TG mice developed the same level of systolic hypertension and hypertrophy as WT littermates after AngII infusion. However, the extent of cardiac fibrosis was significantly greater in TG than WT by ∼2-fold (p&lt;0.05). This was associated with a greater degree of infiltration by CD45+ inflammatory cells (∼1.8-fold, p&lt;0.05) and 30% (p&lt;0.05) more VCAM-1 positive blood vessels in AngII treated TG hearts. Furthermore, isolated TG endothelial cells recruited ∼2-fold (p&lt;0.05) more leukocytes than WT upon AngII treatment. <h3>Conclusion</h3> These results indicate there is a cell-specific role of endothelial Nox2 in the development of fibrosis. Endothelial Nox2 enhances AngII-induced cardiac fibrosis, possibly by enhancing inflammatory cell recruitment and influx via VCAM-1 expression. Although inflammatory cells may be important for the development of fibrosis, our results indicate that Nox2 in these cells is not essential for any pro-fibrotic effect.

MHC class II upregulation and colocalization with Fas in experimental models of immune-mediated bone marrow failure

To test the hypothesis that γ-interferon (IFN-γ) promotes major histocompatibility complex (MHC) class II expression on bone marrow (BM) cell targets that facilitate T-cell-mediated BM destruction in immune-mediated BM failure. Allogeneic lymph node (LN) cells were infused into MHC- or minor histocompatibility antigen-mismatched hosts to induce BM failure. MHC class II and Fas expression and cell apoptosis were analyzed by flow cytometry. MHC class II-Fas colocalization was detected by ImageStream Imaging Flow Cytometry and other cell-to-cell associations were visualized by confocal microscopy. T-cell-mediated BM cell apoptosis and effects of IFN-γ on MHC class II-Fas colocalization on normal BM cells were studied using cell culture invitro followed by conventional and imaging flow cytometry. BM failure animals had significantly upregulated MHC class II expression on CD4(-)CD8(-)CD11b(-)CD45R(-) residual BM cells and significantly increased MHC class II-Fas colocalization on BM CD150(+) and CD34(+) hematopoietic cells. MHC class II(+)Fas(+) BM cells were closely associated with CD4(+) T cells in the BM of affected animals, and they were significantly more responsive to T-cell-mediated cell apoptosis relative to MHC class II(-)Fas(-) BM cells. Infusion of IFN-γ-deficient LN cells into minor histocompatibility antigen-mismatched recipients resulted in no MHC class II-Fas upregulation and no clinically overt BM failure. Treatment with recombinant IFN-γ significantly increased both MHC class II-Fas coexpression and colocalization on normal BM cells. Elevation of the inflammatory cytokine IFN-γ-stimulated MHC class II expression and MHC class II-Fas colocalization, which may facilitate T-cell-mediated cell destruction.

Osteoblast‐secreted factors enhance the expression of dysadherin and CCL2‐dependent migration of renal carcinoma cells

Renal cell carcinoma (RCC) frequently metastasizes to the bone marrow. These metastases are characterized by extensive osteolytic lesions. The mechanism, however, by which RCC cells metastasize to bone marrow remains poorly understood. To unravel the role of bone marrow cells in this context, we performed cell adhesion and migration assays using human RCC cell lines to analyze the influence of resident bone marrow cells on renal tumor cells. The strongest adhesion of RCC cells was observed to osteoblasts. Moreover, conditioned medium of osteoblasts (OB-CM) significantly increased RCC cell migration. By gene expression analysis dysadherin was identified as a transcript whose expression could be elevated more than twofold in RCC cells when exposed to OB-CM. Suppression of dysadherin expression in RCC cells by siRNA reduced their ability to migrate in the presence of OB-CM. Furthermore, the RCC cells secreted high amounts of the chemokine CCL2 when tumor cells migrated under the influence of osteoblast-secreted factors. CCL2 neutralization strongly reduced the migratory ability of the RCC cells. Silencing the expression of dysadherin in RCC cells resulted in a twofold reduction of CCL2 protein expression indicating a dysadherin-dependent expression of the chemokine. Taken together, our data show that osteoblasts are the major cell type of the bone marrow that affect RCC cells by secreting factors that increase the expression of dysadherin and CCL2 in the tumor cells leading to enhanced cell migration. These data suggest an osteoblast-induced autocrine mechanism for a facilitated homing of RCC cells to the bone marrow.