Increased Survival and Migration of CLL B-Cells in the Presence of Marrow Mesenchymal Stromal Cells: Novel Findings for Microenvironment-Targeted Therapies

Abstract Abstract 4571 Background. The accumulation of CD19+/CD5+/CD23+ B cells with a prolonged lifespan in peripheral blood, secondary lymphoid organs and bone marrow (BM) is a peculiar feature of B-cell chronic lymphocytic leukemia (B-CLL). Since CLL cells removed from the in vivo microenvironment and in vitro cultured rapidly undergo spontaneous apoptosis, bidirectional interactions between malignant and by-stander cells may lead to an abnormal microenvironment that confers growth advantages to neoplastic clone. Mesenchymal Stromal Cells (MSCs) are the dominant marrow stromal population in indolent subtype of CLL/small lymphocytic leukemia (SLL) and follicular lymphoma (FL), rather than other aggressive B-cell lymphomas, and are involved in B-CLL cell survival. Despite the phenotypic and cytologic homogeneity, CLL is characterized by extremely variable clinical courses, suggesting that malignant B-cells hold variable degrees of dependency on pro-survival signals coming from the microenvironment. The aim of this study was to assess the role of MSCs in CLL B-cell localization and survival, defining the degree of dependency of leukemic B-cells from external pro-survival signals, with the ultimate goal of identifying patients that mostly benefit microenvironment-targeted therapies. Methods. MSCs isolated from the BM of 47 B-CLL patients were expanded ex vivo and characterized through flow cytometry analysis and differentiation cultures. Fresh isolated CLL peripheral blood mononuclear cells were co-cultured with CLL-MSCs or stromal cells and apoptosis were measured by Annexin V test and western blotting analysis (PARP-1 detection). Chemotactic assays were performed. Results. The survival of neoplastic cells ranged from 13.3% (±13.2) in leukemic cells cultured in medium alone to 58.5% (±17.2) when leukemic cells were cultured in presence of CLL-MSCs (p<0.01). Transwell experiments showed that the anti-apoptotic effect is mediated by soluble factors produced by MSCs. We investigated whether different CLL clones show a different susceptibility to spontaneous apoptosis when co-cultured in presence of MSCs recovered from B-CLL patients. The detection of the 85KDa cleaved PARP fragment in all CLL B-cells cultured in medium alone confirmed that they underwent spontaneous apoptosis. At the same time, the presence or the lacking of the cleaved fragment of PARP-1 on CLL B-cells after 7 day-co-cultures with MSCs discriminated patients into two groups: non-responder (89 kDa Parp fragment detectable) and responder (89 kDa Parp fragment not detectable) to microenvironment pro-survival signals. Finally, chemotaxis tests showed the ability of MSCs to produce and release molecules promoting the migration and the localization of neoplastic B-cells in bone marrow (Migration Index of leukemic cells: 5.1; Migration Index of normal B cells: 1.9; p<0.01). Conclusions. MSCs derived from patients with B-CLL provide survival signals to neoplastic cells, extending their lifespan and producing chemotactic factors that favour their accumulation into BM. On the other hand, CLL cells display heterogeneous responses to environmental pro-survival signals, suggesting that each CLL clone could differently react to the microenvironment protection. The blocking of the cross-talk between malignant clone and accessory cells within the microenvironment might represent an attractive novel strategy for CLL therapy. Our data provide the rationale for tailored therapies which powerfully target the cross-talk with marrow cells, particularly on patients carrying a clone more sensitive to anti-apoptotic signals coming from the microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Bone Marrow Lymphoid Niche Adaptation to Mature B Cell Neoplasms

Frontiers in Immunology ◽

10.3389/fimmu.2021.784691 ◽

2021 ◽

Vol 12 ◽

Author(s):

Erwan Dumontet ◽

Stéphane J. C. Mancini ◽

Karin Tarte

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Tumor Cells ◽

Stromal Cells ◽

Therapeutic Option ◽

B Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Niche Adaptation ◽

Functional Features

B-cell non-Hodgkin lymphoma (B-NHL) evolution and treatment are complicated by a high prevalence of relapses primarily due to the ability of malignant B cells to interact with tumor-supportive lymph node (LN) and bone marrow (BM) microenvironments. In particular, progressive alterations of BM stromal cells sustain the survival, proliferation, and drug resistance of tumor B cells during diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL). The current review describes how the crosstalk between BM stromal cells and lymphoma tumor cells triggers the establishment of the tumor supportive niche. DLBCL, FL, and CLL display distinct patterns of BM involvement, but in each case tumor-infiltrating stromal cells, corresponding to cancer-associated fibroblasts, exhibit specific phenotypic and functional features promoting the recruitment, adhesion, and survival of tumor cells. Tumor cell-derived extracellular vesicles have been recently proposed as playing a central role in triggering initial induction of tumor-supportive niches, notably within the BM. Finally, the disruption of the BM stroma reprogramming emerges as a promising therapeutic option in B-cell lymphomas. Targeting the crosstalk between BM stromal cells and malignant B cells, either through the inhibition of stroma-derived B-cell growth factors or through the mobilization of clonal B cells outside their supportive BM niche, should in particular be further evaluated as a way to avoid relapses by abrogating resistance niches.

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IGHV Unmutated Chronic Lymphocytic Leukemia (CLL) B Cells Are More Susceptible to Spontaneous Apoptosis Than Mutated CLL B Cells and Are Subject to the Anti-Apoptotic Effect of Environmental Signals

Blood ◽

10.1182/blood.v116.21.2431.2431 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2431-2431

Author(s):

Marta Coscia ◽

Francesca Pantaleoni ◽

Chiara Riganti ◽

Candida Vitale ◽

Micol Rigoni ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Board Of Directors ◽

Stromal Cells ◽

Peripheral Blood ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Spontaneous Apoptosis ◽

Advisory Committees

Abstract Abstract 2431 Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IGHV): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival. We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Both M and UM CLL B cells underwent spontaneous apoptosis throughout the culture period. However, the UM CLL B cells showed a significantly higher degree of apoptosis in 7-day cultures as compared to M CLL B cells. In both M and UM CLL B cells, high basal levels of Bcl-2 expression and NF-kB activity were detected. On day 7, the percentage of Bcl-2+ leukemic cells was significantly lower in UM than in M CLL B cells. EMSA test showed that NF-kB was totally inactivated in UM CLL B cells and only partially reduced in M CLL B cells. Quantitative analysis of RelA and RelB subunits showed that NF-kB inactivation in UM CLL B cells consisted in a strong reduction of both RelA and RelB nuclear expression. CD40L, IL-4 and stromal cells significantly improved UM CLL B cells viability and significantly recovered Bcl-2 expression. The protective effect exerted by these stimuli was totally independent from the recovery of NF-kB expression. Indeed, after 7 days of culture, the UM CLL B cells had completely lost the nuclear form of NF-kB, and none of the stimuli was capable of restoring it. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (M or UM PBMC) as compared to purified leukemic cells (M and UM CLL B cells). The reduced apoptosis detected in UM PBMC was accompanied by a retained expression of Bcl-2 and by a restored activity of NF-kB and suggested the presence of a pro-survival element in the peripheral blood of these patients. To investigate the role of NLC in rescuing UM CLL B cells from apoptosis we first evaluated whether M and UM PBMC generated NLC with the same efficiency. Unexpectedly, the former generated significantly higher numbers of NLC than UM PBMC. Despite the lack of generation of NLC, CLL B cells viability was very similar in the non-adherent fraction of M and UM PBMC on day 7 and 14 of culture. This observation ruled out a role for NLC in supporting UM CLL B cells survival. Conversely, a pro-survival effect on UM CLL B cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM CLL B cells were cultured in the presence of autologous peripheral blood T cells (UM CLL B cell/T cell co-cultures). NF-kB activity was completely lost in UM CLL B cells cultured for 7 days in medium alone whereas it was restored in UM CLL B cells / T cells co-cultures. The prosurvival effect of circulating T cells was exerted both in cell-to-cell contact and in trans-well condition and was associated to increased secretions of tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF)-BB and interleukin-8 (IL-8) as detected by analyses of supernatants through a Multiplex system. These data indicate that despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions. Disclosures: Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia: Novartis: Honoraria, Research Funding.

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Low Dose-Radiation of Bone Marrow Stromal Cells Supports Pre-Leukemic Cell Survival Via Adhesion and Inflammatory Signaling Stimulation

Blood ◽

10.1182/blood.v120.21.4610.4610 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4610-4610

Author(s):

Yoko Tabe ◽

Linhua Jin ◽

Yasuhito Hatanaka ◽

Saiko Kazuno ◽

Tsutomu Fujimura ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

B Cells ◽

B Cell ◽

Cdna Microarray ◽

Stromal Cells ◽

Low Dose ◽

Leukemic Cells ◽

Rt Pcr ◽

Inflammatory Signaling

Abstract Abstract 4610 Low-dose irradiation (LDI) exposure is a form of environmental carcinogen, which is of significant interest after the nuclear accident in Japan. In this study, we investigated the LDI-induced molecular alterations of bone marrow (BM) –derived stromal cells (MSCs), and the biological response of pre-leukemic cells neighbor to LDI exposed MSCs. As the model of pre-leukemic cells, we utilized the Epstein- Barr virus (EBV) infected and immortalized B lymphocyte cell line (EBV-B). In MSCs, exposed to 100 mGy irradiation (4MVX rayfrom a LINAC) caused cell growth inhibition (75.8±2.4 % of control, p<0.001, MTT assay) with moderate apoptosis induction (SubG1 %; control 2.3±0.4, radiation 7.3±2.3, p=0.03, PI cell cycle analysis) at 24 hrs. Screening up to 28,869 genes by cDNA microarray (Human Gene 1.0 ST Array, Affymetrix) revealed 48 up-regulated and 45 down-regulated genes (>1.3 fold) in irradiated MSCs (at 24 hrs) compared to non-irradiated MSCs. The functional network analysis of cDNA microarray data by MetaCore (GeneGo) showed an enrichment of the adhesion/ECM remodeling pathways. TaqMan RT-PCR confirmed significant up-regulation of ECM scaffold Sulfatase1 mRNA (2.0±0.1 fold, p=0.004). Proteomic analysis utilized isobaric tags for relative and absolute quantitation (iTRAQ, Applied Biosystems), identified 32 up-regulated and 1 down-regulated proteins (p<0.05) among 1,536 detected proteins comparing irradiated MSCs (at 24 hrs) with non-irradiated MSCs. KEGGontology analysis (Kyoto University) found the activation of focal adhesion and apoptosis pathways in irradiated MSCs; among 32 up-regulated proteins, six are involoved in adhesion/cytoskeleton regulation (Prelamin-A; p<0.001, Catenin beta-1; p=0.03, Keratin type II; p=0.02, Collagen alpha1; p=0.02, alpha actinin1 and 4; p=0.01, p=0.02) and three are involved in apoptosis/senescence (Stress-70 protein l; p=0.02, Endoplasmin; p=0.008, Prohibitin; p=0.04). These changes were accompanied with increased secretion of inflammation and coagulation marker protein plasminogen activator inhibitor 1 (PAI-1) from irradiated MSCs (control 10.1±0.03 ng/mL, radiation >15.0 ng/mL, at 72hrs). We then investigated the effect of LDI exposed MSCs on EBV-B cell proliferation. Under low-serum growth condition (1% FBS), co-culture with pre-irradiated (100 mGy) MSC layers supported EBV-B cell proliferation more prominently than non-irradiated MSCs (118.3±4.0 % viable cell number, p<0.001, 72hrs). Gene expression profiles by cDNA microarray and sequenced MetaCore ontology revealed 53 up-regulated and 39 down-regulated genes showing an enrichment of the coagulation and cell adhesion pathways in EBV-B cells co-cultured with pre-irradiation of MSCs compared to the ones co-cultured with non-irradiated MSCs. Up-regulation of anti-apoptotic BCL2 mRNA (0.5±0.03 fold, p=0.02) and inflammatory IL8 mRNA (2.0±0.03 fold, p<0.001) was further detected by TaqMan RT-PCR. The purity of EBV-B cells separated from MSCs was confirmed by lack of CD90 mRNA expression by PCR. These data suggest that LDI exposure to MSCs leads to latent adhesion and inflammatory signaling in MSCs, which potentially activates pro-survival pathways of co-cultured pre-leukemic EBV-B cells. In summary, we present the biosignature of the response of BM-derived stromal cells to LDI utilizing gene array and quantitative proteomics analysis that may have important implications for risk assessment in environmental medicine. Disclosures: No relevant conflicts of interest to declare.

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Rooting into the Soil, a Model for the Role of Sox4 in Leukemia

Blood ◽

10.1182/blood.v112.11.3794.3794 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3794-3794

Author(s):

Saradhi Mallampati ◽

Baohua Sun ◽

Yun Gong ◽

Enze Wang ◽

M. James You ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Leukemic Cell ◽

Marrow Stromal Cells ◽

Leukemic Cells ◽

Cell Stage

Abstract Development and progression of leukemia requires interaction of leukemia-initiating cells with their bone marrow niches. The niches serve as the nursery and shelter for the leukemic cells, which can result in drug resistance, disease recurrence, and minimal residual disease, the most important causes for the death of patients with leukemia. Therefore, obliteration of the interaction between the leukemic cells and their niches is of utmost importance in eradicating leukemic cells during therapy to cure the disease. However, little is currently known of the molecular mechanisms underlying the interaction of the two types of cells. Sox4, a SRY-related HMG-box containing transcription factor that is vital during development, plays an important role in leukemia. Published mouse studies demonstrated that increased expression of Sox4 was associated with leukemogenesis. We determined the expression levels of Sox4 by real-time RT-PCR in 100 human leukemic samples and found high levels of expression in B- and T-ALL, but not in AML, CML, CLL, Sezary syndrome, or T cell prolymphocytic leukemia. In accordance, 7 of the 8 ALL cell lines (the exception was 697) we tested showed high expression levels of Sox4, but AML cell lines, normal mature B cells, T cells, and bone marrow CD34+ cells had low levels of expression. Since the majority of clinical B-ALL cases correspond to the pre-B cell stage, we investigated the role of Sox4 in a pre-B cell line (Nalm6) by lentivirus-mediated RNAi. Remarkably, knockdown of Sox4 in Nalm6 cells caused 70% reduction in the formation of leukemic cell clusters under the monolayer of co-cultured M2-10B4 bone marrow stromal cells, a phenomenon known as pseudo-emperipolesis. Similar results were obtained with ex vivo cultured bone marrow cells from conditional Sox4 knockout mice that displayed B cell developmental arrest at the transition from pro-B to pre-B cell stage and an absence of pre-B cells. These findings suggested that Sox4 is required for the interaction of the developing B cells or leukemic cells with bone marrow stromal cells, a component of the bone marrow niche. Since CXCR4/SDF1-mediated “homing” is known to be required for pseudo-emperipolesis, we tested the effect of Sox4 on Nalm6 cell migration toward SDF1 gradient and found that Sox4 did not affect the migration, suggesting that Sox4 is not acting through “homing”. Instead, our data indicated that the role of Sox4 in the interaction of leukemic cells with stromal cells is most likely mediated by its ability in enhancing the adhesion of the leukemic cells because we found that lentivirus-medicated overexpression of Sox4 in the 697 B cell line caused the suspension cells to display a spindle and adhesive morphology. In addition, 21% of the putative Sox4 downstream genes that we identified by multiple sets of gene expression microarray experiments are known to be involved in cell adhesion. Moreover, we found that the changes in gene expression profile of leukemic cells upon Sox4 knockdown or overexpression significantly overlap with the changes in response to the presence of bone marrow stromal cells in co-culture, indicating that Sox4 pathways are involved in leukemic cell response to stromal cell signaling. Based on these findings we hypothesize that deletion of Sox4 abolishes the interaction between the developing lymphocytes and their niches during lymphopoiesis. Conversely, overexpression of Sox4 may enforce these cells to over-interact with the niches so that they are overexposed to local growth factor stimuli. If superimposed with other genetic and/or epigenetic changes in the developing lymphocytes, such over-interaction may result in the development of leukemia. In case of established leukemia, such over-interaction may lead to the enhanced protection of leukemic cells by their niches. Therefore, the role of Sox4 in the interaction of developing lymphocytes or leukemic cells with their niches is like “rooting into the soil” of a growing tree, abbreviated as “rooting”.

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The PI3 Kinase δ Inhibitor, CAL-101 (GS-1101), Inhibits Chronic Lymphocytic Leukemia (CLL) Cell Survival in Endothelial and Marrow Stromal Cell Co-Cultures.

Blood ◽

10.1182/blood.v118.21.1769.1769 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1769-1769 ◽

Cited By ~ 1

Author(s):

Stefania Fiorcari ◽

Wells S Brown ◽

Bradley W McIntyre ◽

Susan O'Brien ◽

Mariela Sivina ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Chronic Lymphocytic Leukemia ◽

Cell Survival ◽

Stromal Cells ◽

Lymphocytic Leukemia ◽

Clinical Activity ◽

Spontaneous Apoptosis ◽

Cell Protection ◽

Survival Signals

Abstract Abstract 1769 CLL cells are characterized by their ability to resist apoptosis in vivo, but in vitro they undergo spontaneous apoptosis. This suggests that interactions between CLL cells and accessory cells in the tissue microenvironments, such as mesenchymal stromal cells (MSC), nurselike cells (NLC), T-cells, and endothelial cells are critical for maintaining CLL cell survival. CLL cells display constitutive PI3K pathway activation, presumable due to CLL interactions with the microenvironment. CAL-101 is a potent and selective inhibitor of the p110d PI3K isoform and has shown promising clinical activity in chronic lymphocytic leukemia (CLL) in early stage clinical trials. Here, we investigated the ability of CAL-101 to disrupt interactions between CLL and endothelial cells (EC) or bone marrow stromal cells (BMSC). We tested two EC lines human umbilical vein endothelial cells (HUVEC) and UV-2 mouse vascular endothelial cells, and two BMSC lines, stroma-NKtert derived from human bone marrow, and KUSA-H1, a murine BMSC line. CLL cells were cultured for 72h in presence or absence of EC or BMSC. Fig A displays mean (±SEM) CLL cell viabilities of cells from 7 different patients. We found that both, EC and BMSC rescue CLL cells from spontaneous apoptosis with significantly higher CLL cell viabilities in the presence of EC and BMSC (*P< 0.05; **P< 0.01). For example, after 48h significantly higher CLL cell viabilities were noticed with HUVEC (53.2%±4.3%, p<0.05,), UV2 (61.8%±5.3%, p<0.01), stroma-NKtert (96.7%±5.3%, p<0.01) and KUSA-H1 (93.7%±0.95%, p<0.01), when compared to CLL cultured in medium alone (37.5%±4.1%). To test the effects of CAL-101 on EC- and BMSC-mediated CLL cell protection, CLL cells were cultured on ECs or BMSCs in presence or absence of CAL-101 (0.5μM and 5μM), and CLL cell viabilities were assessed at 24h, 48h and 72h. Viabilities of CAL-101 treated samples were normalized to the viabilities of control samples at the respective timepoints (100%). Fig B depicts the mean relative viabilities of CLL cells co-culture with ECs or BMCSs in presence of 5μM CAL-101, compared to CLL cells in the absence of CAL-101. We found a significant reduction of the viability of CLL cells in co-culture with EC and BMSC with both concentrations of CAL-101 (*P< 0.05; **P< 0.01; n=7). These data demonstrate that marrow stromal and endothelial cells both support the viability and protect CLL cells from apoptosis. When comparing BMSC with EC, we noticed that BMSC were more effective than EC in protecting CLL cells, which may explain why the marrow is a preferred site for residual disease and relapses in patients with CLL. CAL-101 can overcome both, BMSC- and EC-mediated CLL cell protection, indicating that CAL-101 inhibits BMSC- and EC-derived pro-survival signals. Ongoing experiments investigate the role of adhesion molecules on BMSC- and EC-derived survival signals and CLL cell adhesion to BMSC versus EC, and how adhesion molecule function is affected by CAL-101. These studies will give us better insight into the mechanism of action of this interesting new drug. Disclosures: O'Brien: Gilead: Consultancy, Research Support. Lannutti:Gilead Sciences: Employment.

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COMPARISON OF CHROMOSOMAL REARRANGEMENTS IN BONE MARROW CELLS AND BLAST TRANSFORMED B-CELLS IN RELAPSE OF B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA/ SMALL LYMPHOCYTIC LYMPHOMA

Experimental Oncology ◽

10.31768/2312-8852.2017.39(2):141-144 ◽

2017 ◽

Vol 39 (2) ◽

pp. 141-144

Author(s):

S V Andreieva ◽

K V Korets ◽

O E Ruzhinska ◽

I M Skorokhod ◽

O G Alkhimova

Keyword(s):

Bone Marrow ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Chromosomal Rearrangements ◽

Lymphocytic Leukemia ◽

Banding Technique ◽

Small Lymphocytic Lymphoma ◽

Detection Frequency ◽

Cell Chronic Lymphocytic Leukemia

Aim: The genetic mechanisms of resistance to chemotherapy in B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL) are not clear. We aimed to determine the peculiarities of abnormal karyotype formation in bone marrow (BM) cells and peripheral blood (PB) blast transformed B-cells in relapse of B-CLL/SLL. Materials and Methods: Cytogenetic GTG banding technique and molecular cytogenetic in interphase cells (i-FISH) studies of BM cells and PB blast transformed B-lymphocytes were performed in 14 patients (10 males and 4 females) with B-CLL/SLL. Results: The results of karyotyping BM and PB cells revealed the heterogeneity of cytogenetic abnormalities in combined single nosological group of B-CLL/SLL. In PB B-cells, chromosome abnormalities related to a poor prognosis group were registered 2.5 times more often than in BM cells. Additional near tetraploid clones that occurred in 57.1% cases were the peculiar feature of BM cell karyotypes. Chromosomal rearrangements characteristic of the group of adverse cytogenetic prognosis were revealed in all cases from which in 2 cases by karyotyping BM cells, in 6 cases in PB B-cells and in 8 cases by the i-FISH method in BM cells, i.e. their detection frequency was 3 times higher in PB B-cells and 4 times higher when analyzing by i-FISH in BM cells. Conclusions: Mismatch in abnormal karyotypes in BM and PB B-cells by the presence of quantitative and structural chromosomal rearrangements may be indicative of simultaneous and independent processes of abnormal clone formation in the lymph nodes and BM hematopoietic cells. Accumulation the information about previously unidentified chromosomal rearrangements in relapse of the disease may help to understand the ways of resistance formation to chemotherapy.

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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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Aberrant expression of B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: a mechanism for survival

Blood ◽

10.1182/blood-2002-02-0558 ◽

2002 ◽

Vol 100 (8) ◽

pp. 2973-2979 ◽

Cited By ~ 164

Author(s):

Anne J. Novak ◽

Richard J. Bram ◽

Neil E. Kay ◽

Diane F. Jelinek

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

B Lymphocyte ◽

Leukemic Cell ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Aberrant Expression ◽

B Lymphocyte Stimulator

B-cell chronic lymphocytic leukemia (B-CLL) is defined by the accumulation of CD5+ B cells in the periphery and bone marrow. This disease is not characterized by highly proliferative cells but rather by the presence of leukemic cells with significant resistance to apoptosis and, therefore, prolonged survival. B-lymphocyte stimulator (BLyS) is a newly identified tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis and it shares significant homology with another TNF superfamily member, APRIL. The striking effects of BLyS on normal B-cell maintenance and survival raises the possibility that it may be involved in pathogenesis and maintenance of hematologic malignancies, including B-CLL. In this study, we investigated the status of APRIL and BLyS expression, as well as their receptors, in this disease. All B-CLL patient cells studied expressed one or more of 3 known receptors for BLyS; however, the pattern of expression was variable. In addition, we demonstrate for the first time that B-CLL cells from a subset of patients aberrantly express BLyS and APRIL mRNA, whereas these molecules were not detectable in normal B cells. Furthermore, we provide in vitro evidence that BLyS protects B-CLL cells from apoptosis and enhances cell survival. Because these molecules are key regulators of B-cell homeostasis and tumor progression, leukemic cell autocrine expression of BLyS and APRIL may be playing an important role in the pathogenesis of this disease.

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Plasma cells induce apoptosis of pre-B cells by interacting with bone marrow stromal cells

Blood ◽

10.1182/blood.v87.8.3375.bloodjournal8783375 ◽

1996 ◽

Vol 87 (8) ◽

pp. 3375-3383 ◽

Cited By ~ 30

Author(s):

T Tsujimoto ◽

IA Lisukov ◽

N Huang ◽

MS Mahmoud ◽

MM Kawano

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Survival ◽

Cell Lines ◽

Stromal Cells ◽

Plasma Cells ◽

Myeloma Cell ◽

Myeloma Cells ◽

B Cell Survival

By using two-color phenotypic analysis with fluorescein isothiocyanate- anti-CD38 and phycoerythrin-anti-CD19 antibodies, we found that pre-B cells (CD38+CD19+) signifcantly decreased depending on the number of plasma cells (CD38++CD19+) in the bone marrow (BM) in the cases with BM plasmacytosis, such as myelomas and even polyclonal gammopathy. To clarify how plasma cells suppress survival of pre-B cells, we examined the effect of plasma cells on the survival of pre-B cells with or without BM-derived stromal cells in vitro. Pre-B cells alone rapidly entered apoptosis, but interleukin-7 (IL-7), a BM stromal cell line (KM- 102), or culture supernatants of KM-102 cells could support pre-B cell survival. On the other hand, inhibitory factors such as transforming growth factor-beta1 (TGF-beta1) and macrophage inflammatory protein- 1beta (MIP-1beta) could suppress survival of pre-B cells even in the presence of IL-7. Plasma cells alone could not suppress survival of pre- B cells in the presence of IL-7, but coculture of plasma cells with KM- 102 cells or primary BM stromal cells induced apoptosis of pre-B cells. Supernatants of coculture with KM-102 and myeloma cell lines (KMS-5) also could suppress survival of pre-B cells. Furthermore, we examined the expression of IL-7, TGF-beta1, and MIP-1beta mRNA in KM-102 cells and primary stromal cells cocultured with myeloma cell lines (KMS-5). In these cells, IL-7 mRNA was downregulated, but the expression of TGF- beta1 and MIP-1beta mRNA was augmented. Therefore, these results suggest that BM-derived stromal cells attached to plasma (myeloma) cells were modulated to secrete lesser levels of supporting factor (IL- 7) and higher levels of inhibitory factors (TGF-beta1 and MIP-1beta) for pre-B cell survival, which could explain why the increased number of plasma (myeloma) cells induced suppression of pre-B cells in the BM. This phenomenon may represent a feedback loop between pre-B cells and plasma cells via BM stromal cells in the BM.

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Chronic Lymphocytic Leukemia B Cells Are Resistant to the Apoptotic Effects of Transforming Growth Factor-β

Blood ◽

10.1182/blood.v89.3.941 ◽

1997 ◽

Vol 89 (3) ◽

pp. 941-947 ◽

Cited By ~ 64

Author(s):

Raymond S. Douglas ◽

Renold J. Capocasale ◽

Roberta J. Lamb ◽

Peter C. Nowell ◽

Jonni S. Moore

Keyword(s):

B Cells ◽

Growth Factor ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Lymphocytic Leukemia ◽

Western World ◽

Spontaneous Apoptosis

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia of the western world and is characterized by a slowly progressing accumulation of clonal CD5+ B cells. Our laboratory has investigated the role of transforming growth factor-β (TGF-β) and interleukin-4 (IL-4) in the pathogenesis of B-cell expansion in CLL. In vitro addition of TGF-β did not increase spontaneous apoptosis of B cells from most CLL patients, as determined using the TUNEL method, compared with a twofold increase observed in cultures of normal B cells. There was similar expression of TGF-β type II receptors on both CLL B cells and normal B cells. In contrast to apoptosis, CLL B-cell proliferation was variably inhibited with addition of TGF-β. In vitro addition of IL-4, previously reported to promote CLL B-cell survival, dramatically reduced spontaneous apoptosis of CLL B cells compared with normal B cells. CLL B-cell expression of IL-4 receptors was increased compared to normal B cells. Thus, our results show aberrant apoptotic responses of CLL B cells to TGF-β and IL-4, perhaps contributing to the relative expansion of the neoplastic clone.

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