Targeting p62 in Marrow Stromal Cells Is Effective at Inhibiting Myeloma Cell Growth.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 513-513 ◽  
Author(s):  
Noriyoshi Kurihara ◽  
Y. Hiruma ◽  
J. Windle ◽  
C.S. Hong ◽  
J. Shin ◽  
...  
Keyword(s):  
Cell Growth ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Bone Destruction ◽  
Myeloma Cell ◽  
Marrow Stromal Cells ◽  
Bone Microenvironment ◽  
Wild Type ◽  
Myeloma Cells ◽  
Tnf Α

Abstract Release of growth factors from bone by osteoclastic bone destruction and by tumor-stromal cell interactions play critical roles in promoting myeloma cell growth. In particular, expression of the cytokines RANK ligand (RANKL), a potent inducer of osteoclast (OCL) formation, IL-6, tumor necrosis factor-α (TNF-α) and MCP-1 are upregulated in the bone microenvironment in response to myeloma (MM) cells as well as by adhesive interactions between myeloma cells and marrow stromal cells through VCAM-1 on stromal cells. Therefore, agents that can both inhibit OCL formation and block the effects of myeloma stromal cell interactions should have a major impact on both bone destruction and tumor growth. p62 plays a critical role in NF-κB activation induced by TNF-α, RANKL and IL-1 and is involved in multiple signaling pathways that result in enhanced tumor cell survival and bone destruction. It is our hypothesis that inhibiting p62 expression will profoundly diminish osteolytic bone destruction and myeloma growth in patients, by blocking production of RANKL, MCP-1, TNF-α and IL-6 in the tumor-bone microenvironment, and the upregulation of VCAM-1 on stromal cells. Therefore, we used p62 −/− mice to determine the effects of deleting p62 in stromal cells on the growth of myeloma cells. Marrow cells from p62 −/− or wild type mice were used to establish long-term Dexter-type marrow cultures to isolate marrow stromal cells. IL-6 and TNF-α production by p62 −/− stromal cells was decreased compared to WT stromal cells. To determine the effects of the lack of p62 on MM cell growth, GFP-labeled MM.1S cells were co-cultured with p62 −/− stromal cells. Growth of MM.1S cells was decreased by 70% in cocultures of p62 −/− mice, and IL-6 and TNF-α levels were not increased in cocultures of tumor cells with p62 −/− stromal cells. Next, we measured the relative expression levels of VCAM-1 on marrow stromal cells by Western blot in cocultures of human myeloma cells with marrow stromal cells. Stromal cells from the p62 −/− or wild type were cultured with and without MM.1S cells for 3 days in separate experiments. The levels of VCAM-1 in p62 −/− stromal cells were lower than p62 +/− stromal cells. In addition, VCAM-1 levels on p62 −/− bone marrow stromal cells were decreased compared to p62 +/− stromal cells when cocultured with MM cells. The addition of 25 ng/ml mouse TNF-α to p62 −/− stromal cells cocultured with MM.1S cells resulted in enhanced MM.1S growth and VCAM-1 production to similar levels as seen with p62 +/− stromal cells cocultured with MM.1S cells. We then determined the capacity of p62 −/− stromal cells to increase MCP-1 production, a chemoattract for myeloma cells, when they were cocultured with human myeloma cells for 48 hours. The conditioned media were collected after 48 hours of culture. Wild type stromal cells produced increased levels of MCP-1 when cocultured with MM.1S cells. MCP-1 levels in p62 −/− stromal cell conditioned media were decreased compared to wild type stromal cell cultures, regardless of whether MM.1S cells were present in the culture (MCP-1 in p62 +/− stromal cell culture, 980± 70pg/ml vs. p62 −/− 380± 10 pg/ml). These results show that p62 plays an important role in myeloma cell growth through regulation of production of cytokines that are upregulated in the marrow microenvironment in response to myeloma, and suggest that p62 is a novel target for treating myeloma.

Targeting p62ZIP in Marrow Stromal Cells Is Highly Effective at Inhibiting Myeloma Cell Growth and Osteoclast Formation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 630-630
Author(s):  
Noriyoshi Kurihara ◽  
Tadashi Honjo ◽  
Jolene J. Windle ◽  
J. Shin ◽  
G. D. Roodman
Keyword(s):  
Cell Growth ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Critical Role ◽  
Bone Destruction ◽  
Myeloma Cell ◽  
Marrow Stromal Cells ◽  
Wild Type ◽  
Myeloma Cells ◽  
Tnf Α

Abstract Marrow stromal cells play a critical role in osteolytic bone destruction in multiple myeloma, and promote tumor growth. In particular, adhesive interactions between myeloma cells and marrow stromal cells increase RANK ligand (RANKL), a potent inducer of osteoclast formation, IL-6 and TNF-α production by marrow stromal cells. IL-6 enhances the growth and prevents apoptosis of myeloma cells, and TNF increases production of RANKL and IL-6. Recently, a new member of NF-κB signaling pathway, p62ZIP, has been identified. p62ZIP plays a critical role in NF-κB activation induced by TNF-α and RANKL and is involved in multiple signaling pathways that result in enhanced IL-6 production, tumor cell survival and bone destruction. It is our hypothesis that inhibiting p62ZIP expression will profoundly diminish myeloma growth by blocking the effects of IL-6 produced in the tumor-bone microenvironment in response to TNF-α. Therefore, we used p62ZIP−/− mice to determine the effects of deleting p62ZIP in stromal cells on the growth of myeloma cells. Marrow cells from p62ZIP −/− or wild type mice were used to establish long-term Dexter-type marrow cultures to isolate marrow stromal cells. Marrow stromal cells from p62ZIP −/− or wild type mice were cocultured for 48 h with a GFP-labeled human MM.1S myeloma cell line (MM.1S) and murine and human RANKL, IL-6 and TNF-α levels were determined in conditioned media from these cocultures using commercial ELISA assays. Cocultures of MM.1S with wild type marrow stromal cells resulted in much greater upregulation of murine IL-6 than p62−/− stromal cell coculture (IL-6 in p62−/− stromal cell cultures; 114+70 vs. WT 1900+9 pg/ml). In addition, deleting p62ZIP in stromal cells markedly decreased the growth of tumor cells. Coculture with wild type stromal cells induce 1.4-fold greater increase in MM.1S cell growth at 72 h compared to p62−/− stromal cells. Further, addition of neutralizing antibodies to TNF-α and IL-6 to the cocultures of MM.1S cells with WT stromal cells similarly affected the growth of the MM.1S. Since TNF-alpha can increase the expression of adhesion molecules on stromal cells and tumor cells, we measured expression levels of ICAM-1 and VCAM-1 by Western blot. VCAM-1 and ICAM-1 levels on p62ZIP−/− bone marrow stromal cells were not changed compared to WT stromal cells. We then determined the capacity of p62 −/− cells to support OCL formation by normal spleen CFU-blast. OCL formation was decreased about 50 % in cocultures containing p62−/− stromal cells treated with PTH-rp, IL-6 and TNF-α compare with WT stromal cell culture. These results show that p62 plays an important role in myeloma cell growth and OCL formation induced by cytokines that are upregulated in the marrow microenvironment in patients with myeloma.

p62 Signaling Is Increased in Multiple Myeloma Microenvironment.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 669-669
Author(s):  
Yuko Hiruma ◽  
Noriyoshi Kurihara ◽  
David Roodman
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Western Blot ◽  
Stromal Cells ◽  
Bone Destruction ◽  
Marrow Stromal Cells ◽  
Myeloma Patient ◽  
Bone Microenvironment ◽  
Tnf Α ◽  
Human Stromal Cells

Abstract The bone microenvironment plays a critical role in promoting both tumor growth and bone destruction in myeloma (MM). Marrow stromal cells produce factors, which stimulate both the growth of MM cells and osteoclastic bone destruction and are key regulators of these processes. Marrow stromal cells produce these factors in increased amounts when they bind MM cells through adhesive interactions mediated via VCAM-1 on stromal cells and β1 integrins on the surface of MM cells. We have examined the role of sequestosome-1 (p62), a recently described member of the NF-κB signaling pathway, in this process, since it sits at the crossroads of multiple signaling pathways potentially involved in both osteoclastogenesis and MM cell growth. In previous studies, we found that stromal cells lacking p62 minimally supported the growth of MM cells or osteoclast (OCL) formation compared to wild-type p62 containing stromal cells. We have further shown that stromal cells lacking p62 produce much lower levels of TNF-α, interleukin (IL)-6, MCP-1 and RANK ligand (RANKL), factors which increase OCL formation, and express lower levels of VCAM-1. However, the mechanisms responsible for this decreased cytokine production and VCAM-1 expression in multiple myeloma patient stromal cells lacking p62 are unknown. Further, it is unknown that targeting PKCζ, a downstream enzyme activity activated by p62, will have similar effects on myeloma patient stromal cells. Our hypotheses are that inhibiting PKCζ expression will block the effects of cytokines produced in the MM-bone microenvironment in response to TNF-α, decrease VCAM-1 expression in stromal cells, and markedly diminish osteolytic bone destruction and MM growth in MM patients. To test these hypotheses, we established long-term Dexter-type marrow cultures to isolate marrow stromal cells from MM patient and normals and screened for p62 and PKCζ/λ activation in MM marrow. We measured levels of PKCζ and p62 by Western blot analysis in marrow stromal cells from 13 patients and 11 healthy controls. We found significantly elevated levels of phosho-PKCζ/λ and total PKCζ in MM patients although the levels varied greatly among the patients. We next measured the relative expression levels of VCAM-1 on marrow stromal cells by Western blot in these marrow stromal cells. Since inhibiting p62 expression should profoundly diminish osteolytic bone destruction and myeloma growth in patients, by blocking production of RANKL, MCP-1, TNF-α and IL-6 in the tumor-bone microenvironment, and the upregulation of VCAM-1 on stromal cells, we assessed the effects of blocking p62 activity in human stromal cells. p62 siRNA (40 nM) was transduced into human stromal cells using a commercial transfection substrate. We confirmed that p62 expression was decreased by at least 50% in these stromal cells by Western blot analysis using an anti-p62 antibody. Stromal cells from the p62siRNA and control siRNA treatment and non-treatment stromal cells were cultured with or without MM1s cells for 3 days in separate experiments. The levels of VCAM-1 in p62siRNA transducer stromal cells were lower than control siRNA and untreated stromal cells. In addition, IL-6 production by MM1.s was increased when cocultured with control stromal cells but not with p62siRNA treated bone marrow stromal cells. These results show that p62 plays an important role in myeloma cell growth induced by cytokines that are upregulated in the marrow microenvironment and myeloma cells in patients with myeloma. Therefore, p62 is an attractive therapeutic target for MM growth and bone destruction.

IL-3 Inhibits Osteoblast Formation in Multiple Myeloma Via CD45+ Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2347-2347 ◽  
Author(s):  
Lori A. Ehrlich ◽  
Masahiro Ito ◽  
Sun Jin Choi ◽  
G. D. Roodman
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Marrow Stromal Cells ◽  
Inhibitory Effects ◽  
Myeloma Cells ◽  
Primary Mouse

Abstract Normally, osteoclast activation is coupled to an increase in osteoblast activity. In Multiple Myeloma (MM), bone remodeling is uncoupled, and bone destruction occurs both by markedly increased osteoclastic bone destruction and severely impaired osteoblastic bone formation. Although several reports have shown that conditioned media from MM cell lines suppress osteoblast (OBL) differentiation, the identity of the OBL inhibitor(s) is unknown. A recent report by Tian et al (NEJM 2003) has identified DKK1, an inhibitor of the WNT signaling pathway, as a putative OBL inhibitor in MM. However, it is likely that other inhibitors of OBL differentiation are present in the myeloma microenvironment, just as there are several potent stimulators of osteoclast formation produced or induced by myeloma cells. We recently reported that IL-3 levels in bone marrow plasma of patients with MM are increased compared to normal controls and that IL-3 in MM marrow plasma stimulates osteoclast formation. We also demonstrated that IL-3 is produced by primary myeloma cells and that it increased MM cell growth in vitro. However, the effects of IL-3 on OBL are unknown. Therefore, to determine if IL-3 could affect OBL growth and differentiation, we tested the effects of IL-3 on OBL differentiation in primary mouse marrow stromal cells. We found that murine marrow stromal cells and OBL-like cell lines expressed IL-3 receptor alpha (IL-3R) by RT-PCR. Staining for IL-3R in marrow stromal cell cultures after treatment with IL-3 confirmed that the IL-3R was present on thirty percent of the cells. Importantly, treatment of primary murine stromal cell cultures with IL-3 (0.01–10 ng/mL) inhibited basal and BMP-2 stimulated osteoblast formation in a dose dependent manner, without affecting cell growth. At 10 ng/mL IL-3 inhibited OBL differentiation by 80%. Time course studies demonstrated that IL-3 affected the later stages of osteoblast differentiation. Further, the inhibitory effects of IL-3 were not due to induction of TNFalpha, a known OBL inhibitor. TNFalpha levels were very low (0–20pg/ml) in the conditioned media of these cultures, and treating the cultures with anti-mouse TNFalpha did not block the IL-3 effect. IL-3 did not inhibit alkaline phosphatase activity in the osteoblast-like cell lines, MC3T3-E1 and C2C12 suggesting that IL-3 may act indirectly through another cell type in the mixed cell population in the primary mouse osteoblast culture system. Since IL-3 acts primarily on hematopoietic cells, we determined if IL-3 was acting indirectly by increasing CD45+ cells in the cultures. IL-3 increased the number of CD45+ hematopoietic cells in the primary culture from approximately 15% to 30%. Further, depletion of the CD45+ cells abolished the inhibitory effects of IL-3 but not TNFalpha on osteoblasts, and adding back CD45+ cells to the stromal cell cultures restores the inhibitory effects of IL-3. This data suggests that IL-3 is an important mediator of bone destruction in MM by both inducing osteoclast formation and indirectly inhibiting osteoblast formation.

Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2630-2636 ◽  
Author(s):  
Berno Dankbar ◽  
Teresa Padró ◽  
Regine Leo ◽  
Birgit Feldmann ◽  
Martin Kropff ◽  
...  
Keyword(s):  
Growth Factor ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Marrow Stromal Cells ◽  
Vascular Endothelial ◽  
Myeloma Cells ◽  
Marrow Stroma ◽  
Endothelial Growth Factor

Abstract Vascular endothelial growth factor (VEGF), a multifunctional cytokine, potently stimulates angiogenesis including tumor neovascularization. Although well established in solid tumors, the role of VEGF in bone marrow neoangiogenesis and paracrine tumor-stromal cell interactions in lymphohematopoietic malignancies has not been fully elucidated. In multiple myeloma (MM), marrow neovascularization parallels disease progression. This parallel prompted us to investigate the expression and secretion of VEGF by myeloma cells and its potential effects in myeloma-marrow stroma interactions. The biologically active splice variants VEGF165 and VEGF121 were expressed and secreted by myeloma cell lines and plasma cells isolated from the marrow of patients with MM. As shown by immunocytochemistry or RT-PCR, myeloma cells did not express or weakly expressed the VEGF receptors FLT-1 and FLK-1/KDR, indicating that autocrine stimulation is unlikely. In contrast, FLK-1/KDR was abundantly expressed by marrow stromal cells. Therefore, we studied the effects of VEGF on marrow stroma, focusing on the secretion of interleukin-6 (IL-6), a potent growth factor for myeloma cells and an inhibitor of plasma cell apoptosis. Exposure of stromal and microvascular endothelial cells to recombinant human (rh) VEGF165 or VEGF121 induced a time- and dose-dependent increase in IL-6 secretion (14- to 27-fold at 50 ng/mL after 24 hours, P < .001). Conversely, rhIL-6 stimulated VEGF expression and secretion in myeloma cell lines (40%-60%; P < .05) and to a variable degree (up to 5.3-fold; P < .005) in plasma cells purified from the marrow of patients with MM. This mutual stimulation suggests paracrine interactions between myeloma and marrow stromal cells triggered by VEGF and IL-6.

Increased Signaling through p62 in the Marrow Microenvironment Increases Myeloma Cell Growth and Osteoclast Formation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 642-642 ◽  
Author(s):  
Yuko Hiruma ◽  
Noriyoshi Kurihara ◽  
Diane F. Jelinek ◽  
David Roodman
Keyword(s):  
Cell Growth ◽  
Tumor Growth ◽  
Signaling Pathways ◽  
P38 Mapk ◽  
Stromal Cells ◽  
Therapeutic Target ◽  
Bone Destruction ◽  
Marrow Stromal Cells ◽  
Tnf Α ◽  
Attractive Therapeutic Target

Abstract Marrow stromal cells in the bone microenvironment of multiple myeloma (MM) patients play a critical role in promoting both tumor growth and bone destruction. Binding of MM cells to marrow stromal cells, through VCAM-1 on stromal cells and α4β1 integrin on MM cells, results in increased production of TNF-α, receptor activator of NF-KB ligand (RANKL) and IL-6 by marrow stromal cells. These factors in turn increase osteoclast (OCL) formation and the growth of MM cells. Adhesive interaction between MM cells and marrow stromal cells also decrease the sensitivity of MM cells to chemotherapeutic agents. Many of the downstream effects of these adhesive interactions are mediated through the NF-KB, p38 MAPK and JNK signaling pathways. The adapter protein sequestosome-1 (p62) sits at the crossroads of these signaling pathways and mediates the effects of cytokines and other factors that activate NF-KB, p38 MAPK and JNK. However, the role that increased signaling through p62 plays in MM tumor growth and bone destruction is unknown. It is our hypothesis that increased signaling through p62 in marrow stromal cells is necessary for their capacity to increase the growth of MM cells and OCL formation in MM, and thus p62 may be an attractive therapeutic target for MM. To test these hypotheses, we established long-term Dexter-type marrow cultures to isolate marrow stromal cells from MM patients and normals and measured signaling through p62 and PKCζ activation in MM marrow. We found significantly elevated levels of phosho-PKCζ, total PKCζ and VCAM-1 in MM stromal cells. The enhanced IL-6 production resulted from increased p38 MAPK activity and the increased VCAM-1 expression was NF-KB dependent. We then examined the effects of blocking p62 activity in primary patients and normal stromal cells with p62 siRNA (10μg). We confirmed that p62 expression was decreased by at least 90% in both these stromal cells by Western blot analysis. Stromal cells expressing p62siRNA or control siRNA were cultured with or without MM1.S cells for 3 days in separate experiments. Knocking-down p62 in MM derived marrow stromal cells significantly decreased the levels of PKCζ, VCAM-1 and IL-6 in marrow stromal cells and markedly decreased stromal cell support of MM cell growth and OCL formation. Similarly, marrow stromal cells from p62−/− mice produced much lower levels of IL-6, TNF-α, VCAM-1 and RANKL and minimally supported MM cell growth and OCL formation compared to normal cells. Thus, increased signaling through p62 in marrow stromal cells in patients with MM plays an important role in the increased tumor growth and OCL formation in MM, and support p62 as an attractive therapeutic target for MM.

Blocking the ZZ Domain of Sequestosome 1/p62 Suppress the Enhancement of Myeloma Cell Growth and Osteoclast Formation by Marrow Stromal Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 888-888 ◽  
Author(s):  
Jumpei Teramachi ◽  
Kyaw Ze Yar Myint ◽  
Rentian Feng ◽  
Xiangqun Xie ◽  
Jolene J. Windle ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Mononuclear Cells ◽  
Apoptotic Cell ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Marrow Stromal Cells ◽  
Sequestosome 1

Abstract Abstract 888 The marrow microenvironment enhances both tumor growth and bone destruction in multiple myeloma (MM) through MM cell-induced activation of multiple signaling pathways in bone marrow stromal cells (BMSC) by TNFα. We reported that sequestosome-1 (p62) acts as a signaling hub for NF-kB, MAPK and PI3K activation in BMSC of MM patients and enhances MM growth and osteoclast (OCL) formation. p62 is composed of 5 domains that are involved in protein–protein interactions required for formation of these signaling complexes, but which domain of p62 mediates increase MM growth and OCL formation is unclear. Therefore, deletion constructs of p62 that lacked each of the 5 domains (PB1, ZZ, p38, TBS or UBA) were transfected into a p62−/− stromal cell line. We found that the ZZ domains mediated BMSC enhancement of MM cell growth, IL-6 production, VCAM-1 expression and OCL formation. Using virtual modeling of the ZZ domain, we identified 6 candidate p62-ZZ inhibitory molecules and tested them for their capacity to block enhanced MM cell growth, OCL formation, IL-6 production, and VCAM-1 expression on BMSC induced by TNFα. When MM1.S, RPMI8266, ANBL6 MM cell-lines or CD138+ primary MM cells were cultured in the absence of BMSC with p62-ZZ inhibitor #3 (10mM), this inhibitor directly induced cell death. The p62-ZZ inhibitor-induced cell death was characterized by an increase in reactive oxygen species (ROS) production by inhibiting NF-kB activation, and apoptotic cell death by triggering the activation of caspases 3, 7, and 9. This inhibitor had an IC50 of 4.6mM for MM1.S survival. In contrast, CFU-Blast formation by human CD34+ cells was not inhibited by p62-ZZ inhibitor #3 (10mM). p62-ZZ inhibitor #3 (10mM) treatment of human OCL precursors derived from CFU-GM, inhibited OCL formation by blocking precursor proliferation. To examine the specificity of the p62-ZZ inhibitor #3, we tested its effects on OCL formation by CD11b+ mononuclear cells from wild type (WT) and p62−/− mice cultured with TNFα for 7 days. p62-ZZ inhibitor #3 blocked WT OCL formation but did not block p62−/− OCL formation. The p62-ZZ inhibitor also blocked VCAM-1 expression and IL-6 production by normal and MM patient stromal cells induced by TNFα compared to vehicle. Importantly, the inhibitor (10mM) did not block stromal cell proliferation. Further, the inhibitor blocked TNFα induced PKCζ phosphorylation in stromal cells and MM1.S when the cells were pretreated with the p62-ZZ inhibitor for 3 hours. These results demonstrate that p62-ZZ inhibitor #3 specifically blocks both stromal cells independent and dependent MM cell growth and OCL formation but does not affect hematopoietic or stromal cell growth. These results support p62 as a potential novel therapeutic target for MM. Disclosures: Roodman: Amgen: Consultancy; Millennium: Consultancy.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1373-1373
Author(s):  
Kristine Misund ◽  
Katarzyna Anna Baranowska ◽  
Toril Holien ◽  
Christoph Rampa ◽  
Dionne Klein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Stromal Cell ◽  
Drug Sensitivity ◽  
Myeloma Cell ◽  
Marrow Stromal Cells ◽  
Myeloma Cells

Abstract Abstract 1373 The aim of this work was to establish a robust and simple method for the measurement of drug sensitivity in myeloma cells under conditions mimicking aspects of the bone marrow microenvironment. In particular we wanted to measure drug sensitivity in myeloma cells cultivated in the presence of stromal cells. The tumor microenvironment can profoundly affect tumor cell survival as well as alter antitumor drug activity, and it is generally believed that growth and survival of myeloma cells is critically dependent on the bone marrow microenvironment. Bone marrow stromal cells (BMSC) have been shown to protect myeloma cells from common cytostatic or cytotoxic drugs in vitro. Common in vitro assays used for high-throughput drug screening cannot easily discriminate between stromal and tumor cell responses in co-cultures. Although a few recent studies have overcome this problem (Ramasamy K. et al., 157(5):564–79,2012, McMillin D. et al., 16(4):483–9, 2010), the application of stable transfection for labeling of cells limits the practical application of these co-culture studies to cell lines, excluding primary myeloma cells that inherently may be hard to transduce even by retroviral vectors. Here, we analyzed survival of myeloma cells co-cultured with BMSC using an automated fluorescence microscope, ScanR. ScanR is a microscope based screening station. By staining the cell nuclei with DRAQ5, we were able to discriminate 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 main advantages of this method are the non-necessity of cell manipulation before co-culture and the low number of myeloma cells (5000 primary cells) that are needed per measurement, which makes the method ideal for experiments with primary myeloma cells. In fact, the analysis was easier and more robust when using slowly growing cells, i.e. by using primary myeloma cells compared to more rapidly proliferating myeloma cell lines. This method should be well-suited for high throughput analysis, as the cells are stained in situ with no washing, centrifugation, or fixation steps before analysis. The method was compared to a conventional method for detecting cell viability; flow cytometry where annexin V labeling was used to detect apoptotic cells. As shown in figure 1, the dose-response curves obtained for ANBL-6 cells treated with different doses of melphalan were similar and showed the same trends for both methods. However, the effects of melphalan treatment were more evident analyzed by the ScanR system than by flow cytometry (EC50 YO-PRO-1 = 11μM versus EC50Annexin V= 15μM). The stromal cell population applied in this study was able to support IL-6 dependent myeloma cell lines without addition of IL-6. This as IL-6 dependent INA-6 cells cultivated in the presence of BMSC survived in the absence of added IL-6. 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 was also observed with this method. For instance, experiments with primary myeloma cells from patient MM7, showed that in the presence of BMSC, the EC50 for the common myeloma drug cyclophosphamide was increased from 5 μM to approximately 10 μM (figure 2). Figure 1 Figure 1. Figure 2 Figure 2. Disclosures: No relevant conflicts of interest to declare.

Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2630-2636 ◽  
Author(s):  
Berno Dankbar ◽  
Teresa Padró ◽  
Regine Leo ◽  
Birgit Feldmann ◽  
Martin Kropff ◽  
...  
Keyword(s):  
Growth Factor ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Marrow Stromal Cells ◽  
Vascular Endothelial ◽  
Myeloma Cells ◽  
Marrow Stroma ◽  
Endothelial Growth Factor

Vascular endothelial growth factor (VEGF), a multifunctional cytokine, potently stimulates angiogenesis including tumor neovascularization. Although well established in solid tumors, the role of VEGF in bone marrow neoangiogenesis and paracrine tumor-stromal cell interactions in lymphohematopoietic malignancies has not been fully elucidated. In multiple myeloma (MM), marrow neovascularization parallels disease progression. This parallel prompted us to investigate the expression and secretion of VEGF by myeloma cells and its potential effects in myeloma-marrow stroma interactions. The biologically active splice variants VEGF165 and VEGF121 were expressed and secreted by myeloma cell lines and plasma cells isolated from the marrow of patients with MM. As shown by immunocytochemistry or RT-PCR, myeloma cells did not express or weakly expressed the VEGF receptors FLT-1 and FLK-1/KDR, indicating that autocrine stimulation is unlikely. In contrast, FLK-1/KDR was abundantly expressed by marrow stromal cells. Therefore, we studied the effects of VEGF on marrow stroma, focusing on the secretion of interleukin-6 (IL-6), a potent growth factor for myeloma cells and an inhibitor of plasma cell apoptosis. Exposure of stromal and microvascular endothelial cells to recombinant human (rh) VEGF165 or VEGF121 induced a time- and dose-dependent increase in IL-6 secretion (14- to 27-fold at 50 ng/mL after 24 hours, P < .001). Conversely, rhIL-6 stimulated VEGF expression and secretion in myeloma cell lines (40%-60%; P < .05) and to a variable degree (up to 5.3-fold; P < .005) in plasma cells purified from the marrow of patients with MM. This mutual stimulation suggests paracrine interactions between myeloma and marrow stromal cells triggered by VEGF and IL-6.

Anti-α4 integrin antibody suppresses the development of multiple myeloma and associated osteoclastic osteolysis

Blood ◽  
2004 ◽  
Vol 104 (7) ◽  
pp. 2149-2154 ◽  
Author(s):  
Yoshihisa Mori ◽  
Nobuaki Shimizu ◽  
Mark Dallas ◽  
Maryla Niewolna ◽  
Beryl Story ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
Stromal Cells ◽  
Bone Destruction ◽  
Firefly Luciferase ◽  
Myeloma Cell ◽  
Tumor Burden ◽  
Cell Interactions ◽  
Myeloma Cells ◽  
Therapeutic Administration

Abstract Supporting roles of stromal cells in preferential colonization of myeloma cells in bone marrow and development of associated osteoclastic osteolysis through cell-cell interactions have been indicated. Here we examined the effects of a monoclonal antibody to α4 integrin (anti-α4 Ab) that disrupts myeloma cell-stromal cell interactions mediated via α4β1 integrin and vascular cell adhesion molecule-1 (VCAM-1) on myeloma cell growth in bone marrow and accompanying osteolysis. The anti-α4 Ab decreased VCAM-1-stimulated 5TGM1/luc cell growth in culture. The 5TGM1 murine myeloma cells stably transfected with the firefly luciferase (5TGM1/luc) were inoculated from tail vein in bg/xid/nd mice. Preventative administration of the anti-α4 Ab suppressed the elevation of serum IgG2b levels, decreased 5TGM1/luc tumor burden with increased apoptosis in bone and spleen, reduced bone destruction with diminished number of osteoclasts, and prolonged survival of 5TGM1/luc-bearing mice. In contrast, therapeutic administration of the antibody failed to show these effects. However, therapeutic administration of the antibody combined with melphalan significantly suppressed serum IgG2b levels and tumor burden in bone. Our results suggest that the interactions with stromal cells via α4β1/VCAM-1 are critical to the development of myeloma and associated osteolysis and that disruption of these interactions using anti-α4 Ab is a potential therapeutic approach for myeloma. (Blood. 2004;104:2149-2154)

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.

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