scholarly journals The PI3-Kinase Delta Inhibitor Idelalisib (GS-1101) Targets Integrin-Mediated Adhesion of Chronic Lymphocytic Leukemia (CLL) Cell to Endothelial and Marrow Stromal Cells

PLoS ONE ◽  
2013 ◽  
Vol 8 (12) ◽  
pp. e83830 ◽  
Author(s):  
Stefania Fiorcari ◽  
Wells S. Brown ◽  
Bradley W. McIntyre ◽  
Zeev Estrov ◽  
Rossana Maffei ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Pi3 Kinase ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells

Standardizing Co-Culture Conditions Between Chronic Lymphocytic Leukemia Cells and Marrow Stromal Cells: Towards a Reliable and Reproducible System to Assess Cell Adhesion-Mediated Drug Resistance

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3149-3149
Author(s):  
Antonina Kurtova ◽  
Maite P. Quiroga ◽  
William G. Wierda ◽  
Michael Keating ◽  
Jan A. Burger
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells ◽  
Hpv E6 ◽  
Induced Apoptosis

Abstract Contact between chronic lymphocytic leukemia (CLL) cells and accessory stromal cells in tissue microenvironments is considered to play a major role in regulating CLL cell survival and disease progression. Stromal cells of various origins and species, and variable stromal-CLL cell ratios have been used in the past to study CLL-stromal cell interactions and to assess cell-adhesion mediated drug resistance (CAM-DR). Because of the heterogeneity of the currently used in vitro systems to study CLL-MSC interactions, and the importance of these co-culture systems for development and testing of novel agents, we tested a panel of murine and human MSC lines for their capacities to support CLL cell survival and CAM-DR, using various CLL-MSC ratios and fludarabine (F-ara-A) to induce CLL cell apoptosis. We tested four murine, non-transformed MSC lines derived from bone marrow: M210B4, KUM4, ST-2 and KUSA-H1. Also, we tested three human transformed cell lines: Stroma-NKtert, derived from bone marrow and immortalized by human telomerase reverse transcriptase (hTERT), UE6E7-T2 derived from bone marrow and transformed with human papilloma viruses (HPV) E6, E7 and hTERT, and UCB408E6E7Tert33 derived from umbilical cord blood and transformed with hTERT and HPV E6, E7. CLL cells were isolated from peripheral blood of untreated patients and each cell line was tested with at least three different patients according to the following protocol: viability of CLL was tested after 24, 48 and 72 hours by flow cytometry after staining with DiOC6 and propidium iodide. The following conditions were assayed on each of the MSC lines: CLL cells in suspension culture, CLL cells in suspension culture with 10 mM F-ara-A, CLL cells in co-culture with MSC, and CLL cells in co-culture with MSC and with 10 mM F-ara-A. Firstly, we performed titration experiments in order to identify the most appropriate ratio between stromal and CLL cells, using CLL-MSC ratios of 5:1, 10:1, 20:1, 50:1 and 100:1. We found a decline in MSC-derived CLL cell protection at the highest ratio of 100:1, suggesting that ratios of 50:1 or lower provide optimal conditions for in vitro assays. Results shown in Table 1 were assayed using a 20:1 ratio and represented relative viabilities when compared to untreated controls (mean±SEM). Regarding the protective effect of different MSC, we found that all MSC lines demonstrated remarkable protection of CLL cells from spontaneous and F-ara-A-induced apoptosis. We also found that stromal cells that had round shape morphology and easily formed confluent monolayer (M210B4, KUSA-H1, Stroma-NKTert) showed more prolonged protective effect in comparison to cell lines with more spindle shaped morphology (ST-2, KUM4, UE6E7-T2). The failure of UE6E7-T2 and UCB408E6E7Tert33 to demonstrate long-term protection of CLL cells could be related to their own sensitivity to F-ara-A. In this comparative study we demonstrated that both murine and human MSC provide substantial and comparable levels of protection from spontaneous and drug-induced apoptosis. CLL:MSC ratios of 50:1 or lower can be considered ideal for co-culture experiments. Further experiments have to be done to determine the levels of MSC-derived protection in a larger series of CLL samples and in different laboratories for validation. Collectively, in these co-culture assays we can study CLL-MSC interactions and CLL drugs under more standardized conditions that may allow us to evaluate the efficacy of new treatments that target the CLL microenvironment. Time points 24 hours 48 hours 72 hours +Flu + MSC + MSC +Flu +Flu + MSC + MSC +Flu +Flu +MSC + MSC +Flu M210B4 85.2±2.4 117.2±5.0 110.5±4.9 30.8±12.6 138.1±9.5 113.0±2.2 5.2±3.1 138.1±5.1 120.4±3.4 ST-2 93.6±3.0 99.9±2.6 103.1±0.5 51.6±9.4 111.9±2.6 89.8±8.7 13.9±6.3 112.6±5.7 87.0±16.4 KUM-4 93.6±3.0 106.4±1.8 104.2±1.9 51.6±9.4 112.4±2.6 100.8±2.8 13.9±6.3 111.8±6.7 88.5±11.4 KUSA-H1 79.4±7.4 125.1±3.7 118.2±2.0 33.9±10.9 136.0±3.6 107.2±7.0 11.3±6.1 133.6±5.4 84.9±7.6 Stroma-NKTert 79.3±7.0 118.6±7.0 111.0±7.0 30.5±9.5 130.7±9.5 115.6±8.0 7.1±4.3 133.0±11.5 122.7±9.0 UE6E7-T2 79.3±7.0 113.4±3.9 109.3±3.0 30.5±9.5 118.4±4.8 85.0±7.1 7.1±4.3 119.2±6.9 51.0±10.1 UCB408 E6E7Tert33 81.5±7.2 120.2±5.4 111.8±2.7 36.7±9.4 123.7±6.3 86.7±7.7 8.5±6.7 119.7±6.1 50.8±13.0 Table 1. Flu: fludarabine (10mM/ml), MSC: marrow stromal cells

Chronic Lymphocytic Leukemia B Cells Express Functional CXCR4 Chemokine Receptors That Mediate Spontaneous Migration Beneath Bone Marrow Stromal Cells

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3658-3667 ◽  
Author(s):  
Jan A. Burger ◽  
Meike Burger ◽  
Thomas J. Kipps
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Pertussis Toxin ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells

Chemokines play a central role for lymphocyte trafficking and homing. The mechanisms that direct the tissue localization of B cells from patients with chronic lymphocytic leukemia (B-CLL) are unknown. We found that CLL B cells express functional CXCR4 receptors for the chemokine stromal cell-derived factor-1 (SDF-1), as demonstrated by receptor endocytosis, calcium mobilization, and actin polymerization assays. Moreover, CLL B cells displayed chemotaxis to this chemokine that could be inhibited by monoclonal antibodies (MoAbs) against CXCR4, pertussis toxin, or Wortmannin, a phosphatidylinositol 3-kinase inhibitor. That this chemotaxis may be involved in the homing of CLL cells is argued by studies in which CLL B cells were cocultured with a murine marrow stromal cell line that secretes SDF-1. Within 2 hours, CLL B cells spontaneously migrated beneath such stromal cells in vitro (pseudoemperipolesis). This migration could be inhibited by pretreatment of CLL B cells with anti-CXCR4 MoAbs, SDF-1, or pertussis-toxin. Furthermore, we noted strong downmodulation of CXCR4 on CLL B cells that migrated into the stromal cell layer. These findings demonstrate that the chemokine receptor CXCR4 on CLL B cells plays a critical role for heterotypic adherence to marrow stromal cells and provide a new mechanism to account for the marrow infiltration by neoplastic B cells.

Chronic Lymphocytic Leukemia B Cells Express Functional CXCR4 Chemokine Receptors That Mediate Spontaneous Migration Beneath Bone Marrow Stromal Cells

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3658-3667 ◽  
Author(s):  
Jan A. Burger ◽  
Meike Burger ◽  
Thomas J. Kipps
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Pertussis Toxin ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells

Abstract Chemokines play a central role for lymphocyte trafficking and homing. The mechanisms that direct the tissue localization of B cells from patients with chronic lymphocytic leukemia (B-CLL) are unknown. We found that CLL B cells express functional CXCR4 receptors for the chemokine stromal cell-derived factor-1 (SDF-1), as demonstrated by receptor endocytosis, calcium mobilization, and actin polymerization assays. Moreover, CLL B cells displayed chemotaxis to this chemokine that could be inhibited by monoclonal antibodies (MoAbs) against CXCR4, pertussis toxin, or Wortmannin, a phosphatidylinositol 3-kinase inhibitor. That this chemotaxis may be involved in the homing of CLL cells is argued by studies in which CLL B cells were cocultured with a murine marrow stromal cell line that secretes SDF-1. Within 2 hours, CLL B cells spontaneously migrated beneath such stromal cells in vitro (pseudoemperipolesis). This migration could be inhibited by pretreatment of CLL B cells with anti-CXCR4 MoAbs, SDF-1, or pertussis-toxin. Furthermore, we noted strong downmodulation of CXCR4 on CLL B cells that migrated into the stromal cell layer. These findings demonstrate that the chemokine receptor CXCR4 on CLL B cells plays a critical role for heterotypic adherence to marrow stromal cells and provide a new mechanism to account for the marrow infiltration by neoplastic B cells.

Marrow Stromal Cells Induce TCL1 Expression in Chronic Lymphocytic Leukemia B Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2347-2347
Author(s):  
Mariela Sivina ◽  
Elena Hartmann ◽  
Michael Keating ◽  
William G Wierda ◽  
Andreas Rosenwald ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Stromal Cells ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells ◽  
Immunoglobulin Gene ◽  
Prolymphocytic Leukemia

Abstract Abstract 2347 Poster Board II-324 The human T cell leukemia/lymphoma 1 (TCL1) oncogene was initially identified as a target of chromosomal translocations and inversions at the 14q32.1 chromosome breakpoint region in T-cell prolymphocytic leukemia (T-PLL). Increased TCL1 expression is seen in follicular lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, and chronic lymphocytic leukemia (CLL). Transgenic mice over-expressing TCL1 under control of the mu immunoglobulin gene enhancer develop a CD5+ B cell lymphoproliferative disorder that mimics human CLL, indicating that TCL1 plays a central and/or causal role in the pathogenesis of CLL. However, chromosome aberrations that constitutively activate TCL1 have not (yet) been identified in the vast majority of CLL patients, and therefore the oncogenic mechanism(s) of TCL1 activation in CLL remain unclear. There is growing evidence that external signals from the microenvironment control and regulate the survival and proliferation of CLL cells. Marrow stromal cells (MSC) are highly effective in protecting CLL cells from spontaneous and drug-induced apoptosis, and are used as a model system to study the marrow microenvironment. In order to explore the molecular cross talk between CLL cells and MSC, we co-cultured CLL cells with different MSC and analyzed gene expression changes induced by co-cultures with MSC, an approach similar to our recent study with nurselike cells (Blood 113:3050-8, 2009). For this, RNA was extracted from 19-purified CLL cells from 10 different patients (baseline expression, day 0). Also, the same patients' samples were co-cultured on stroma cells (KUSA-H1, NK-Tert) for 2 and 7 days. At these time points, RNA again was isolated after CD19-purification. Then, gene expression was determined using HG U133 plus 2.0 oligonucleotide arrays from Affymetrix. Gene expression changes were analyzed in individual patients' samples, comparing baseline samples' gene expression to samples after 2 and 7 of co-culture on MSC. We observed relatively homogeneous gene expression changes in CLL cells after co-culture with MSC. We found that TCL1 was among the top 5 genes that were most highly up-regulated by MSC, based on at least 3-fold up-regulation in at least 6 of the paired samples. We also found an up-regulation of TCL1 at the protein level when assessed by immunoblotting and flow cytometry in CLL samples after co-culture with MSC. These findings indicate that MSC can induce and regulate TCL1 expression in CLL, suggesting that the microenvironment plays an even greater role in the pathogenesis of this disease than previously recognized. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Circulating microvesicles in B-cell chronic lymphocytic leukemia can stimulate marrow stromal cells: implications for disease progression

Blood ◽  
2010 ◽  
Vol 115 (9) ◽  
pp. 1755-1764 ◽  
Author(s):  
Asish K. Ghosh ◽  
Charla R. Secreto ◽  
Traci R. Knox ◽  
Wei Ding ◽  
Debabrata Mukhopadhyay ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Microenvironment ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Disease Progression ◽  
Stromal Cells ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells ◽  
Akt Activation ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Microvesicles (MVs) released by malignant cancer cells constitute an important part of the tumor microenvironment. They can transfer various messages to target cells and may be critical to disease progression. Here, we demonstrate that MVs circulating in plasma of B-cell chronic lymphocytic leukemia (CLL) patients exhibit a phenotypic shift from predominantly platelet derived in early stage to leukemic B-cell derived at advanced stage. Furthermore, the total MV level in CLL was significantly greater compared with healthy subjects. To understand the functional implication, we examined whether MVs can interact and modulate CLL bone marrow stromal cells (BMSCs) known to provide a “homing and nurturing” environment for CLL B cells. We found that CLL-MV can activate the AKT/mammalian target of rapamycin/p70S6K/hypoxia-inducible factor-1α axis in CLL-BMSCs with production of vascular endothelial growth factor, a survival factor for CLL B cells. Moreover, MV-mediated AKT activation led to modulation of the β-catenin pathway and increased expression of cyclin D1 and c-myc in BMSCs. We found MV delivered phospho-receptor tyrosine kinase Axl directly to the BMSCs in association with AKT activation. This study demonstrates the existence of separate MV phenotypes during leukemic disease progression and underscores the important role of MVs in activation of the tumor microenvironment.

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