The FLK2/FLT3 ligand synergizes with interleukin-7 in promoting stromal- cell-independent expansion and differentiation of human fetal pro-B cells in vitro

Abstract The effects of a novel cytokine FLK2/FLT3 ligand (FL) on human fetal bone marrow-derived CD34+CD19+ pro-B cells were analyzed in a stromal- cell-independent, serum-deprived culture system. FL, like interleukin-3 (IL-3), synergized with IL-7 in promoting pro-B cell growth, and differentiation of these cells into CD34-CD19+clgM+slgM- pre-B cells, whereas a small proportion of these cells even differentiate into more mature slgM+ B cells. In contrast, KIT ligand (KL) and granulocyte- macrophage colony-stimulating factor (GM-CSF) were ineffective in promoting IL-7-dependent pro-B cell growth and differentiation. Maximal levels of pro-B cell expansion, generally resulting in 15- to 30-fold increases in cellularity, were obtained in cultures supplemented with optimal doses of FL + IL-7 + IL-3. The addition of mouse bone marrow stromal cells further enhanced the proliferation and differentiation of pro-B cells obtained in the presence of these three cytokines. Under these conditions, cultures could be maintained for more than 4 weeks, and in general 40- to 50-fold increases in cell numbers were observed by 3 weeks of culture. The percentages of clgM+ and slgM+ B cells increased 1.5- to 3-fold and 2-fold, respectively, suggesting that stromal cells may provide additional costimulatory signals for human B- cell growth and differentiation that are different from IL-7, IL-3, and FL. Collectively, our results indicate that FL, in contrast to KL, strongly promotes long-term expansion and differentiation of human pro- B cells in the presence of IL-7 or in combination of IL-7 and IL-3, which is a novel property of this hematopoietic growth factor.

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The FLK2/FLT3 ligand synergizes with interleukin-7 in promoting stromal- cell-independent expansion and differentiation of human fetal pro-B cells in vitro

Blood ◽

10.1182/blood.v87.5.1881.bloodjournal8751881 ◽

1996 ◽

Vol 87 (5) ◽

pp. 1881-1890 ◽

Cited By ~ 2

Author(s):

R Namikawa ◽

MO Muench ◽

JE de Vries ◽

MG Roncarolo

Keyword(s):

Bone Marrow ◽

B Cells ◽

Cell Growth ◽

B Cell ◽

Stromal Cells ◽

Stromal Cell ◽

Mouse Bone Marrow ◽

Flt3 Ligand ◽

Cell Growth And Differentiation

The effects of a novel cytokine FLK2/FLT3 ligand (FL) on human fetal bone marrow-derived CD34+CD19+ pro-B cells were analyzed in a stromal- cell-independent, serum-deprived culture system. FL, like interleukin-3 (IL-3), synergized with IL-7 in promoting pro-B cell growth, and differentiation of these cells into CD34-CD19+clgM+slgM- pre-B cells, whereas a small proportion of these cells even differentiate into more mature slgM+ B cells. In contrast, KIT ligand (KL) and granulocyte- macrophage colony-stimulating factor (GM-CSF) were ineffective in promoting IL-7-dependent pro-B cell growth and differentiation. Maximal levels of pro-B cell expansion, generally resulting in 15- to 30-fold increases in cellularity, were obtained in cultures supplemented with optimal doses of FL + IL-7 + IL-3. The addition of mouse bone marrow stromal cells further enhanced the proliferation and differentiation of pro-B cells obtained in the presence of these three cytokines. Under these conditions, cultures could be maintained for more than 4 weeks, and in general 40- to 50-fold increases in cell numbers were observed by 3 weeks of culture. The percentages of clgM+ and slgM+ B cells increased 1.5- to 3-fold and 2-fold, respectively, suggesting that stromal cells may provide additional costimulatory signals for human B- cell growth and differentiation that are different from IL-7, IL-3, and FL. Collectively, our results indicate that FL, in contrast to KL, strongly promotes long-term expansion and differentiation of human pro- B cells in the presence of IL-7 or in combination of IL-7 and IL-3, which is a novel property of this hematopoietic growth factor.

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Mesenchymal Stromal Cells Orchestrate Follicular Lymphoma Cell Niche Through the CCL2-Dependent Recruitment and Polarization of Monocytes

Blood ◽

10.1182/blood.v118.21.1566.1566 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1566-1566

Author(s):

Fabien Guilloton ◽

Gersende Caron ◽

Cédric Ménard ◽

Céline Pangault ◽

Patricia Amé-Thomas ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Cell Growth ◽

Follicular Lymphoma ◽

B Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Gene Set Enrichment Analysis ◽

Cell Niche

Abstract Abstract 1566 Accumulating evidence indicates that infiltrating stromal cells contribute directly and indirectly to tumor growth in a wide range of solid cancers and hematological malignancies. In follicular lymphoma (FL), malignant B cells are found admixed with heterogeneous lymphoid-like stromal cells within invaded lymph nodes and bone marrow (BM). In addition, in vitro functional studies have underlined that mesenchymal cells recruit malignant FL B cells and protect them from spontaneous and drug-induced apoptosis. In particular, we have previously demonstrated that mesenchymal stromal cells (MSC) efficiently support in vitro FL B-cell survival, especially after their engagement towards lymphoid differentiation through treatment with TNF-α and Lymphotoxin-α1β2 (TNF/LT) or after coculture with malignant B cells. However, the mechanisms of this supportive activity remain largely unknown. In this study, we used Affymetrix U133 Plus 2.0 microarrays, to compare the gene expression profile (GEP) of bone marrow-derived MSC (BM-MSC) obtained from 10 FL patients at diagnosis versus 6 age-matched healthy donors (HD). In these conditions, neither the CFU-F concentration in the BM nor the cumulative population doubling of BM-MSC significantly differed between HD and FL patients. Unsupervised analysis was able to perfectly segregate FL-MSC from HD-MSC and we identified, using supervised analyzes, a list of 408 probesets defining FL-MSC signature, including 320 nonredundant genes upregulated in FL-MSC compared to HD-MSC. We then defined the GEP of human lymphoid-like stroma using HD-MSC treated in vitro by TNF/LT and demonstrated, by a Gene Set Enrichment Analysis (GSEA) approach, that the FL-MSC signature is significantly enriched for genes associated with a lymphoid-like commitment. Interestingly, CCL2 was strongly overexpressed by FL-MSC, was upregulated in HD-MSC by coculture with malignant B cells, and was detected at a higher level in FL BM plasma compared to normal BM plasma (504.4 pg/mL [23.8-4413] versus 33.9 pg/mL [5-126.1]; P <.01). In agreement, FL-MSC triggered a more potent CCL2-dependent monocyte migration than HD-MSC. Moreover, FL-MSC and macrophages cooperated to sustain malignant B-cell growth through both protection from apoptosis and enhancement of cell proliferation. Finally, FL-MSC promoted monocyte differentiation towards a proangiogenic LPS-unresponsive phenotype close to that of tumor-associated macrophages. We unraveled a key role for the Notch pathway in this process and identified an overexpression of JAGGED1 in FL-MSC compared to HD-MSC. Altogether, these results highlight the complex role of FL stromal cells that promote direct tumor B-cell growth and orchestrate FL cell niche. The identification and characterization of this intricate network of cell interactions may provide novel therapeutic targets in this disease. Disclosures: No relevant conflicts of interest to declare.

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Mesenchymal stromal cells orchestrate follicular lymphoma cell niche through the CCL2-dependent recruitment and polarization of monocytes

Blood ◽

10.1182/blood-2011-08-370908 ◽

2012 ◽

Vol 119 (11) ◽

pp. 2556-2567 ◽

Cited By ~ 75

Author(s):

Fabien Guilloton ◽

Gersende Caron ◽

Cédric Ménard ◽

Céline Pangault ◽

Patricia Amé-Thomas ◽

...

Keyword(s):

B Cells ◽

Cell Growth ◽

Follicular Lymphoma ◽

B Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Specific Gene ◽

Wide Range ◽

Cell Niche

Abstract Accumulating evidence indicates that infiltrating stromal cells contribute directly and indirectly to tumor growth in a wide range of cancers. In follicular lymphoma (FL), malignant B cells are found admixed with heterogeneous lymphoid-like stromal cells within invaded lymph nodes and BM. In addition, mesenchymal stromal cells (MSCs) support in vitro FL B-cell survival, in particular after their engagement toward lymphoid differentiation. We show here that BM-MSCs obtained from patients with FL (FL-MSCs) display a specific gene expression profile compared with MSCs obtained from healthy age-matched donors (HD-MSCs). This FL-MSC signature is significantly enriched for genes associated with a lymphoid-like commitment. Interestingly, CCL2 could be detected at a high level within the FL-cell niche, is up-regulated in HD-MSCs by coculture with malignant B cells, and is overexpressed by FL-MSCs, in agreement with their capacity to recruit monocytes more efficiently than HD-MSCs. Moreover, FL-MSCs and macrophages cooperate to sustain malignant B-cell growth, whereas FL-MSCs drive monocyte differentiation toward a proangiogenic and lipopolysaccharide-unresponsive phenotype close to that of tumor-associated macrophages. Altogether, these results highlight the complex role of FL stromal cells that promote direct tumor B-cell growth and orchestrate FL-cell niche, thus emerging as a potential therapeutic target in this disease.

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Galectin-1–expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow

Blood ◽

10.1182/blood-2010-12-323113 ◽

2011 ◽

Vol 117 (24) ◽

pp. 6552-6561 ◽

Cited By ~ 51

Author(s):

Frédéric Mourcin ◽

Caroline Breton ◽

Julie Tellier ◽

Priyanka Narang ◽

Lionel Chasson ◽

...

Keyword(s):

Bone Marrow ◽

B Cell ◽

Stromal Cells ◽

Stromal Cell ◽

Close Contact ◽

Cell Receptor ◽

Cell Development ◽

Mouse Bone Marrow ◽

Reticular Cells ◽

B Cell Subsets

Abstract In the bone marrow (BM), stromal cells constitute a supportive tissue indispensable for the generation of pro-B/pre-BI, pre-BII, and immature B lymphocytes. IL-7–producing stromal cells constitute a cellular niche for pro-B/pre-BI cells, but no specific stromal cell microenvironment was identified for pre-BII cells expressing a functional pre-B cell receptor (pre-BCR). However expression of the pre-BCR represents a crucial checkpoint during B-cell development. We recently demonstrated that the stromal cell derived-galectin1 (GAL1) is a ligand for the pre-BCR, involved in the proliferation and differentiation of normal mouse pre-BII cells. Here we show that nonhematopoietic osteoblasts and reticular cells in the BM express GAL1. We observed that pre-BII cells, unlike the other B-cell subsets, were specifically localized in close contact with GAL1+ reticular cells. We also determined that IL-7+ and GAL1+ cells represent 2 distinct mesenchymal populations with different BM localization. These results demonstrate the existence of a pre-BII specific stromal cell niche and indicate that early B cells move from IL-7+ to GAL1+ supportive BM niches during their development.

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Stromal Rescue of Drug Exposed CLL Cells Can Be Overcome by the Green Tea Extract Epigallocatechin 3 Gallate

Blood ◽

10.1182/blood.v116.21.3610.3610 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3610-3610

Author(s):

Connie 1Lesnick ◽

Neil E. Kay ◽

Betsy LaPlant ◽

Tait Shanafelt

Keyword(s):

Bone Marrow ◽

B Cells ◽

Phase I ◽

Green Tea ◽

Stromal Cells ◽

Stromal Cell ◽

Antagonistic Effects ◽

Primary Bone ◽

Primary Bone Marrow

Abstract Abstract 3610 Purpose Chronic Lymphocytic Leukemia (CLL) is incurable with conventional chemotherapy treatments. Nurturing of leukemic CLL B cells by marrow microenvironment appears to be one important method of chemoresistance (NEJM 2005). Epigallocatechin 3 gallate (EGCG), the major catechin in green tea, induces caspase-dependent death CLL B-cells and down regulates anti-apoptotic proteins (Mcl-1; XIAP) critical for enhanced apoptosis resistance of CLL B-cells (Blood 2004). In Phase I and II clinical testing, we found EGCG induced a decline in absolute lymphocyte count and/or lymphadenopathy in the majority of Rai stage 0-II CLL patients (JCO 2009; ASCO 2010). In these phase I/II studies, trough plasma EGCG levels as high as 8 mM were achieved. Since it does not cause myelosuppression, EGCG has the potential to be combined with other chemotherapeutic agents used for CLL. We previously reported that pharmacologically achievable plasma levels of EGCG have an additive or synergistic effect when combined with fludarabine (F), chlorambucil (C), and FC in the majority of patients (ASH 2009). In this study, we evaluated i) the effect of pharmacologically achievable doses of EGCG when combined with bendamustine (B) and ii) the ability of pharmacologically achievable doses of EGCG to overcome in vitro stromal rescue of CLL B-cells exposed to B or FC. Methods We first evaluated the effects of EGCG combined with B to establish if this partnership would be additive or synergistic. Primary CLL cells were treated with B (12.5-100 mM) with or without 4 uM EGCG. After 48 hours cells were stained with annexin/PI, and viability analyzed by flow cytometry. Data were analyzed using the CalcuSyn program (Chou & Talay method) to determine the combination index (indicating antagonistic, additive or synergistic effects). Next, we evaluated the ability of 4 mM EGCG to overcome stromal rescue of CLL cells treated with B or FC. To eliminate the cytotoxic effects of FC and B on stromal cells, we treated CLL cells with FC or B for 24 hours, washed with PBS, and then cultured these drug exposed cells directly on the HS-5 human stromal cell line with or without 4 mM EGCG for 48 hours (FC) or 24 hours (B). Cells were harvested and viability of CD19+ cells was assessed by annexin/PI staining. To specifically evaluate the ability of 4 mM EGCG to overcome stromal protection, co-culture samples in which no stromal protection was observed were excluded. Similar experiments were conducted using primary bone marrow mesenchymal stromal cells (MSC) from CLL patients (Leuk Res, 2007). In these experiments the fludarabine/chlorambucil combination was used as an approximation of fludarabine/cyclophosphamide since the active form of cyclophosphamide (4-hydroxycyclophosphamide) rapidly breaks down in aqueous buffer and is not readily available for in vitro use and. Results We previously observed additive or synergistic FC and EGCG interactions in a majority of patient samples (ASH 2009). 4 mM EGCG was less consistently beneficial when combined with increasing doses of B in the absence of stroma. Synergistic or mixed synergistic/additive effect was seen in 5 patients, mixed additive/antagonistic effects in 3, and antagonistic effects in 2. Given previously observed synergy with FC and additive/synergistic interactions with at least some B treated samples, we next explored the effects of EGCG on stromal cell rescue. Co-culture with HS-5 stromal cells significantly reduced both B and FC mediated cell death. However the addition of 4 μM EGCG significantly attenuated stromal cell rescue of CLL B-cells exposed to B and FC therapy (Fig. 1A and 1B respectively). Remarkably in the case of FC, 4 mM EGCG almost completely abrogated stromal rescue. Experiments using primary bone marrow MSC cultures from CLL patients yields similar results (n=3, data not shown). Conclusions Pharmacologically achievable doses of EGCG appear to reduce stromal rescue of CLL cells treated with B or combinations of purine nucleoside analog/alkylating agents. This occurs in the absence of detectable stromal cell kill by EGCG. These findings, coupled with the favorable toxicity profile and lack of myelosuppression in phase I/II trials of EGCG make it an attractive agent to test in combination with bendamustine or purine analogue/alkylating agent chemo-immunotherapy for patients with CLL. Disclosures: Shanafelt: Polyphenon Pharma: Research Funding.

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Human Placenta-Derived Stromal Cells Rescue Mice from Radiation-Induced Bone Marrow Failure: A Cytof-Based Mechanistic Analysis

Blood ◽

10.1182/blood.v128.22.2677.2677 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2677-2677 ◽

Cited By ~ 1

Author(s):

Hoshea Allen ◽

Noa Sher ◽

Shiran Rekhes ◽

Lena Pinzur ◽

Tal Prezma ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Mechanism Of Action ◽

Stromal Cells ◽

Stromal Cell ◽

Early Stage ◽

Fold Increase ◽

Hematopoietic System ◽

Cell Product

Abstract Acute Radiation Syndrome (ARS) is a set of health effects involving damage to multiple organs caused by exposure to high dose ionizing radiation over a short period of time. Even low doses damage the radio-sensitive hematopoietic system (causing H-ARS). We probed the mechanism of action by which a 3D-expanded placenta-derived stromal cell product designated for the treatment of hematological disorders alleviates symptoms in the H-ARS mouse model. These cells have been shown in vitro to secrete hematopoietic proteins, stimulate colony formation, and induce bone marrow (BM) migration. Previous studies show that cells administered intramuscularly to C3H/HeN and C57BL/6 mice, 1 and 5 days after (LD70/30) total body irradiation, rescue radio-induced weight loss, survival, and peripheral blood (PB) and BM cellularity. The injected cells transiently secrete haematopoiesis related proteins to enhance reconstitution of the hematopoietic system and further induce endogenous cells to secrete a panel of cytokines. Analysis of PB and BM taken from the experimental endpoint (day 23) of cell-treated irradiated mice indicated rescue of blood lineages to levels near those of naïve mice. CyTOF analysis of BM cells indicated that in the myeloid lineage, the percentages of neutrophils and monocytes were consistently higher in cell-treated irradiated mice than in naïve mice (2.6±0.08 fold for neutrophils and 1.4±0.1 fold for monocytes). There was also a 1.5±0.5 fold increase in the percentage of the lymphoid lineage pDCs and a 2.8±0.9 fold reduction in the percentage of T cells in the BM of the treated compared to naïve mice. On the other hand, the percentage of B cells and NK cells were more similar when compared to naïve mice (1.2±0.4 fold for B cells and 1.1±0.2 fold for NK cells). Neutrophils and monocytes were also elevated in the PB of cell-treated irradiated mice (1.3±0.09 fold for neutrophils and 1.7±0.2 fold for monocytes), and within the monocyte population, percentages of classical and non-classical monocytes were essentially identical to naïve mice (0.97±0.05 fold for classical and 1.1±0.1 fold for non-classical). In cell-treated mice, B cells showed evidence of active recovery from pro-B to the immature stage of development when compared to naïve mice (4.4±0.7 fold increase for pro-B cells in BM and 5.5±0.8 fold increase for immature B cells in blood). In addition, CD8+ effector memory T cells showed a 1.9±0.3 fold increase in treated compared to naïve mice. The effect of placenta-derived stromal cells on human BM migration in vitro was also analyzed by CyTOF. Results indicated that the stromal cell product may induce significant maturation of early-stage neutrophils to late-stage neutrophils and significantly enhance the migration of neutrophils 17.9±1.4 fold compared to control. Active induction of migration of monocytes (2.4±0.0004 fold), eosinophils (2.6±0.1 fold), and endothelial cells (2.1±0.2 fold) was also observed. A number of these beneficial effects may be ascribed to cytokines known to be secreted by the cell product in high concentration. IL-8 and osteopontin are highly chemotactic toward neutrophils and MCP1 is highly chemotactic toward monocytes. These cytokines may contribute to the observed migration of human BM neutrophils and monocytes in vitro as well as to the increased percentage of neutrophils and monocytes in the blood of the cell-treated irradiated mice. In addition, cytokines known to be involved in neutrophil granulopoiesis, i.e. SCF, G-CSF, GM-CSF, IL-3 and IL-6, are present in the secretome of the cell product and may contribute to the observed maturation of early-stage neutrophils in human BM. Taken together, placenta-derived stromal cells have the capacity to alleviate symptoms of BM failure in the H-ARS model and rescue multiple blood lineages via a complex mechanism of action based on the secretion of multiple proteins acting on multiple hematopoietic lineages. Due to this combined mechanism of action involving the induction of cell migration, proliferation and differentiation, as well as an adaptive secretion profile based on the changing environment within the animal, placenta-derived cells are able to rescue from BM failure multiple cell lineages of the hematopoietic system to near-normal levels. Disclosures Allen: Pluristem: Employment. Sher:Pluristem: Employment. Rekhes:Pluristem: Employment. Pinzur:Pluristem: Employment. Prezma:Pluristem: Employment. Gorodetsky:Pluristem: Consultancy. Volk:Pluristem: Consultancy. Aberman:Pluristem: Employment. Ofir:Pluristem: Employment.

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Co-Transplantation of Stromal Cells Enhances Engraftment Ability of MDS-Derived Hematopoietic Precursors in NOD/SCID-b2microglobulin-Deficient (b2mnull) Mice.

Blood ◽

10.1182/blood.v106.11.3429.3429 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3429-3429

Author(s):

Daniella B. Kerbauy ◽

Vladimir Lesnikov ◽

Eileen Bryant ◽

Nissa Abbasi-Shafer ◽

Beverly Torok-Storb ◽

...

Keyword(s):

Bone Marrow ◽

Cell Growth ◽

Injection Site ◽

Stromal Cells ◽

Human Cells ◽

Cell Contact ◽

Fish Cell ◽

Mouse Bone Marrow

Abstract Propagation of hemopoietic clones derived from MDS marrow in vitro or in vivo has proven difficult. We recently showed engraftment of clonal cells identified by fluorescence in situ hybridization (FISH) markers, in b2mnull mice using MDS bone marrow mononuclear cells (BMMC) injected intramedullarly along with the human stroma-derived cell lines, HS5 and HS27a. To define the role of stromal cells for transplant success, we conducted additional in vitro and in vivo studies. Methods: Using NOD/SCID-b2mnull mice (irradiated with 350cGy), MDS BMMC (107cells) or purified CD34+ MDS-derived hematopoietic precursors (106cells) were transplanted into the right femur with or without the addition of HS5 and HS27a (2x105) stroma cells. Engraftment was defined as presence of ≥ 0.2% human CD45+ cells in peripheral blood. To document the presence of human stroma, mice were transplanted with GFP transduced HS5 and HS27a cells, and sacrificed 1h or 24h after transplant. Marrow cells from the femora were evaluated for the presence of GFP signals by flow cytometry. MDS-derived hematopoietic precursors from patients with identifiable FISH markers, were sorted from the blast gate (CD45 dim, low side scatter [ SSC]), and plated on stromal feeder layers (either HS5 or HS27a, irradiated with 1800cGy), in contact or transwell cultures. After one week, cells were recovered and analyzed by FISH, and also plated in methocult. Colonies were plucked at two weeks and also analyzed by FISH. Results: 11 of 13 mice transplanted without co-injection of stroma cells showed engraftment of human cells. At six weeks, clonal cells were detected in four of six mice with a FISH marker [−5 and del(5q)], suggesting that stromal cells were not an absolute requirement for the propagation of clonal cells. However, in mice transplanted with cells from the same marrow source with or without stromal cells, the engraftment rate was higher with stroma (4/7, 57%) than without stroma (2/6, 33%), as was the proportion of human hemopoietic cells in marrow (6.4±2.7% vs 2.2±0.4%, respectively). The proportion of human cells was higher at the injection site than in the contralateral femur. Furthermore, clonal (FISH+) cells were detected in 3 of 4 mice in the stroma group (del(5q) and +8), but not in two mice that were not injected with stroma but showed engraftment of “normal” human cells. Among mice transplanted with cells from a patient with +8, only those in the stroma group showed engraftment (clonal and non-clonal cells). Nine mice transplanted with CD34+ MDS cells (7 with and 2 without addition of stroma) did not show engraftment, suggesting that not only stroma but also other accessory cells are important for the tranplant success. GFP+ stromal cells were recognized in mouse bone marrow obtained from the injection site, but not contralaterally. In vitro, clonal cells (−7) were recovered from short-term assays on HS5 or HS27a cells, from contact and trans-well cultures. However, colonies were formed only from cells in contact cultures; HS5 stroma (a rich source of cytokines) favored clonal (FISH+) cell growth. Conclusions: Thus, the intramedullary co-transplantation of stromal cells enhanced the engraftment ability of normal and clonal cells from human MDS marrow. In vitro data suggest that cell-to-cell contact is necessary, and that clonal cell growth may have an advantage in the presence of HS5 cells.

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Dietary Products Induce Apoptosis in CLL B Cells and Reveal Potential as a Therapeutic Combination That Can Overcome Stromal Cell Mediated Protection.

Blood ◽

10.1182/blood.v110.11.3130.3130 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3130-3130

Author(s):

Asish K. Ghosh ◽

Tait D. Shanafelt ◽

Charla Secreto ◽

Neil E. Kay

Keyword(s):

B Cells ◽

B Cell ◽

Stromal Cells ◽

Stromal Cell ◽

Combined Treatment ◽

Flow Cytometric Analysis ◽

Physical Contact ◽

Flow Cytometric ◽

Induced Apoptosis

Abstract Background: B-cell chronic lymphocytic leukemia (CLL) is characterized by defects in the level of apoptosis rather than accelerated leukemic cell proliferation. We have been interested in identification of naturally occurring substances that induce apoptosis in CLL B cells with potential for therapeutic application with a favorable toxicity profile. Two such agents, epigallocatechin-3-gallate (EGCG) in green tea and curcumin from the spice turmeric have recently been studied by our laboratory. We have shown previously that in vitro treatment of CLL B cells with EGCG induces apoptosis [Blood, 2004, 104:788] and is now being tested in a Phase I/II clinical trial. In this study, we explored the possibility of using curcumin in the induction of CLL B cell apoptosis either alone or in combination with EGCG. We also tested their ability to induce CLL B cell apoptosis in the presence of human stromal cells. Results: In vitro treatment of CLL B cells with curcumin induces apoptosis in a dose (5–20 μM) and time dependent manner. Further analysis suggested that curcumin treatment inhibited the effects of the constitutively active proteins STAT3, AKT and NF-κB and suppressed expression of XIAP in CLL B cells. Interestingly, we observed that curcumin/EGCG combination was more effective in the induction of apoptosis in CLL B cells as compared to the corresponding individual dose of the drug alone. We next evaluated the effects of curcumin when CLL B cells were co-cultured with the human stromal cell line HS-5. Curcumin-induced apoptosis was significantly inhibited when CLL B cells were co-cultured in physical contact with HS-5 cells. We also found protection against curcumin-induced cell death, albeit less, when CLL B cells were co-cultured with HS-5 separated by transwells. However, higher doses of curcumin can overcome HS-5 mediated apoptosis-protection of CLL B cells in transwells but, did not overcome the protective effect of physical contact with HS-5. Further analysis of CLL B cells co-cultured with HS-5 in transwells demonstrated elevated levels of STAT3 with increased phosphorylation at serine-727 and increased expression of Mcl-1 and XIAP. At this stage, we employed combined therapeutic approach to evaluate whether combining curcumin with EGCG could overcome stromal cell-mediated protection. Interestingly, combined treatment of CLL B cells could significantly overcome stromal cell-mediated protection against apoptotic cell death, although the effect was more pronounced when CLL B cells were separated by transwells (see figure below). Conclusion: Together, these results suggest that curcumin induces apoptosis in CLL B cells in vitro and may mediate its effects by suppressing the constitutively active signaling pathways that are known to be important in apoptosis resistance This work also demonstrates that stromal cells and their soluble products can resist drug-induced apoptosis of leukemic CLL B cells. Importantly, the apoptosis-inducing activity of curcumin and EGCG is not impaired completely in the presence of stromal cells, suggesting that the combination of curcumin/EGCG may warrant clinical testing in patients with CLL. Figure. Combined treatment of CLL B cells with curcumin/EGCG reduces stromal cell mediated protection against apoptosis. CLL B cells were cultured alone or co-cultured with HS-5 cells and treated with the indicated doses of curcumin (C) and/or EGCG (E). After 24h, cells were collected for Annexin/PI staining for flow cytometric analysis. Mean percent of annexin/PI positive cells are presented with standard error bars. Figure. Combined treatment of CLL B cells with curcumin/EGCG reduces stromal cell mediated protection against apoptosis. CLL B cells were cultured alone or co-cultured with HS-5 cells and treated with the indicated doses of curcumin (C) and/or EGCG (E). After 24h, cells were collected for Annexin/PI staining for flow cytometric analysis. Mean percent of annexin/PI positive cells are presented with standard error bars.

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Interactions with the Microenvironment Protect Lymphoma B-Cells From Rituximab Induced Apoptosis and Could Represent a Therapeutic Target

Blood ◽

10.1182/blood.v116.21.3115.3115 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3115-3115

Author(s):

Marek Mraz ◽

Clive S. Zent ◽

Amy K Church ◽

Nisar A Baig ◽

Diane F. Jelinek ◽

...

Keyword(s):

Bone Marrow ◽

Cell Adhesion ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Stromal Cells ◽

Research Funding ◽

Stromal Cell ◽

Plastic Surface ◽

Induced Apoptosis

Abstract Abstract 3115 Background. Rituximab significantly improves the outcome of patients with B-cell non-Hodgkin lymphomas, but it does not completely eradicate residual B-cell populations in the bone marrow (BM) and lymph nodes (LN). The architecture and gene expression profile of LN stromal cells in diffuse large cell lymphoma correlates with outcome following treatment with R-CHOP (Lenz et al. 2008). Interestingly, multiple studies have demonstrated the importance of B-cells adhesion and pro-survival signaling mediated by integrin alfa-4-beta-1 (VLA-4/CD49d), which is constitutively expressed on malignant B-cells. Although several studies have demonstrated that adhesion to stromal cell cultures or ligand coated surfaces can protect malignant B-cells from apoptosis induced by chemotherapy drugs (cell adhesion-mediated drug resistance (CAM-DR); Dalton, 2002), a similar mechanism of resistance to rituximab has not, to our knowledge, been described. Hypothesis. In this study we tested the hypothesis that interactions with the microenvironment protect malignant B-cells from rituximab induced apoptosis, and that targeting these interactions with natalizumab, a humanized monoclonal anti-alfa-4 antibody approved for treatment of multiple sclerosis and Crohn's disease, can overcome this protection. Methods. Lymphoma B-cell lines were cultured on either a plastic surface, confluent HS-5 cells or a fibronectin (alfa-4-beta-1 ligand) coated surface. HS-5 is an immortalized human bone marrow stromal cell line and a well characterized model for a component of the BM microenvironment (Roecklein and Torok-Storb, 1995). Cells were treated with rituximab (10 ug/mL; Genentech), natalizumab (10ug/mL; Biogen IDEC), control IgG (10ug/mL, Sigma-Aldrich). Cell viability was measured by flow cytometry using Annexin V-FITC, propidium iodide in malignant B-cell population (anti-CD19 antibody, BD PharMingen). Results. We studied ten CD20-positive B-cell lymphoma cell lines and the CD20-positive MEC-1 cell line (derived from prolymfocytoid transformation of CLL) for rituximab induced apoptosis. The four most rituximab sensitive cell lines were used for further experiments (Karpas-422, Raji, DOHH2, SUDHL-4). Cell lines were co-cultured for 24hrs with confluent HS-5 stromal cells and subsequently treated for 24hrs with rituximab. The percentage of apoptotic cells was significantly lower (17-23% vs. 30–42%, p<0.05) for cells co-cultured with HS-5 cells compared to control cells cultivated under the same conditions on the plastic surface for all four cell lines. Similar results were obtained for several primary CLL samples (3-5% vs. 13–18%, p<0.05). We next examined if natalizumab could disrupt cell adhesion to fibronectin and overcome the stromal cell-mediated resistance to rituximab. Natalizumab reduced the number of lymphoma cells that adhered to fibronectin by 75–95% (p<0.05). Moreover, adherent cells cultivated on fibronectin coated surface completely lost the adhesion morphology after the addition of natalizumab to the cultivation media. The combination of natalizumab and rituximab versus rituximab alone increased by 26–32% (p<0.05) the number of apoptotic B-cells in co-culture with HS-5 for three of four rituximab-responding cell lines. Conclusion. We have shown that human bone marrow stromal cells (HS-5) protect lymphoma B cells from rituximab induced apoptosis suggesting existence of stromal cell adhesion-mediated antibody resistance (CAM-AR) analogous to CAM-DR. Targeting integrin alfa-4-beta-1 with natalizumab partially overcomes this cell adhesion-mediated resistance to rituximab. Research supported by P50CA97274-8 LYMPHOMA SPORE, Genentech, MSMT-MSM0021622430 and IGAMZCR NT11218-3/2010. Disclosures: Zent: Genentech: Research Funding; Genzyme: Research Funding; Novartis: Research Funding.

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Modulated Expression of the Epidermal Growth Factor-Like Homeotic Protein dlk Influences Stromal-Cell–Pre-B-Cell Interactions, Stromal Cell Adipogenesis, and Pre-B-Cell Interleukin-7 Requirements

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5247 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5247-5255 ◽

Cited By ~ 46

Author(s):

Steven R. Bauer ◽

María José Ruiz-Hidalgo ◽

Eva K. Rudikoff ◽

Julia Goldstein ◽

Jorge Laborda

Keyword(s):

B Cells ◽

Growth Factor ◽

Cell Growth ◽

Epidermal Growth Factor ◽

B Cell ◽

Stromal Cells ◽

Stromal Cell ◽

Fetal Liver ◽

Interleukin 7 ◽

Epidermal Growth

ABSTRACT A close relationship exists between adipocyte differentiation of stromal cells and their capacity to support hematopoiesis. The molecular basis for this is unknown. We have studied whether dlk, an epidermal growth factor-like molecule that intervenes in adipogenesis and fetal liver hematopoiesis, affects both stromal cell adipogenesis and B-cell lymphopoiesis in an established pre-B-cell culture system. Pre-B-cell cultures require both soluble interleukin-7 (IL-7) and interactions with stromal cells to promote cell growth and prevent B-cell maturation or apoptosis. We found that BALB/c 3T3 fibroblasts express dlk and function as stromal cells. Transfection of these cells with antisense dlk decreased dlk expression and increased insulin-induced adipocytic differentiation. When antisense transfectants were used as stroma, IL-7 was no longer required to support the growth of pre-B cells and prevent maturation or apoptosis. Antisense dlk transfectants of S10 stromal cells also promoted pre-B-cell growth in the absence of IL-7. These results show that modulation of dlk on stromal cells can influence their adipogenesis and the IL-7 requirements of the pre-B cells growing in contact with them. These results indicate that dlk influences differentiation signals directed both to the stromal cells and to the lymphocyte precursors, suggesting that dlk may play an important role in the bone marrow hematopoietic environment.

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