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DNA methylation, a major biological process regulating the transcription, contributes to the pathophysiology of hematologic malignancies, and hypomethylating agents are commonly used to treat myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). In these diseases, bone marrow mesenchymal stromal cells (MSCs) play a key supportive role through the production of various signals and interactions. The DNA methylation status of MSCs, likely to reflect their functionality, might be relevant to understand their contribution to the pathophysiology of these diseases. Consequently, the aim of our study was to analyze the modifications of DNA methylation profiles of MSCs induced by MDS or AML. MSCs from MDS/AML patients were characterized via5-methylcytosine quantification, gene expression profiles of key regulators of DNA methylation, identification of differentially methylated regions (DMRs) by methylome array, and quantification of DMR-coupled genes expression. MDS and AML-MSCs displayed global hypomethylation and under-expression of DNMT1and UHRF1. Methylome analysis revealed aberrant methylation profiles in all MDS and in a subgroup of AML-MSCs. This aberrant methylation was preferentially found in the sequence of homeobox genes, especially from the HOXfamily (HOXA1, HOXA4, HOXA5, HOXA9, HOXA10, HOXA11, HOXB5, HOXC4, and HOXC6), and impacted on their expression. These results highlight modifications of DNA methylation in MDS/AML-MSCs, both at global and focal levels dysregulating the expression of HOXgenes well known for their involvement in leukemogenesis. Such DNA methylation in MSCs could be the consequence of the malignant disease or could participate in its development through defective functionality or exosomal transfer of HOX transcription factors from MSCs to hematopoietic cells.

The bone marrow (BM) niche impacts the progression of acute myeloid leukemia (AML) by favoring the chemoresistance of AML cells. Intimate interactions between leukemic cells and BM mesenchymal stromal cells (BM-MSCs) play key roles in this process. Direct intercellular communications between hematopoietic cells and BM-MSCs involve connexins, components of gap junctions. We postulated that blocking gap junction assembly could modify cell–cell interactions in the leukemic niche and consequently the chemoresistance. The comparison of BM-MSCs from AML patients and healthy donors revealed a specific profile of connexins in BM-MSCs of the leukemic niche and the effects of carbenoxolone (CBX), a gap junction disruptor, were evaluated on AML cells. CBX presents an antileukemic effect without affecting normal BM-CD34+progenitor cells. The proapoptotic effect of CBX on AML cells is in line with the extinction of energy metabolism. CBX acts synergistically with cytarabine (Ara-C) in vitro and in vivo. Coculture experiments of AML cells with BM-MSCs revealed that CBX neutralizes the protective effect of the niche against the Ara-C-induced apoptosis of leukemic cells. Altogether, these results suggest that CBX could be of therapeutic interest to reduce the chemoresistance favored by the leukemic niche, by targeting gap junctions, without affecting normal hematopoiesis.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders with an inherent tendency for transformation in secondary acute myeloid leukemia. This study focused on the redox metabolism of bone marrow (BM) cells from 97 patients compared with 25 healthy controls. The level of reactive oxygen species (ROS) was quantified by flow cytometry in BM cell subsets as well as the expression level of 28 transcripts encoding for major enzymes involved in the antioxidant cellular response. Our results highlight increased ROS levels in BM nonlymphoid cells and especially in primitive CD34posCD38low progenitor cells. Moreover, we identified a specific antioxidant signature, dubbed “antioxidogram,” for the different MDS subgroups or secondary acute myeloblastic leukemia (sAML). Our results suggest that progression from MDS toward sAML could be characterized by 3 successive molecular steps: (1) overexpression of enzymes reducing proteic disulfide bonds (MDS with <5% BM blasts [GLRX family]); (2) increased expression of enzymes detoxifying H2O2 (MDS with 5% to 19% BM blasts [PRDX and GPX families]); and finally (3) decreased expression of these enzymes in sAML. The antioxidant score (AO-Score) defined by logistic regression from the expression levels of transcripts made it possible to stage disease progression and, interestingly, this AO-Score was independent of the revised International Scoring System. Altogether, this study demonstrates that MDS and sAML present an important disturbance of redox metabolism, especially in BM stem and progenitor cells and that the specific molecular antioxidant response parameters (antioxidogram, AO-Score) could be considered as useful biomarkers for disease diagnosis and follow-up.

The test setup in Fig 1 determines the ac frequency response of a mechanical system. In this case, the mechanical system includes a dc motor, switch-mode driver, and the motor's [...]

Molecular mechanisms leading to mobilization of hematopoietic cells from bone marrow (BM) to peripheral blood (PB) involve modulation of adhesion molecule expression on these cells that probably result in changes in adhesion capacity to the microenvironment. However, it is not clear whether these changes involve different stages or lineages of progenitor cells. In this study, we compared the capacity of mature and immature clonogenic progenitor cells from granulocyte colony-stimulating factor (G-CSF)-mobilized PB and normal BM CD34+ cells to adhere to complete marrow stroma. This functional capacity was assessed concurrently with molecular expression on CD34+ cells of integrins VLA-4 (α4/β1), VLA-5 (α5/β1), and LFA-1 (αL/β2) by interindividual (between mobilized PB and normal BM) and intraindividual (between mobilized PB and steady-state BM and PB in the same patient) analysis. The proportion of adherent clonogenic progenitor cells was significantly lower in PB than in BM, not only for total progenitor cells but also for mature and immature progenitor cells, and the difference was found for granulocytic and particularly for erythroid lineages. The lower adhesion capacity of PB CD34+ cells to stroma was associated with decreased expression (signal/noise MFI ratio) of integrin α4, β1, αL, and β2 chains whereas that of α5 chain did not differ from BM cells with the lowest expression level. Similar differences in integrin expression levels were also found between mobilized PB and steady-state BM CD34+ cells in the same patient except for the αL chain. Moreover, we demonstrated for the first time a strong positive correlation between mobilizing capacity and expression levels on mobilized CD34+ cells for the LFA-1 αL chain but not for VLA-4 or VLA-5. In conclusion, the decreased adhesion capacity of mobilized PB progenitor cells to stroma involves different maturation stages and different lineages. This is associated with down-regulation of integrins VLA-4 and LFA-1, but mobilizing capacity appears positively correlated with LFA-1 levels.

The aim of this study was to examine whether the severe prolonged deficiency in marrow clonogenic progenitor cells reported after autologous stem cell transplantation (ASCT) is associated with impairment of the primitive progenitor cell compartment. We performed Dexter-type marrow cultures and limiting dilution assays with CD34+ cells from patients 1 year and/or later after autografting with peripheral blood stem cells for non-Hodgkin's lymphoma (NHL). Flow cytometric analysis was used to assess the CD38 antigen expression and apoptotic state (7-ADD-/annexin-V+ cells) of the CD34+ cell population. We found a dramatic decrease in both clonogenic progenitor cell production and frequency of long term culture-initiating cells (LTC-IC) in all the patients tested at 1 year, even in those displaying normal progenitor cell frequency. Surprisingly, the clonogenic capacity of each LTC-IC was not increased. Flow cytometric analysis of the CD34+ cell population confirmed this quantitative defect, with a reduction in the CD38dim/neg cell population but no increase in apoptosis. This defect did not improve over time up to 4 years after transplantation. In addition, qualitative abnormalities were revealed, demonstrated by decreased CD34 antigen expression, together with impaired differentiating properties of LTC-IC toward erythroid lineage at 1 year. This study indicates that both quantitative and qualitative abnormalities of the primitive progenitor cell compartment are a constant feature up to 4 years after autologous stem cell transplantation.

Colony counting remains an important source of variation in colony-forming unit-granulocytemacrophage (CFU-GM) assays performed in methylcellulose or agar. We studied the reliability of colony scoring of CFU-GM assays carried out with collagen, a matrix that allows gel collection on glass slides and in situ cellular morphology. Fourteen slides were exchanged among laboratories, and two rounds of colony (CFU-GM and burst-forming units-erythrocyte [BFU-E]) counting were performed by 11 (first counting), then 8 (second counting) different laboratories, the majority of which had no previous experience of collagen gel cultures and reading. Two-way analysis of variance (ANOVA) of the first round of colony counting showed significant differences among centers in CFU-GM counts (p = 0.023) but not in BFU-E counts (p = 0.163). Coefficients of variation for the 14 slides ranged from 22% to 50% (median 28%) for CFU-GM counts and from 12% to 74% (median 23%) for BFU-E counts. After a 3h session of collective colony reading attended by members of 8 laboratories, a second round of colony counting was performed. This time, ANOVA showed no significant difference among centers for CFU-GM (p = 0.533) and BFU-E (p = 0.328) counts, and coefficients of variation were significantly improved, with medians of 17% for CFU-GM counts and 20% for BFU-E counts. In addition, when data from the second round of readings were analyzed without the 2 centers counting consistently low (center 8) or consistently high (center 5), variance among centers was further improved for both CFU-GM (p = 0.798) and BFU-E (p = 0.619). In summary, this study shows for the first time that reproducible BFU-E and CFU-GM scoring can be achieved using collagen-based semisolid medium (now commercially available) as long as adequate training in colony identification is provided.

The influence of zinc stearate on the dehydrogenation of atactic polypropylene (APP) by sulfur has been studied by thermal differential analysis. the stearate is quickly converted into ZnS which then reacts with the biradicals S8, giving a polysulfide, which dehydrogenates APP, this process being catalyzed by ZnS. A reaction sequence for the overall process is proposed.

The reaction kinetics of atactic polypropylene (APP) in bulk with dicumyl peroxide (DCP) as a radical generator have been studied by means of thermal differential analysis. Hydrogen abstraction from APP by cumyl radicals gives (polypropylen)yl radicals which undergo transfer to DCP, chain scission, disproportionation, and crosslinking, as well as reaction with cumyl radicals. Chain scission is shown to be the faster and crosslinking the slower of these two processes, even at temperatures of 473 K and above. Activation parameters are determined, and an analysis of the different processes is made on their basis.

Hematopoiesis after autologous bone marrow transplantation (BMT) is characterized by a prolonged and severe deficiency of marrow progenitors for several years, especially of erythroid and megakaryocyte progenitors, while the peripheral blood cells and marrow cellularity have reached relatively normal values within a few weeks. These anomalies are comparable to those reported for allogeneic BMT, despite the absence of any allo-immune reaction or post-graft immunosuppressive therapy. Post-graft hematopoietic impairment is the consequence of quantitative and qualitative changes involving both stem cell and stromal compartments which are expressed by an impaired capacity of stem cell self-renewal and commitment towards erythroid and megakaryocytic lineages. Besides the toxicity of conditioning regimens, hematopoietic reconstitution using autologous grafts is particularly dependent on a combination of factors related to the patient, such as underlying disease and pre-graft chemotherapy regimens, and to the graft processing itself, such as in vitro purging with chemotherapeutic agents.

The original version of this Article omitted the following from the Acknowledgements: This research was also supported by grants to KZ (UL and L-CNRS). This has now been corrected in both the PDF and HTML versions of the Article.

No relevant conflicts of interest to declare.

Introduction: Myelodysplastic syndromes (MDS) are heterogeneous group of blood diseases with varying degrees of severity, which can be classified into one of several subgroups based on features of the abnormal cells. The progression of MDS to secondary AML (sAML) is a good example of the multi-step theory of leukemogenesis in which a series of mutations occur in an initially normal cell and transform it into a cancer cell. Reactive Oxygen Species (ROS) are highly involved in normal hematopoiesis, a low amount being associated with quiescence and self-renewal, while a higher level is requested for ROS signaling pathways. The level of intracellular ROS is finely tuned by the expression of ROS-related enzymes that are suggested as major players in human MDS / AML. Altogether, these elements highlight the potential implication of redox metabolism in the pathophysiology of MDS / sAML.

The contribution of the stromal cell compartment to leukemogenesis remains poorly understood. Several studies have described abnormalities involving this compartment in acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS) including proliferative defect of mesenchymal stem/stromal cells (MSCs) as reported by our group in AML patients (Domenech et al., Haematologica 2012, EHA meeting, abstr. 37). Recently, evidence of an involvement of MSCs in the leukemic process has been provided in murine models (Walkley et al., Cell 2007; Raaijmakers et al. Nature 2010). In the present study, we investigated potential modifications of human AML blast cell biology induced in vitro by MSCs from healthy individuals and AML patients.Bone marrow MSCs from 6 AML patients (3 M0, 3 M1 [FAB classification]) were compared to those from 6 normal individuals. All the MSCs were analyzed at the end of the second passage of culture. Capacity of MSCs to influence leukemic cell behavior was assessed by co-culture with immature leukemia cells from KG1a line and with heterologous or autologous primary blast cells from the AML patients. Apoptotic cell frequencies, cell cycle phase distributions, and presence of DNA double-strand breaks of leukemia cells with or without MSCs were performed by flow cytometry.In co-cultures, no difference in leukemia cell adhesion were found on AML and normal MSCs. The presence of MSCs reduced apoptosis of primary blast cells (-30%, p=0.002) although no effect was observed on campthotecin-induced apoptosis of KG1a cells, as compared to MSC-free culture conditions. Cell cycle analysis revealed that G0/G1 ratios of KG1a cells were strongly increased by the presence of MSCs considering the whole cells (+128%, p=0.005) as well as the cell fraction adherent to MSCs (+138%, p=0.006). Comparable results were obtained with primary blast cells considering all the cells (+51%, p=0.117), especially within the adherent cell fraction (+229%, p=0.004). In addition, direct contact with MSCs was associated with a slight decrease in the proportion of blast cells with DNA double-strand breaks compared to MSC-free cultures (-10%, p= 0.034). No significant differences were found for all these results when co-cultures with AML and normal MSCs were compared. Likewise, no differences existed between co-cultures with M0 and M1 AML MSCs or between co-cultures of autologous and heterologous primary blast cells with AML MSCs.This study shows that MSCs influence leukemic cell behavior, irrespective of their healthy or leukemic origin. In particular, they protect blast cells from apoptosis and induce their quiescence, (mainly by direct contact) which could contribute to decreased yields of DNA double-strand breaks, a source of genetic instability. Further experiments are in progress to evaluate potential changes in the capacity of AML MSCs to support normal hematopoiesis.Gyan: FRESENIUS KABI: Consultancy, Research Funding. Domenech:Celgene Corporation: Research Funding.

TLDA analysis of gene expression pattern of MSC from CML patients (n=11) as compared to normal MSCs (n=3) identified 6 genes significantly over-expressed in CML-MSC: PDPN (10-Fold Increase), V-CAM and MITF (∼8 Fold increase), MET, FOXO3 and BMP-1 (∼ 5 Fold increase). To confirm these results we have performed Q-RT-PCR in a cohort of CML-MSC (n= 14, including the 11 patients as analyzed in TLDA) as compared to normal MSC. High levels of PDPN (Podoplanin, ∼8 fold increase), MITF (Microphtalmia Associated Transcription factor, 4-Fold) and VCAM (Vascular Cell Adhesion Protein, 2 fold increase) mRNA were again observed on CML MSCs. Our second strategy (co-culture of normal MSC with BCR-ABL-expressing UT7) revealed an increase of IL-8 and TNFR mRNA expression in co-cultured MSCs (∼5-fold ) whereas there was a major decrease in the expression of DHH (∼ 25-fold) upon contact with BCR-ABL-expressing cells. No modification of the expression of PDPN, MITF or VCAM was noted in normal MSC after this 3-day co-culture strategy using UT7-BCR-ABL cells. Current experiments are underway to determine if primary CD34+ cells from CML patients at diagnosis could induce a specific gene expression pattern in normal MSC after 5 days of co-culture. PDPN is a glycoprotein involved in cell migration and adhesion, acting downstream of SRC. It has been shown to promote tumor formation and progression in solid tumor models and is highly expressed in CAFs. MITF is a bHLH transcription factor involved in the survival of melanocyte stem cells and metastatic melanoma. Finally, high VCAM1 mRNA expression by MSCs from CML patients could be involved in increased angiogenesis known to be present on CML microenvironment. In conclusion, our results demonstrate an abnormal expression pattern of 3 important genes (PDPN, MITF and VCAM1) in MSC isolated in CP-CML patients at diagnosis. The mechanisms leading to an increased mRNA expression (instructive or not instructive by leukemic cells) and their relevance to CML biology are under evaluation. Our results, confirming previous data, suggest strongly the existence of a molecular cross-talk between leukemic cells and the leukemic niche. The elucidation of such aberrant pathways in the microenvironment could lead to the development of “niche-targeted” therapies in CML.Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.

No relevant conflicts of interest to declare.

Abstract 4743

Acute Myeloid Leukemia (AML) represents a life-threatening myeloid malignancy for which standard chemotherapy remains the cornerstone of therapy. Chemosensitizing agents are needed to improve the therapeutic results of chemotherapy. Both Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) belong to the n-3 polyunsaturated fatty acid (n-3-PUFA) family and display chemosensitizing properties in solid tumors both in vitro and in vivo. AIMS: To explore the effect of the combination of DHA with standard chemotherapy in vitro, and the effect of n-3-PUFA- containing fish oil emulsion on AML cell lines and primary blasts in vitro. METHODS: Two parenteral nutrition lipidic emulsions, OMEGAVEN (OM, containing a high level of n-3 PUFAs) and INTRALIPIDE (IL, containing a very low level of n-3 PUFAs, used as a control) were purchased from Fresenius Kabi GMBH. After 24h and 48h of culture, viable cells were counted with Trypan blue exclusion dye by an automated cell counter (TC10TM, BIO-RAD, Hercules, CA, USA). Assessement of phosphatidylserine exposure was performed with AnnexinV-FITC/7-AAD Kit (PN IM3614, Beckman Coulter), and mitochondrial membrane depolarization by flow cytometry DioC6(3) cellular uptake quantification. p62 and GRP98 expression were assessed by Western Blotting. Primary blasts were retrieved from the blood of patients with hyperleucocytic refractory AML, with informed consent. RESULTS: OM, DHA, and EPA consistently inhibited cell growth in U937, ML-2, HL-60, and THP-1 cell lines in a dose-dependent manner, whereas oleic acid (OA) or IL controls did not interfere with cell growth. OM also displayed a dose-dependent proliferation inhibition on AML cell lines, but did not interfere with normal granulopoiesis in vitro. U937 cell killing with OM was associated with p62 and GRP98 overexpression, suggesting the involvement of autophagy and endoplasmic reticulum stress, and apoptosis mechanisms. The cell-kiling effect of OM on AML primary blasts was associated with increased phosphatidylserine exposure and mitochondrial membrane depolarization. The supplementation of IL with DHA and EPA reproduced the effect of OM on the U937 cell line. At therapeutic concentrations of daunorubicin, cytarabine, or both, OM addition induced significant cytotoxity compared to IL in vitro. The isobologram assay displayed a pattern consistent with an additivity between OM and cyarabine (AraC) in vitro. DISCUSSION: This study confirms previous findings of n-3 PUFAs on AML cell line cytotoxicity both in murine and human models. We show for the first time that this effect is observed with primary blasts, is additive with chemotherapy, and can be obtained with commercially available fish oil emulsions. CONCLUSION: Altogether, these results indicate that DHA and EPA are the contributors of the cytotoxicity of a commercially available n-3 PUFA emulsion and provide a strong rationale for exploring the combination of parenteral n-3 fatty acid supplementation during AML chemotherapy.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 2264

Abstract 1058

The bone marrow (BM) hematopoietic stem cell (HSC) niche is a specialized structure of the microenvironment, which supports survival and regulates HSC function (i.e. the HSC control of the self-renewal/differentiation balance and migration). The supportive cells involved in the HSC niche are usually named as “stromal cells” but their precise nature remains a matter of debate (in particular, to know whether these cells belong to osteoblastic or to vascular smooth muscle lineage). Mesenchymal stem cells (MSCs) that are present into the BM are characterized by a broad differentiation potential including adipocytic (A), osteoblastic (O) and vascular smooth muscle (V) pathways. Although MSCs are believed to be at the origin of stromal cells, their real function within the niche is unknown. The aim of this study was to investigate in vitro the hematopoietic function (HSC support and migration) of cultured adult BM MSCs non-differentiated and during induced differentiation along A, O and V lineages. MSCs were obtained from BM nucleated cells of patients undergoing orthopedic surgery by culture in expansion medium (alpha-MEM medium with 10% FCS and 1 ng/mL FGF-2). The MSCs were tested before (cultured in expansion medium) and during differentiation induction in appropriate medium for A, O or V lineages (from 3 to 21 days). Interestingly, non-differentiated MSCs already co-expressed O (PTH-receptor), A (leptin) and V (ASMA) markers as assessed by Western blotting. Capacity of MSCs to support hematopoiesis was evaluated by long-term cultures (for 5 wks) with BM CD34+ cells in limiting dilution (CAFC assay), and capacity to control CD34+ cell migration by using Transwells seeded with MSCs (trans-stromal migration assay). We showed that non-differentiated MSCs have the most important capacity to support hematopoiesis (5-week CAFC frequency) and that this capacity was quickly and dramatically lost from 3 days of differentiation towards A (36±2% of non-differentiated values), O (40±3%) and V (38±1%) lineages. This capacity was almost abolished after 14 days of A, O and V differentiation (<5%). In parallel, CD34+ cell migration was clearly reduced through 3-day A and O differentiated MSCs, while it was increased through 3-day V differentiated MSCs (5 fold). These results show that MSCs maintained in vitro in non-differentiated state (although already expressing some A, O and V markers), display the strongest hematopoietic supportive activity compared to MSCs induced to differentiate into adipocytic, osteoblastic, or vascular smooth muscle lineages. Therefore, the stromal cell function could be supported by a cell close to a non-differentiated MSC in endosteal or perivascular niches as well. In contrast, vascular smooth muscle differentiated MSCs at advanced stages could be devoted rather to HSC migration control than to HSC support.

The number of peripheral white blood cells (WBC) shows a wide range in acute myeloblastic leukemia (AML) patients. A high WBC count constitutes an adverse prognosis factor when at presentation and (myelo)monocytic subtypes were more frequently associated with extramedullar tumor sites. Marrow endothelial cells express CD31 and CD31/CD31 interaction is known to promote the migration in physiological mechanisms, as for example transendothelial migration of neutrophiles and monocytes in diapedesis. CD31 is a specific receptor of CD38 which is associated with numerous molecules on the surface of blood cells. Moreover, CD38 could interact with hyaluronate, a component of the extracellular matrix, 2 hyaluronate-binding sites having been reported in its extracellular domain. These elements lead us to postulate that CD31 and CD38 are colocalized on AML cells and that an excess of CD31 promotes the egress of the cells from the marrow compartment, while an excess of CD38 favors their anchorage to marrow microenvironment. The CD38/CD31 colocalization was demonstrated using FRET and cocapping strategies. FRET experiments were performed with a 488 nm laser flow cytometer, Cy3-conjugated anti-CD38 mAb and FITC-conjugated mAb against CD31. For cocapping experiments, we induced capping of CD31 with anti-CD31 mAb at 37°C; the cells were then fixed and labeled with anti-CD38 mAb. Transendothelial migration has been studied using anti-CD31 mAb and anti-CD38 blocking mAbs in Transwell® experiments performed with TrHBMEC cell line. To study the CD38/hyaluronate interaction, cells were treated or not with all-trans retinoic acid to induce CD38 overexpression just before plating the cells onto hyaluronate-coated dish. The in vivo influence of CD31 and CD38 coexpression level was evaluated by studying the phenotype of marrow leukemic cells (S/N ratio of the MFI) and the WBC count of 78 consecutive patients with newly diagnosed de novo AML. In all experiments, P value <0.05 has been considered as statistically significant. FITC-conjugated anti-CD31 induced increase in MFI of Cy-3 by 108.4% and we observed a CD31/CD38 cocapping. The inhibition of migration by anti-CD31 blocking mAb ranged from 2% to 66% of control and was correlated to the CD31 expression level (R=0.9236; P<0.0001), while migration in presence of anti-CD38 blocking mAb was about 80% of the control without correlation with CD38 expression level, suggesting a lesser role in the transendothelial migration process. The blasts exposed to ATRA exhibited a 1.3±0.1-fold increase in the CD38 expression (S/N of MFI) and a 40±10 % increase in adhesion to hyaluronate. The expression of CD31 and CD38 on the membrane of marrow leukemic cells of the AML patients were correlated (R=0.6621; P<0.0001). Moreover, peripheral WBC count was correlated with the CD31/CD38 ratio (R=0.5286; P=0.0129) and an excess of CD31 antigen was associated with an increased peripheral WBC count when comparing patients with ratio >1 to those with ratio <1 (P<0.0001). Interestingly, CD31/CD38 ratio was higher in AML (myelo)monocytic subtypes when comparing M4/M5 to all other FAB subtypes (1.2±0.1 and 0.7±0.1, respectively; P<0.0001). These findings suggest that extramedullar dissemination of AML cells depends on CD31 and CD38 coexpression level and give new insights into novel strategies to reduce the number of extramedullar tumor sites, notably in AML (myelo)monocytic subtypes.

Bone marrow (BM) mesenchymal stem cells (MSCs) are characterized by their wide differentiation potential into adipose, bone, cartilage, muscle, and neural tissues. This capacity makes MSCs potentially useful for cell therapy in case of tissue repair. The therapeutic efficacy of such cells depends on their capacity to migrate to damaged tissues in response to pro-migratory signals produced locally. In this study, we investigated the in vitro migration capacity of human BM MSCs (hMSCs) evaluating their response to several potential chemotactic factors, including chemokines and mesenchymal growth factors (MGF). BM cells obtained from patients undergoing orthopedic surgery, were cultured in alpha-MEM medium with 10% fetal calf serum (FCS). Non adherent cells were discarded after 2–3 days. Layers were passaged once or twice to deplete in hematopoietic cells. The three-lineal (adipogenic, osteoblastic, and chondrocytic) differentiation potential of CD45- cells was verified. hMSCs were then induced to migrate (37°C, 5% CO2 for 16 hours) through Transwells (8-μm pore filters), in response to purified chemotactic agents at optimal concentrations, as compared to that of medium with 30% FCS (positive control) and to that of medium alone (negative control). In parallel, the pattern of chemokine and growth factor receptor expression was assessed by flow cytometry and transcripts for cytokines, chemokines and receptors were quantified using Taqman Low Density Arrays® (RQ-PCR). Among MGF, the best pro-migratory activity was observed with PDGF-AB and IGF-1 (mean values of 64.9 and 37.9% of the positive control, respectively), while EGF and HGF activity was 20%–30% of the positive control and VEGF and FGF2 activity was comparable to that of the negative control (10%). Among chemokines, only MCP-1, Eotaxin-2 and MDC had significant activity (about 20 % of the control), while activity of RANTES and Eotaxin-1 was between 10 and 20% and that of SDF-1, Fractalkine, MIP-1alpha and GROalpha was below 10%. CCR1, CCR3, CCR4, CXCR4, PDGF-R, HGF-R, EGF-R and IGF1-R were expressed by hMSCs, as demonstrated by flow cytometry. RQ-PCR analysis indicated that some chemokines were expressed at high level (X-Y level of the GAPDH housekeeping gene), either constitutively (MCP-1, GROalpha), or after IL-1/TNF-alpha stimulation (RANTES, SDF-1), while others were expressed at intermediate (Fractalkine) or low (MIP-1alpha) level. Chemokine receptors were usually expressed at very low level (X-Y level of GAPDH), either constitutively (CCR3, CX3CR1) or only after stimulation (CCR1, CCR4, CXCR4 and CXCR5), while CCR2, CXCR-1 and CXCR-2 were not expressed at all. Transcripts for all MGF assayed in vitro and for their receptors were detected at intermediate/high level. In conclusion, mesenchymal growth factors appear to be better chemoattractants in vitro for BM hMSCs than chemokines. The reduced pro-migratory activity of exogenous chemokines might be related to the low level of their receptors, themselves downregulated by endogenous chemokines autocrinally produced.

Bone marrow (BM) mesenchymal stem cells (MSCs) are characterized by their wide differentiation potential into adipose, bone, cartilage, muscle, and neural tissues. This capacity makes MSCs potentially useful for cell therapy in case of tissue repair. The therapeutic efficacy of such cells depends on their capacity to migrate to damaged tissues in response to pro-migratory signals produced locally. In this study, we investigated the in vitromigration capacity of human BM MSCs (hMSCs) evaluating their response to several potential chemotactic factors, including chemokines and mesenchymal growth factors (MGF). BM cells obtained from patients undergoing orthopedic surgery, were cultured in alpha-MEM medium with 10% fetal calf serum (FCS). Non adherent cells were discarded after 2–3 days. Layers were passaged once or twice to deplete in hematopoietic cells. The three-lineal (adipogenic, osteoblastic, and chondrocytic) differentiation potential of CD45- cells was verified. hMSCs were then induced to migrate (37°C, 5% CO2for 16 hours) through Transwells (8-μm pore filters), in response to purified chemotactic agents at optimal concentrations, as compared to that of medium with 30% FCS (positive control) and to that of medium alone (negative control). In parallel, the pattern of chemokine and growth factor receptor expression was assessed by flow cytometry and transcripts for cytokines, chemokines and receptors were quantified using Taqman Low Density Arrays® (RQ-PCR). Among MGF, the best pro-migratory activity was observed with PDGF-AB and IGF-1 (mean values of 64.9 and 37.9% of the positive control, respectively), while EGF and HGF activity was 20%–30% of the positive control and VEGF and FGF2 activity was comparable to that of the negative control (10%). Among chemokines, only MCP-1, Eotaxin-2 and MDC had significant activity (about 20 % of the control), while activity of RANTES and Eotaxin-1 was between 10 and 20% and that of SDF-1, Fractalkine, MIP-1alpha and GROalpha was below 10%. CCR1, CCR3, CCR4, CXCR4, PDGF-R, HGF-R, EGF-R and IGF1-R were expressed by hMSCs, as demonstrated by flow cytometry. RQ-PCR analysis indicated that some chemokines were expressed at high level (X-Y level of the GAPDH housekeeping gene), either constitutively (MCP-1, GROalpha), or after IL-1/TNF-alpha stimulation (RANTES, SDF-1), while others were expressed at intermediate (Fractalkine) or low (MIP-1alpha) level. Chemokine receptors were usually expressed at very low level (X-Y level of GAPDH), either constitutively (CCR3, CX3CR1) or only after stimulation (CCR1, CCR4, CXCR4 and CXCR5), while CCR2, CXCR-1 and CXCR-2 were not expressed at all. Transcripts for all MGF assayed in vitroand for their receptors were detected at intermediate/high level. In conclusion, mesenchymal growth factors appear to be better chemoattractants in vitro for BM hMSCs than chemokines. The reduced pro-migratory activity of exogenous chemokines might be related to the low level of their receptors, themselves downregulated by endogenous chemokines autocrinally produced.

The mechanism of G-CSF-induced hematopoietic progenitor/stem cell (HPC/HSC) mobilization from bone marrow microenvironment to peripheral blood has been related to local production by neutrophils of proteases, including elastase, cathepsin G and metalloproteinases (MMPs). We previously showed that in vitro migration of hematopoietic cells across a layer of human marrow stromal cells (MSC) is induced when stimulated by G-CSF. In the present study, we investigated the role of MMPs produced by MSC used for the trans-stromal migration of MO7e line and marrow CD34+ cells. Normal human MSC were previously grown to confluence on Transwell® filters (pore diameter 5-μm) and then stimulated or not by IL-1 (15 U/mL/Day) or G-CSF (15 ng/mL/Day) for three consecutive days. In a second step, MO7e or marrow CD34+ cells, put in the upper chamber, were allowed to migrate through the layers (4 hrs, SDF-1 100 ng/mL in the bottom chamber). The contribution of MMPs was evaluated in this in vitro trans-stromal migration assay using specific blocking monoclonal antibodies (mAb), anti-MMP-1, anti-MMP-2 and anti-MMP-9 (each at 5 μg/mL). The amounts of antigenic MMP-2 and MMP-9 produced by MSC were evaluated in cell supernatants by enzyme-linked immunosorbent assay (ELISA) and gelatinolytic activity of MMPs by zymography. mRNA expression of MMPs in MSC was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). None of the MSC layers used contained detectable hematopoietic cells since they were all CD45-. Migration of MO7e cells across unstimulated or G-CSF-stimulated stromal cells was significantly inhibited by specific mAb. Percentages of inhibition were 25+/−5 % and 54+/−9 % for anti-MMP-2 mAb, and 18+/−6 % and 53+/−9 % for anti-MMP-9 mAb, respectively. Using the combination of anti MMP-2 and anti-MMP-9, inhibition reached 71+/−24 % across stimulated layers. In contrast, anti-MMP-1 mAb did not show any significant inhibition. Migration of CD34+ across G-CSF-stimulated stromal cells was similarly inhibited with percentages of 38+/−18 % for anti-MMP- 2 mAb, and 34+/−11 % for anti-MMP-9 mAb, while migration was not inhibited again, using anti-MMP-1 mAb. In parallel, we demonstrated that MSC did express G-CSF receptor by RT-PCR. Production of MMPs by unstimulated and G-CSF-stimulated MSC was detected by ELISA, zymography and RT-PCR for MMP-2, and by zymography and RT-PCR for MMP-9. In conclusion, G-CSF-induction of HPC trans-stromal migration involves MMP-2 and MMP-9 but not MMP-1. These findings suggest that microenvironment, in the absence of neutrophils, could have a significant role in G-CSF mobilization of HPC/HSC. A local production of MMP-2 and MMP-9 by marrow stromal cells could be critical in the egress of HSC out of the hematopoietic niche.

Our group has reported an association between the mobilizing capacity of normal hematopoietic progenitor cells and polymorphism at position 801 (G to A transition) in CXCL12, the SDF-1-encoding gene (Benboubker et al, Br J Haematol 2001, 113: 247). The ability of leukemic cells to exit from the bone marrow microenvironment, circulate in the peripheral blood and anchor in extramedullary locations might thus depend on the CXCL12 genotype. Eighty six Caucasian patients with newly diagnosed de novo AML were included in this study. Cytogenetic risk groups were classically defined as favourable (t(8;21), t(15;17) and inv(16)), unfavourable (−5, −7, del(5q), del(7q), abnormal 11q23 and complex karyotype) and intermediate (normal and all other structural/numerical abnormalities). Leukemic cell dissemination and tissue infiltration were evaluated by leukemic blood cell (LBC) count and the presence of at least one extramedullary tumor site. Genomic DNA was extracted from marrow samples and CXCL12-G801A- polymorphism was determined with a PCR-RFLP assay. The results were expressed as median [range] and number [percentage] of patients. Age, gender, FAB subtype, cytogenetic group, leukemic marrow cell percentage, LBC count, extramedullary tumor sites and CXCL12-G801A polymorphism were considered as variables in univariate analysis (Mann-Whitney U test, χ2 test and z-test for non-zero correlation). In order to evaluate the influence of independent factors on the risk of extramedullary tumor sites, a multivariate analysis was performed using the multiple regression method, including the variables with p<0.10 in the univariate tests. The level of significance was set at 0.05. The LBC count was highly correlated with the leukemic marrow cell percentage (p=0.0007), which was not different in CXCL12-3′A carrier and G/G patients (80 [23–98]% and 76 [17–98]%, respectively, p=0.7239). Moreover, the presence of the CXCL12-3′A allele was associated with an increased LBC count when comparing CXCL12-3′A carriers to G/G patients (10.4 [0.1–94.1] vs 2.6 [0–137.1] LBC/mL, respectively, p=0.0309). The patients presenting extramedullary tumor site(s) were characterized by a lower mean age (39 [18–78] vs 53 [16–75] years, p=0.0050), a higher LBC count (10.9 [0.1–137.1] vs 2.5 [0–99.3] LBC/mL, p=0.0020), and a more frequent CXCL12-3′A allele (59.4% vs 33.3%, p=0.0184). CXCL12-3′A carrier status was indeed highly associated with extramedullary locations, which were found in 51.4% of CXCL12-3′A carriers (66.7% and 48.4% of A/A and A/G patients, respectively) and in 26.5% of G/G patients, with an odds ratio of 2.92 (95% CI 1.18–7.21). Age, LBC count, CXCL12-3′A carrier status and leukemic marrow cell percentages (87% [17–98] vs 75% [23–98] in patients presenting or not tissue infiltration, respectively, p=0.0904) were included in the multivariate analysis, and the independent variables found to be associated with risk of extramedullary tumor site(s) were LBC count (p=0.0122), age (p=0.0289) and CXCL12-G801A polymorphism (p=0.0416). In conclusion, we report that CXCL12-G801A polymorphism is a genetic determinant involved in the clinical presentation of acute myeloid leukemia. This description constitutes the first report of an association between this polymorphism and the risk of tissue infiltration by tumor cells.

The mechanism of G-CSF-induced hematopoietic progenitor/stem cell (HPC/HSC) mobilization from bone marrow microenvironment to peripheral blood has been related to local production by neutrophils of proteases, including elastase, cathepsin G and metalloproteinases (MMPs). We previously showed that in vitromigration of hematopoietic cells across a layer of human marrow stromal cells (MSC) is induced when stimulated by G-CSF. In the present study, we investigated the role of MMPs produced by MSC used for the trans-stromal migration of MO7e line and marrow CD34+cells. Normal human MSC were previously grown to confluence on Transwell® filters (pore diameter 5-μm) and then stimulated or not by IL-1 (15 U/mL/Day) or G-CSF (15 ng/mL/Day) for three consecutive days. In a second step, MO7e or marrow CD34+cells, put in the upper chamber, were allowed to migrate through the layers (4 hrs, SDF-1 100 ng/mL in the bottom chamber). The contribution of MMPs was evaluated in this in vitrotrans-stromal migration assay using specific blocking monoclonal antibodies (mAb), anti-MMP-1, anti-MMP-2 and anti-MMP-9 (each at 5 μg/mL). The amounts of antigenic MMP-2 and MMP-9 produced by MSC were evaluated in cell supernatants by enzyme-linked immunosorbent assay (ELISA) and gelatinolytic activity of MMPs by zymography. mRNA expression of MMPs in MSC was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). None of the MSC layers used contained detectable hematopoietic cells since they were all CD45-. Migration of MO7e cells across unstimulated or G-CSF-stimulated stromal cells was significantly inhibited by specific mAb. Percentages of inhibition were 25+/−5 % and 54+/−9 % for anti-MMP-2 mAb, and 18+/−6 % and 53+/−9 % for anti-MMP-9 mAb, respectively. Using the combination of anti MMP-2 and anti-MMP-9, inhibition reached 71+/−24 % across stimulated layers. In contrast, anti-MMP-1 mAb did not show any significant inhibition. Migration of CD34+across G-CSF-stimulated stromal cells was similarly inhibited with percentages of 38+/−18 % for anti-MMP- 2 mAb, and 34+/−11 % for anti-MMP-9 mAb, while migration was not inhibited again, using anti-MMP-1 mAb. In parallel, we demonstrated that MSC did express G-CSF receptor by RT-PCR. Production of MMPs by unstimulated and G-CSF-stimulated MSC was detected by ELISA, zymography and RT-PCR for MMP-2, and by zymography and RT-PCR for MMP-9. In conclusion, G-CSF-induction of HPC trans-stromal migration involves MMP-2 and MMP-9 but not MMP-1. These findings suggest that microenvironment, in the absence of neutrophils, could have a significant role in G-CSF mobilization of HPC/HSC. A local production of MMP-2 and MMP-9 by marrow stromal cells could be critical in the egress of HSC out of the hematopoietic niche.

Our group has reported an association between the mobilizing capacity of normal hematopoietic progenitor cells and polymorphism at position 801 (G to A transition) in CXCL12, the SDF-1-encoding gene (Benboubker et al, Br J Haematol 2001, 113: 247). The ability of leukemic cells to exit from the bone marrow microenvironment, circulate in the peripheral blood and anchor in extramedullary locations might thus depend on the CXCL12genotype.