Stroma-Activated Integrin-Linked Kinase (ILK) Supports Survival of Leukemic Cells Via Stimulation of Notch-Hes Signaling: New Therapeutic Opportunities.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2466-2466
Author(s):  
Yoko Tabe ◽  
Linhua Jin ◽  
Nobuko Tanaka ◽  
Michael Andreeff ◽  
Marina Konopleva
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Survival ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Leukemic Cells ◽  
Nb4 Cells ◽  
Hes1 Expression ◽  
Integrin Linked Kinase

Abstract We have previously demonstrated that the BM microenvironment plays a crucial role in the pathogenesis of AML by influencing tumor growth, survival, and drug resistance. Integrin-linked kinase (ILK) has been shown to directly interact with β integrins and phosphorylate AKT in a PI3-kinase (PI3K)-dependent manner to promote cell survival and proliferation. HES-1 encodes a basic helix-loop-helix transcription factor downstream of the Notch receptor, and functions as a positive regulator of hematopoietic and neuronal stem cell self-renewal. Direct co-culture of human mesenchymal stem cell (MSC) and leukemic NB4 cells results in activation of PI3K/ILK/AKT signaling (elevated phospho(p)-Akt, p-GSK3β and nuclear-localized β-catenin), increased expression of Notch1 and Hes1 proteins and upregulation of p-STAT3 detected by Western blot and confocal microscopic analyses. Both, PI3K inhibitor LY294002 (20 μM) and ILK inhibitor QLT0254 (10 μM) specifically inhibited stroma-induced activation of AKT and Stat-3 signaling, suppressed GSK phosphorylation and decreased Notch 1 and HES1 expression. This resulted in massive induction of apoptosis which was not abrogated by stromal co-culture (AnnexinV positivity %, MSC(-) vs MSC(+); control 33.8±2.5 vs 27.3±1.9 p=0.02, QLT 51.4±2.5 vs 55.8±3.5 p=0.26, LY 47.0±8.1 vs 47.9±6.1 p=0.85, 48hrs). In contrast, GSK3b inhibitor BIO (0.1 μM) prevented the serum-withdrawal-induced apoptosis of NB4 cells (AnnexinV positivity %, control 38.1±4.0 vs BIO 25.9±3.4 p=0.003, 48hrs) with marked increase in Notch1 and Hes1 expression detected by confocal microscopy. These observations indicate that Notch signaling is involved in leukemic cell survival stimulated by BM stromal interactions via activation of the ILK-AKT-GSK3β pathway. We have next investigated the effects of leukemic cells on stroma cells. Coculture with NB4 cells caused significant increase in Hes1 and Bcl2 proteins in MSC along with phosphorylation of STAT3 and Akt, which were all abrogated by the treatment with QLT0254 or LY294002. In summary, these results demonstrate that interactions of leukemic and bone marrow stromal cells result in activation of PI3K/ILK/AKT and Notch-Hes signaling in both, leukemic and stromal cells. Disruption of these interactions by specific ILK inhibitors represents a novel therapeutic approach to eradicate leukemia in the bone marrow microenvironment via direct effects on leukemic cells and by targeting activated bone marrow stromal cells.

scholarly journals A Method for Measurement of Drug Sensitivity of Myeloma Cells Co-Cultured with Bone Marrow Stromal Cells

2013 ◽  
Vol 18 (6) ◽  
pp. 637-646 ◽  
Author(s):  
Kristine Misund ◽  
Katarzyna A. Baranowska ◽  
Toril Holien ◽  
Christoph Rampa ◽  
Dionne C. G. Klein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Survival ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Large Scale ◽  
Stromal Cell ◽  
Marrow Stromal Cells ◽  
Cell Nuclei ◽  
Myeloma Cells

The tumor microenvironment can profoundly affect tumor cell survival as well as alter antitumor drug activity. However, conventional anticancer drug screening typically is performed in the absence of stromal cells. Here, we analyzed survival of myeloma cells co-cultured with bone marrow stromal cells (BMSC) using an automated fluorescence microscope platform, ScanR. By staining the cell nuclei with DRAQ5, we could distinguish between BMSC and myeloma cells, based on their staining intensity and nuclear shape. Using the apoptotic marker YO-PRO-1, the effects of drug treatment on the viability of the myeloma cells in the presence of stromal cells could be measured. The method does not require cell staining before incubation with drugs, and less than 5000 cells are required per condition. The method can be used for large-scale screening of anticancer drugs on primary myeloma cells. This study shows the importance of stromal cell support for primary myeloma cell survival in vitro, as half of the cell samples had a marked increase in their viability when cultured in the presence of BMSC. Stromal cell–induced protection against common myeloma drugs is also observed with this method.

Multiple stem cell traits of expanded rat bone marrow stromal cells

2006 ◽  
Vol 199 (2) ◽  
pp. 416-426 ◽  
Author(s):  
Jung Yeon Lim ◽  
Sin-Soo Jeun ◽  
Kyung-Jin Lee ◽  
Ji Hyun Oh ◽  
Seong Muk Kim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Rat Bone

Muc1 Expression Identifies Human Self-Renewing Stem/Progenitor Populations and Is Elevated in AML CD34+ Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4040-4040
Author(s):  
Szabolcs Fatrai ◽  
Simon M.G.J. Daenen ◽  
Edo Vellenga ◽  
Jan J. Schuringa
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Fold Increase ◽  
Marrow Stromal Cells ◽  
Muc1 Expression ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Mucin1 (Muc1) is a membrane glycoprotein which is expressed on most of the normal secretory epithelial cells as well as on hematopoietic cells. It is involved in migration, adhesion and intracellular signalling. Muc1 can be cleaved close to the membrane-proximal region, resulting in an intracellular Muc1 that can associate with or activate various signalling pathway components such as b-catenin, p53 and HIF1a. Based on these properties, Muc1 expression was analysed in human hematopoietic stem/progenitor cells. Muc1 mRNA expression was highest in the immature CD34+/CD38− cells and was reduced upon maturation towards the progenitor stage. Cord blood (CB) CD34+ cells were sorted into Muc1+ and Muc1− populations followed by CFC and LTC-IC assays and these experiments revealed that the stem and progenitor cells reside predominantly in the CD34+/Muc1+ fraction. Importantly, we observed strongly increased Muc1 expression in the CD34+ subfraction of AML mononuclear cells. These results tempted us to further study the role of Muc1 overexpression in human CD34+ stem/progenitor cells. Full-length Muc1 (Muc1F) and a Muc1 isoform with a deleted extracellular domain (DTR) were stably expressed in CB CD34+ cells using a retroviral approach. Upon coculture with MS5 bone marrow stromal cells, a two-fold increase in expansion of suspension cells was observed in both Muc1F and DTR cultures. In line with these results, we observed an increase in progenitor counts in the Muc1F and DTR group as determined by CFC assays in methylcellulose. Upon replating of CFC cultures, Muc1F and DTR were giving rise to secondary colonies in contrast to empty vector control groups, indicating that self-renewal was imposed on progenitors by expression of Muc1. A 3-fold and 2-fold increase in stem cell frequencies was observed in the DTR and Muc1F groups, respectively, as determined by LTC-IC assays. To determine whether the above mentioned phenotypes in MS5 co-cultures were stroma-dependent, we expanded Muc1F and DTR-transduced cells in cytokine-driven liquid cultures. However, no proliferative advantage or increase in CFC frequencies was observed suggesting that Muc1 requires bone marrow stromal cells. In conclusion, our data indicate that HSCs as well as AML cells are enriched for Muc1 expression, and that overexpression of Muc1 in CB cells is sufficient to increase both progenitor and stem cell frequencies.

Partial Characterization and In Vitro Expansion of Putative CLL Precursor/Stem Cells Which Are Dependent on Bone Marrow Microenvironment for Survival

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2433-2433
Author(s):  
Medhat Shehata ◽  
Rainer Hubmann ◽  
Martin Hilgarth ◽  
Susanne Schnabl ◽  
Dita Demirtas ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Healthy Persons

Abstract Abstract 2433 Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of B lymphocytes which typically express CD19 and CD5. The disease remains incurable and recurrence often occurs after current standard therapies due to residual disease or probably due to the presence of therapy-resistant CLL precursors. Based on the growing evidence for the existence of leukemia stem cells, this study was designed to search for putative CLL precursors/stem cells based on the co-expression of CLL cell markers (CD19/CD5) with the hematopoietic stem cell marker (CD34). Forty seven CLL patients and 17 healthy persons were enrolled in the study. Twenty four patients had no previous treatment and 23 had pre-therapy. Twenty two patients were in Binet stage C and 25 patients in B. Twenty two patients had unmutated and 18 mutated IgVH gene (7: ND). Cytogenetic analysis by FISH showed that 14 patients had del 13q, 8 had del 11q, 4 had del 17p and 9 had trisomy 12. Peripheral blood and bone marrow mononuclear cells were subjected to multi-colour FACS analysis using anti-human antibodies against CD34, CD19 and CD5 surface antigens. The results revealed the presence of triple positive CD34+/CD19+/CD5+ cells in CLL samples (mean 0.13%; range 0.01–0.41) and in healthy donors (0.31%; range 0.02–0.6) within the CD19+ B cells. However, due to the high leukocyte count in CLL patients, the absolute number of these cells was significantly higher in CLL samples (mean: 78.7; range 2.5–295 cells /μL blood) compared to healthy persons (mean: 0.45: range 0.04–2.5 cells/μl)(p<0,001). These triple positive “putative CLL stem cells” (PCLLSC) co-express CD133 (67%), CD38 (87%), CD127 (52%), CD10 (49%), CD20 (61%), CD23 (96%), CD44 (98%) and CD49d (74%). FISH analysis on 4 patients with documented chromosomal abnormalities detected the corresponding chromosomal aberrations of the mature clone in the sorted CD34+/CD5+/CD19+ and/or CD34+/CD19-/CD5- cells but not in the CD3+ T cells. Multiplex RT-PCR analysis using IgVH family specific primer sets confirmed the clonality of these cells. Morphologically, PCLLSC appeared larger than lymphocytes with narrow cytoplasm and showed polarity and motility in co-culture with human bone marrow stromal cells. Using our co-culture microenvironment model (Shehata et al, Blood 2010), sorted cell fractions (A: CD34+/19+/5+, B: CD34+/19-/5- or C: CD34-/CD19+/5+) from 4 patients were co-cultured with primary autologous human stromal cells. PCLLSC could be expanded in the co-culture to more than 90% purity from fraction A and B but not from fraction C. These cells remained in close contact or migrated through the stromal cells. PCLLSC required the contact with stromal cells for survival and died within 1–3 days in suspension culture suggesting their dependence on bone marrow microenvironment or stem cell niches. RT-PCR demonstrated that these cells belong to the established CLL clone. They also eexpress Pax5, IL-7R, Notch1, Notch2 and PTEN mRNA which are known to play a key role in the early stages of B cells development and might be relevant to the early development of the malignant clone in CLL. Using NOD/SCID/IL2R-gamma-null (NOG) xenogeneic mouse system we co-transplanted CLL cells from 3 patients (5 million PBMC/mouse) together with autologous bone marrow stromal cells (Ratio: 10:1). The percentage of PCLLSC in the transplanted PBMC was 0.18% (range 0.06–0.34%). Using human-specific antibodies, human CD45+ cells were detected in peripharal blood of the mice (mean 0.9 % range 0.47–1.63%) after 2 months of transplantation. More than 90% of the human cells were positive for CD45 and CD5. Among this population, 26% (range 15–35%) of the cells co-expressed CD45, CD19, CD5 and CD34 and thus correspond to the PCLLSC. In conclusion, our data suggest the existence of putative CLL precursors/stem cells which reside within the CD34+ hematopoietic stem cell compartment and carry the chromosomal aberrations of the established CLL clone. These cells could be expanded in vitro in a bone marrow stroma-dependent manner and could be engrafted and significantly enriched in vivo in NOG xenotransplant system. Further characterization and selective targeting and eradication of these cells may pave the way for designing curative therapeutic strategies for CLL. Disclosures: No relevant conflicts of interest to declare.

Protection of ALL Cells by Bone Marrow Stromal Cells and the Underlying Molecular Mechanism.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2587-2587
Author(s):  
Yang Yang ◽  
Baohua Sun ◽  
Saradhi Mallampati ◽  
Zhen Cai ◽  
Xiaoping Sun
Keyword(s):  
Bone Marrow ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Stromal Cells ◽  
Cell Apoptosis ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Leukemic Cells ◽  
New Therapies ◽  
Induced Apoptosis

Abstract Abstract 2587 Acute lymphoblastic leukemia (ALL) is one of the fastest-growing hematological malignancies affecting patients with all ages, particularly children. Significant advances have been made in recent years in our understanding of the disease and the development of new therapies, which have led to a greatly improved outcome. Nevertheless, in a significant number of patients with ALL, the disease relapse and become resistant to treatment, causing death of the patients. Increasing evidence suggests that relapse of the disease and resistant to treatment are largely attributed to the protection of the leukemic cells by various components in the microenvironment, such as bone marrow stromal cells. However, the cross-talk between leukemic cells and their microenvironment remains poorly understood. Therefore, better understanding the mechanisms underlying the protection of ALL cells by the microenvironment is of ultimate importance in developing new therapies targeting such protection and eventually eradicating all the leukemic cells to cure the disease. In this study, we used a coculture system with leukemic cells and bone marrow stromal cells (MSC) to mimic the in vivo interaction between the two cell types to explore the molecular events that might be responsible for the protection of ALL cells from Ara-C induced apoptosis. We cocultured human primary ALL cells with hTERT-immortalized normal human MSC and evaluated ALL cell apoptosis by FACS after staining with Annexin V and propidium iodide. In all 8 cases, the MSC provided significant protection of ALL cells from both spontaneous and Ara-C induced apoptosis. For example, the mean Ara-C induced apoptosis of ALL cells cultured without MCS was 42.7% (range, 27–54%), whereas it was 19.1% (range, 8–27%) with MSC. Similar results were obtained with human leukemia cell lines Reh, SEMK2 and RS4.11. We also found that the murine MSC line M210B4 could provide similar protection to ALL cells, whether the ALL cells are primary or cell lines. The reduced apoptosis in the coculture were confirmed by Western blot which showed that MSC could protect ALL cells from Caspase-3 and PARP cleavage. Furthermore, our results showed no significant Ara-C induced reduction in S phase when cocultured with MSC. This phenomenon was associated with decreased cyclinA and CDK2 expression. In addition, we found that cocultured with MSC resulted in phosphorylation of AKT in ALL cells and PI3K inhibitor LY294002 specifically inhibited MSC-induced activation of AKT and promoted ALL cell apoptosis. In addition, beta-catenin and c-myc had increased expression in ALL cells cocultured with MSC, suggesting that Wnt pathway could play a role in MSC-mediated protection. To identify candidate molecules potentially involved in the protection of ALL cells by MSC, we performed gene expression microarray analyses with ALL cells exposed to Ara-C in presence or absence of MSC. Our data indicated that several signaling pathways might be involved in this process, including apoptosis signaling and cell cycle checkpoint control, which confirmed above findings. The top expressed genes identified in the microarray studies were confirmed by RT-PCR. Collectively, our results demonstrated that MSC can protect ALL cells from Ara-C induced apoptosis by multiple signaling pathways, such as those involving PI3K/AKT and Wnt signaling. Hence, targeting these pathways may become potential novel therapeutic strategies to disrupt the support of the microenvironment to ALL cells and to eventually eradicate leukemic cells. Disclosures: No relevant conflicts of interest to declare.

Green Tea Extract EGCG Induces Apoptosis in CLL Cells and Overcomes the Supportive Effect of Primary Bone Marrow Stromal Cells Through the Regulation of PI3K/Akt Cascade and Proteasome Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3916-3916 ◽  
Author(s):  
Elena Ponath ◽  
Susanne Schnabl ◽  
Martin Hilgarth ◽  
Dita Demirtas ◽  
Marlies Reiter ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Viability ◽  
Green Tea ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Cell Killing ◽  
Marrow Stromal Cells ◽  
Proteasome Activity ◽  
Leukemic Cells ◽  
Tea Extract

Abstract Abstract 3916 There is accumulating evidence that green tea extract EGCG [(-)-epigallocatechin-3-gallate] may exert a preventive or a direct anti-tumor effect in several tumor types including chronic lymphocytic leukemia (CLL) and clinical trials with EGCG are already on-going. However, EGCG has a broad spectrum of activities and downstream targets. Therefore, it would be necessary to precisely characterize the key targets of this compound and identify the CLL patients who would most likely profit from EGCG. Therefore, the aim of this study was to evaluate the effect of EGCG on the viability of CLL cells in a well characterized cohort of patients and to get insight into its mechanism of action in CLL. Peripheral blood mononuclear cells (PBMC) of 27 CLL patients were included in this study. Patients were characterized according to the Rai/Binet stage, IgVH mutation status and cytogenetics (13q-del, 11q-del, 17p-del, trisomy-12). The percentage of the leukemic cells (CD19+/CD5+) ranged between 60–98%. CLL cells were exposed to a wide range of concentrations of EGCG (0.1 – 200μM) and cell viability was evaluated by cell titer blue (CTB) assays and FACS analysis after 4 hours, 1, 2 and 3 days. Treatment with EGCG was performed in suspension cultures and under co-culture with primary human bone marrow stromal cells (BMSC). Cell viability assays demonstrated a dose and time dependent decrease in the cell viability after the exposure to EGCG with an IC50 ranging between 50–80μM (25–50μg/ml). A moderate variation in the response to EGCG was observed between patients demonstrating the heterogeneity of the disease. No clear correlation between the in vitro response to EGCG and the clinical background and prognostic markers could be observed in this cohort of patients. Annexin V/propidium iodide (Anx/PI) staining showed that EGCG increased the percentage of early apoptotic (Anx+/PI-) and late apoptotic/necrotic cells (Anx+/PI+). These data suggest that EGCG exerts a pro-apoptotic effect and activates other cell killing mechanisms in CLL cells. The leukemic cells (CD19/CD5) were relatively more sensitive to the compound compared to T cells and monocytes. Co-culture experiments showed that EGCG effectively overcomes the supportive effect of BMSC and induces apoptosis/cell killing in CLL cells. BMSC were less sensitive to the compound and a toxic effect was observed at a concentration of 200 μM or higher. RT-PCR showed a downregulation of the catalytic domain p110a and the regulatory domain p85 of phosphoinositide 3-kinases (PI3K) as well as Bcl-2 and Mcl-1 mRNA expression after exposure to EGCG. Western blotting analysis demonstrated a decrease in the phosphorylation of Akt particularly at pThr308 residue and de-phosphorylation of the tumor suppressor PTEN at pSer380 residue in parallel to the induction of PARP cleavage. In addition, EGCG induced a decrease in the protein expression of the activation marker CD23 and the adhesion molecule CD44. Furthermore, proteasome assays showed that EGCG inhibits the chymotrypsin-like activity within 4 hours of incubation in parallel to induction of early apoptosis. This effect was more remarkable after 24 hours. However, EGCG was less effective in proteasome inhibition compared to Bortezomib. In conclusion, these data demonstrate that EGCG induces cell death in CLL cells through a complex mechanism which may involve the inactivation of PI3K/Akt signaling cascade and inhibition of proteasome activity. The results also point to a potential therapeutic effect of EGCG in CLL which warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

Phase 1 Trial Of Bone Marrow Stromal Cells (Bone Marrow-derived MSCS) To Treat Tissue Damage In Allogeneic Stem Cell Transplant Recipients: Biological Markers Correlate With Clinical Responses and Survival

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3282-3282
Author(s):  
Minoo Battiwalla ◽  
Fang Yin ◽  
Sawa Ito ◽  
Xingmin Feng ◽  
Fariba Chinian ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Tissue Injury ◽  
Marrow Stromal Cells ◽  
Fixed Dose ◽  
Cell Transplant ◽  
Clinical Responses ◽  
Steroid Refractory

Abstract Bone marrow stromal cells (BMSC, also known as bone marrow-derived “mesenchymal stem cells”) have been used to treat acute graft-versus-host disease (GVHD) and other complications following allogeneic hematopoietic stem cell transplantation (SCT). We conducted a phase I trial using third party, early passage, BMSC for patients with steroid-refractory liver or gastrointestinal GVHD, tissue injury or marrow failure following SCT to investigate safety and clinical responses following BMSC infusion. To identify mechanisms of BMSC immunomodulation and tissue repair, patients were monitored for plasma GVHD biomarkers, cytokines, growth factors, and lymphocyte phenotype before and after BMSC infusion. BMSCs were prepared from marrow aspirates from healthy volunteers with the expansion of 3 passages. Ten subjects were infused a fixed dose of 2 x 106 BMSCs /kg weekly for 3 doses. There was no treatment related toxicity (primary endpoint). Eight subjects were evaluable for response assessment at 4 weeks after the last infusion. Five of the seven patients with steroid-refractory acute GVHD achieved complete remission (CR), two of two patients with tissue injury (pneumomediastinum/ pneumothorax) achieved resolution but there was no response in two subjects with delayed marrow failure. Rapid reductions in inflammatory cytokines occurred after the first BMSC infusion (fig1). Clinical responses correlated with a fall in biomarkers (Reg 3α, CK18, and Elafin) relevant for the site of GVHD, or CK18 for tissue injury. The GVHD complete responders survived significantly longer (>300 days vs a median of 33 days), had higher baseline absolute lymphocyte and central memory CD4 and CD8 counts but there was no clear difference in natural or induced Tregs. Cytokine changes also segregated with survival. These results confirm that BMSC are associated with rapid clinical responses and biomarker normalization in steroid-refractory GVHD and PM. However BMSC were ineffective in patients with more aggressive GVHD with lower lymphocyte counts, which suggest that effective GVHD control by BMSC, requires a relatively intact immune system. Early detection and BMSC treatment appear important in patients with refractory GVHD. Disclosures: No relevant conflicts of interest to declare.

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