Establishment of Cell Lines from Both Myeloma Bone Marrow and Plasmacytoma: SNU_MM1393_BM and SNU_MM1393_SC from a Single Patient

Purpose.We tried to establish clinically relevant human myeloma cell lines that can contribute to the understanding of multiple myeloma (MM).Materials and Methods.Mononuclear cells obtained from MM patient’s bone marrow were injected via tail vein in an NRG/SCID mouse. Fourteen weeks after the injection, tumor developed at subcutis of the mouse. The engraftment of MM cells into mouse bone marrow (BM) was also observed. We separated and cultured cells from subcutis and BM.Results.After the separation and culture of cells from subcutis and BM, we established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). Karyotype of the two newly established MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In contrast to SNU_MM1393_BM, cell proliferation of SNU_MM1393_SC was IL-6 independent. SNU_MM1393_BM and SNU_MM1393_SC showed high degree of resistance against bortezomib compared to U266 cell line. SNU_MM1393_BM had the greater lethality compared to SNU_MM1393_SC.Conclusion.Two cell lines harboring different site tropisms established from a single patient showed differences in cytokine response and lethality. Our newly established cell lines could be used as a tool to understand the biology of multiple myeloma.

Download Full-text

Genomic Characterization Of Newly Established Two Distinct Patient-Driven Multiple Myeloma Cell Lines (SNU_MM1393_BM and SNU_MM1393_SC) From a Single Patient

Blood ◽

10.1182/blood.v122.21.5361.5361 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5361-5361

Author(s):

Youngil Koh ◽

Kwang-Sung Ahn ◽

Chansu Lee ◽

Woo June Jung ◽

Hyun Jung Lee ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Lines ◽

Somatic Mutations ◽

Mononuclear Cells ◽

Cultured Cells ◽

Conflicts Of Interest ◽

Clonal Evolution ◽

Myeloma Cell ◽

Single Patient

Abstract To establish patient-derived multiple myeloma (MM) cell lines, mononuclear cells obtained from a MM patient’s bone marrow were directly injected via tail vein into a NRG/SCID mouse. Fourteen weeks after injection, tumor developed at subcutis and bone marrow (BM) in the same mouse. We separated and cultured cells from these two sites (subcutis and BM) and established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). In cytogenetic analysis, karyotype of newly established two MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In FACS analysis, the expression of CD138 and CD45 was detected in both cell lines. Response to IL-6 and soluble IL-6 receptor was different between the two cell lines. Moreover, SNU_MM1393_SC showed higher degree of resistance against proteasome inhibitor (bortezomib) compared to SNU_MM1393_BM. However, two cell lines were both sensitive to histone deacetylase inhibitor (panobinostat). When whole exome sequencing was performed using the DNA of these two cell lines, a set of somatic mutations involving Wnt signaling and NF-kB pathway were detected in both cell lines. Additional somatic mutations of JAK1, PLCG1, IRS2, and HGS which are known to interact with JAK/STAT pathway were detected in SNU_MM1393_BM. Whereas, additional somatic mutations of EGFR, HSP90AB1, CFDR, and CALML5, which are known to interact with growth factor cell signaling pathway were detected in SNU_MM1393_SC. These findings highlight the importance of interactions between tumor and tumor microenvironment as the myeloma progresses and will pave a way to more effective selection of targeted agents according to specific tumor characteristics obtained in the process of disease progression. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Role of interleukin-6 in the proliferation of human multiple myeloma cell lines OCI-My 1 to 7 established from patients with advanced stage of the disease

Blood ◽

10.1182/blood.v78.8.1996.1996 ◽

1991 ◽

Vol 78 (8) ◽

pp. 1996-2004 ◽

Cited By ~ 59

Author(s):

JK Hitzler ◽

H Martinez-Valdez ◽

DB Bergsagel ◽

MD Minden ◽

HA Messner

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Lines ◽

Interleukin 6 ◽

Myeloma Cell ◽

Receptor Expression ◽

Enzyme Linked Immunosorbent Assay ◽

Cell Populations ◽

Thymidine Uptake ◽

Auxiliary Cell

Abstract Interleukin-6 (IL-6) has been shown to stimulate the proliferation of multiple myeloma cells purified to a high degree from human bone marrow. IL-6 production in multiple myeloma has been attributed to cells belonging to the myeloma clone, thus supporting a mechanism of autostimulation. In addition, it has been shown that IL-6 may be produced by auxiliary cell populations of the bone marrow that are not part of the myeloma clone. A definitive separation of both putative sources for IL-6 may be difficult to achieve in fresh patient IL-6 growth requirement and production by pure myeloma cell populations using seven human myeloma cell lines (OCI-My 1 to 7) that were established from patients with advanced disease. The proliferative response of each line to recombinant IL-6 was measured in a clonogenic assay providing human plasma and methylcellulose as a viscous support and by 3H-thymidine uptake in liquid suspension culture. We observed marked heterogeneity, ranging from IL-6-dependent colony formation by OCI-My 4, to IL-6-independent growth. All lines expressed mRNA for the IL-6 receptor. Expression of IL-6 mRNA was analyzed after amplification by polymerase chain reaction and was present in five of seven lines. IL- 6 protein was detected by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of two lines (OCI-My 3 and 2). Its functional activity was confirmed in a bioassay using the IL-6-dependent murine hybridoma line B 13.29. This activity was neutralized by anti-IL-6 antibody. Two lines did not express mRNA for IL-6. The remaining three lines expressed mRNA for IL-6, but did not secrete IL-6 protein. Immunoprecipitation experiments with lysates of one of these three lines did not detect the presence of IL-6 protein. These results suggest that autocrine stimulation by IL-6 may occur in some cell lines derived from patients with multiple myeloma. However, it does not represent a universal mechanism in myeloma cell growth.

Download Full-text

Nocodazole Induces Myeloma Cell Death by Disrupting the Microtubular Network, Resulting in G2/M Arrest

Blood ◽

10.1182/blood.v112.11.3673.3673 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3673-3673

Author(s):

Rentian Feng ◽

Jorge A Rios ◽

Markus Mapara ◽

Suzanne Lentzsch

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Cell Death ◽

Cell Lines ◽

Myeloma Cell ◽

Caspase 8 ◽

Inhibition Rate ◽

Growth And Survival ◽

Microtubular Network

Abstract Patients with relapsed multiple myeloma (MM) previously treated with bortezomib and lenalidomide often fail to respond to further therapies. To identify potential new treatment approaches for MM, we used Luminex technology to screen a library of 1,120 compounds provided by the Multiple Myeloma Research Foundation. By multiplex cytokine array, we identified benzimidazoles including the anthelmintics mebendazole, fenbendazole, albendazole, nocodazole and pyrvinium pamoate, as inhibiting the production of cytokines essential for MM cell growth and survival, such as IL-6 (inhibition rate 40–70%), MIP-1α (inhibition rate 65–75%), VEGF (inhibition rate 75%), and soluble IL-6R (inhibition rate 40–52%). Consequently, these anthelmintics demonstrated dose-dependent inhibition of myeloma cell (RPMI-8226, H929, U266 and MM1S) proliferation. The lead compound, nocodazole, caused nuclear fragmentation and caspase-8 activation in MM cell lines and primary CD138+ cells in dose- and time-dependent fashion (IC50: 30–60 nM). Importantly, growth and survival signals provided by bone marrow stromal cells in bone marrow co-cultures failed to protect MM cells from nocodazole-induced cell death. In the apoptotic cells, caspase-8 was more activated than caspase-9, suggesting that mitochondrial signaling is not a major apoptotic pathway. Cell cycle analysis indicated that G2/M cell cycle arrest reached a peak at 17 hr. Sub-G1 proportion was strongly increased after treatment for 24 hr in all tested cell lines. Electron microscope (EM) and nuclear staining studies consistently showed the accumulation of metaphase cells, and morphologic elongation at 7 hr, at which time G2/M arrest was obvious. Most of the elongated cells had only one nucleus, suggesting that they failed to progress to mitosis due to overall microtubular network disarray. We conclude that nocodazole exposure induced microtubular network disarray with cell elongation, and G2/M arrest with a late stage mitotic block resulting in cell death. Benzimidazoles including nocodazole, traditionally used as antihelmintic drugs, have shown antitumor activity against hepatocellular, lung and adrenocortical carcinoma, and melanoma. In our study, we identified the anthelmintic compound nocodazole as a new anti-myeloma agent. Nocodazole warrants further investigation for its anti-MM effects in vitro and in vivo.

Download Full-text

A Role for Syntenin-1 in Multiple Myeloma Cell Survival

Blood ◽

10.1182/blood-2018-99-113594 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1008-1008

Author(s):

Tyler Moser-Katz ◽

Catherine M. Gavile ◽

Benjamin G Barwick ◽

Sagar Lonial ◽

Lawrence H. Boise

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Death ◽

Cell Survival ◽

Cell Lines ◽

Plasma Cells ◽

Surface Expression ◽

Myeloma Cell ◽

Myeloma Cells ◽

Rpmi 8226

Abstract Multiple myeloma is the second most common hematological malignancy in the U.S. with an estimated 30,700 new diagnoses in 2018. It is a clonal disease of plasma cells that, despite recent therapeutic advances, remains incurable. Myeloma cells retain numerous characteristics of normal plasma cells including reliance on survival signals in the bone marrow for long term viability. However, malignant transformation of plasma cells imparts the ability to proliferate, causing harmful bone lesions in patients, and in advanced stages independence of the bone-marrow microenvironment. Therefore, we are investigating the molecular mechanisms of myeloma cell survival that allow them to become extramedullary. We identified syntenin-1 (SDCBP) as a protein involved in myeloma cell survival and a potential therapeutic target. Syntenin-1 is an adapter protein that has been shown to regulate surface expression of several transmembrane proteins by binding with membrane phospholipids and mediating vesicular trafficking of proteins throughout the cell. Syntenin-1 regulates the surface expression of CD138, a plasma/myeloma cell marker. Syntenin-1 has been shown to regulate apoptosis in numerous cancer cell lines including breast cancer, glioma, and pancreatic cancer but its role in multiple myeloma survival has not been studied. To determine if syntenin-1 expression has an effect on myeloma cell survival, we utilized the CoMMpass dataset (IA12), a longitudinal study of myeloma patients that includes transcriptomic analysis throughout treatment. We found that patients with the highest expression of syntenin-1 mRNA (top quartile) had significantly worse overall survival, progression-free survival, and a shorter response duration than those in the bottom quartile of expression. To determine if syntenin-1 has a role in myeloma cell survival, we used short hairpin RNA to knock down syntenin-1 (shsyn) in RPMI 8226 and MM1.s myeloma cell lines. We then determined the amount of cell death using Annexin-V staining flow cytometry four days following lentiviral infection. We found increased cell death in syntenin-1-silenced cells compared to our empty vector control in both RPMI 8226 (control=42.17%, shsyn=71.53%, p=0.04) and MM1.s cell lines (control=8.57%, shsyn=29.9%, p=0.04) suggesting that syntenin-1 is important for myeloma cell survival. Syntenin-1 contains two PDZ domains that allow it to bind to receptor proteins via their corresponding PDZ-binding motifs. We therefore wanted to look at correlation of syntenin-1 expression with CD138 and CD86, two PDZ-binding domain containing proteins expressed on the surface of myeloma cells. Using the CoMMpass dataset, we found patients with high expression of syntenin-1 had a median expression of CD86 that was twice as high as the total population (P<0.0001) while syntenin-1-low patients expressed CD86 at levels that were half as much as the population (P<0.0001). In contrast, there was no clear relationship between syntenin-1 and CD138 mRNA expression. Indeed if one takes into account all patients, there is a positive correlation between CD86 and syntenin-1 expression (r=0.228, P<0.0001) while there is a negative correlation between CD138 and syntenin-1 (r=-0.1923, P<0.0001). The correlation with CD86 but not CD138 suggests a previously undescribed role for syntenin-1 in myeloma cells. Our lab has previously shown that expression of CD86 is necessary for myeloma cell survival, and signals via its cytoplasmic domain to confer drug resistance. Silencing syntenin-1 results in a decrease in CD86 surface expression. However, there is no change in CD86 transcript or total cellular CD86 protein levels in our shsyn treated cells. Moreover, knockdown of CD86 resulted in increased protein expression and transcript levels of syntenin-1. Taken together, these data suggest that syntenin-1 may regulate CD86 expression on the cell surface. Our data supports a novel role for syntenin-1 in myeloma cell viability and as a potential regulator of CD86 surface expression. The role of syntenin-1 has not previously been explored in multiple myeloma and determining its molecular function is warranted as it may be an attractive target for therapeutic treatment of the disease. Disclosures Lonial: Amgen: Research Funding. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.

Download Full-text

Mcl-1 Is Overexpressed in Multiple Myeloma and Correlates with Disease Severity.

Blood ◽

10.1182/blood.v104.11.1419.1419 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1419-1419

Author(s):

Soraya Wuilleme-Toumi ◽

Nelly Robillard ◽

Patricia Gomez-Bougie ◽

Philippe Moreau ◽

Steven Le Gouill ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Disease Severity ◽

Cell Lines ◽

Plasma Cells ◽

Myeloma Cell ◽

Lymphocytic Leukemia ◽

Myeloma Cells

Abstract Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (>20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.

Download Full-text

Upregulation of MMP-2 & 9 by Co-Culture of Human Myeloma Cell Lines and Endothelial Cells May Be Responsible for Protection Against Cytotoxic Drugs.

Blood ◽

10.1182/blood.v108.11.5080.5080 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5080-5080

Author(s):

Shankaranarayana Paneesha ◽

Raghu Adya ◽

Hemali Khanji ◽

Ed Leung ◽

C. Vijayasekar ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Endothelial Cells ◽

Mrna Expression ◽

Cell Lines ◽

Myeloma Cell ◽

Myeloma Cells ◽

Human Myeloma Cell

Abstract Multiple myeloma is a clonal lymphoproliferative disorder characterised by the proliferation of plasma cells in the bone marrow. Inspite of good initial response, it is associated with universal relapse. We hypothesise this is due to sanctuary provided to myeloma cells by the endothelium. Matrix metalloproteinases (MMPs) are shown play a role in cell growth, invasion, angiogenesis, metastasis and bone degradation. We show here the protection offered by endothelial cells to human myeloma cell lines in in-vitro co-culture with upregulation of MMP-2 & 9 and the role of GM6001 MMP inhibitor (Ilomastat) in overcoming this protection. Human myeloma cell lines (H929, RPMI 8226, U266 & JJN3) with or without endothelial cells (human umbilical vein endothelial cells and EaHy 926 cell line) in-vitro co-culture were treated with melphalan, dexamethasone, arsenic trioxide and Ilomastat. Cytotoxicity/proliferation were assessed by the alamarBlue™ assay (Serotec) and validated by Annexin V-FITC apoptosis detection Kit (Calbiochem) and BrDU proliferation assay (BD Pharmingen™). Gelatin Zymography was used to demonstrate activity of MMP-2 & 9 in the supernatant. MMP-2 and 9 mRNA expression was quantified by Real Time Quantitative PCR (ROCHE). Co-culture of human myeloma cell lines with endothelial cells lead to increase in the proliferation of myeloma cell lines and also protected them from the cytotoxicity of chemotherapeutic agents. MMP-2 & 9 activity was upregulated by the co-culture. MMP-2 mRNA expression in human myeloma cell lines increased following 4 hr co-culture. Treatments with Ilomastat lead to the suppression of proliferation in co-culture in a dose dependent manner, associated with a reduction of MMP-2 and 9 activity. Our study shows endothelial cells offer protection to human myeloma cell lines in the presence of cytotoxic agents. This may result in the sanctuary of myeloma cells in bone marrow leading to ultimate relapse of disease. Our study also demonstrates the upregulation of MMP-2 and 9 by co-culture and increased cytotoxicity achieved by the inhibition of MMPs. Further studies are needed to determine the exact role of MMPs in myeloma biology as MMP inhibition may be an interesting therapeutic target and help in averting relapse in multiple myeloma.

Download Full-text

Effects of Integrin-Linked Kinase (ILK) Inhibitor QLT0267 on Multiple Myeloma Cells and Evaluation of ILK as a Therapeutic Target in Multiple Myeloma.

Blood ◽

10.1182/blood.v110.11.3529.3529 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3529-3529

Author(s):

Thorsten Stühmer ◽

Torsten Steinbrunn ◽

Evelyn Grella ◽

Ralf C. Bargou

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cells ◽

Cell Lines ◽

Mononuclear Cells ◽

Treatment Options ◽

Therapeutic Window ◽

Culture Dish ◽

Peripheral Blood Mononuclear ◽

Myeloma Cells

Abstract Multiple myeloma (MM) is a fatal plasma cell tumor that accounts for about 1% of cancers. A hallmark of the disease is its location in the bone marrow where the tumor cells receive prosurvival support from the microenvironment and cause extensive osteolytic damage. Novel drugs are currently being developed into a range of new treatment options. However, because the problems of cancer relapse and eventual selection of therapy-resistant offspring remain, additional therapeutic targets should still be investigated. ILK is a multifunctional protein that, as an adaptor and/or as a kinase, may relay adhesion- and growth factor receptor-mediated signals to downstream signaling cascades that promote growth and survival. We have analysed the expression of ILK in MM cells and have tested the effects of a novel small molecule ILK-inhibitor (QLT0267; QLT Inc., Vancouver, Canada) in MM cell lines, primary MM tumor cells and healthy cells, respectively. ILK expression at either cDNA or protein level was detectable in virtually every MM sample tested. Treatment with QLT0267 for up to 3 days resulted in extensive apoptotic death in MM cell lines (EC50 values below 10 microM in 8/9 MM cell lines tested) and in a majority of primary (anti-CD138-purified) MM samples (EC50 values below 10 microM in 8/14 primary MM samples tested). Drug treatment led to rapid decreases in the levels of phospho-STAT3, phospho-GSK3beta and total Akt protein, whereas levels of ILK and of phospho-ERK were unaffected or, in the latter case, showed a slight increase. Similar to other current pharmacologic approaches, targeting ILK may have several detrimental impacts on the signaling network that sustains MM cells. Such pleiotropic effects could prove valuable for combination treatments. The survival of peripheral blood mononuclear cells and of bone marrow stromal cells (BMSCs) at 20 microM QLT0267 was just slightly affected, indicating that the scope for establishment of a therapeutic window in MM might exist. High (20 microM) concentrations of QLT0267 gradually (and reversibly) promoted detachment of BMSCs from the culture dish, indicating that the drug might be useful to temporarily impair their effectiveness to support myeloma cells. Taken together, these experiments provide a rationale to further explore the utility of ILK-inhibition for the treatment of MM.

Download Full-text

Survival Signals, Growth Cytokines, Immunosuppressive Factors and Their Cellular Perpetrators in the Myeloma Tumor Microenvironment.

Blood ◽

10.1182/blood.v112.11.1664.1664 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1664-1664

Author(s):

Jayakumar R Nair ◽

Louise M Carlson ◽

Noreen Ersing ◽

Asher Alban Chanan-Khan ◽

Kelvin P. Lee

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Microenvironment ◽

Cell Lines ◽

Plasma Cells ◽

Immune Escape ◽

Human Monocyte ◽

Fold Increase ◽

Myeloma Cell ◽

Myeloma Cells

Abstract Multiple myeloma (MM) is an incurable neoplasia of terminally differentiated plasma cells in the bone marrow. Essential interactions of MM cells with host bone marrow stromal cells (BMSC) induce growth factors essential for MM progression and pathogenesis, as well as induce an immunosuppressive environment that inhibits endogenous and therapeutically-induced immune responses against the MM cells. However, despite their importance, little is known about the identity of these BMSC cells or the molecular basis of their interaction with myeloma cells. A potential MM surface protein that could be involved in these interactions is CD28, based on its known pro-survival role in T cells. Clinical studies have shown that expression of CD28 in multiple myeloma highly correlates (p=0.006) with myeloma disease progression. Moreover, CD28+ MM cells invariably express the CD28 ligand CD86. A survival role for MM-CD28 might involve interactions with cellular partners that express the B7 (CD80/CD86) ligands. Potential candidates would include CD86+ myeloma cells themselves or B7+ dendritic cells (DC) that are known to be closely associated with myeloma cells in the patient bone marrow. When myeloma-myeloma interactions were disrupted by using the high affinity CD80/CD86 blocker CTLA4Ig (Abatacept®), increased sensitivity to arsenic trioxide (ATO) and melphalan (MEL) was observed in all the three MM cell lines U266, RPMI8226 and MM1S. For U266 viability was 93% in media alone, 84% with CTLA4Ig (100 μg/ml) alone, 86% with 2 μM ATO alone and was significantly reduced to 36% with CTLA4Ig + ATO. Similar drops in viability were observed with 25 μM MEL in combination with CTLA4Ig (33% as opposed to 71–74 % with CTLA4Ig or MEL alone). Our data suggests that this does not involve the downregulation of anti-apoptotic proteins Bcl-2, Bcl-xL or Mcl-1, commonly associated with drug resistance in myeloma. In the second part of the study, we demonstrate that myeloma cell lines or primary CD138+ myeloma cells can enhance via direct contact the ability of human monocyte derived immature DC to produce the immunosuppressive tryptophan depleting enzyme indoleamine 2,3 dioxygenase (IDO, as estimated by kynurenine (Kyn) (a tryptophan catabolite) levels in the supernatant) and also the pro-plasma cell survival cytokine IL-6. In co-cultures of IFNg treated immature DCs with either MM cell lines or with primary CD138+ myeloma cells from patient BM aspirates, the activity of IDO was enhanced ~ 2–8 fold (81 mM kyn with U266 and 20–43mM with primary cells) over that observed in control IFNg-treated DCs (9.7 mM Kyn). Western analysis also demonstrated increased IDO expression relative to IFNg activated DC controls. Blocking MM-CD28 with (Fab)2 fragments of anti-hCD28 mAb 9.3 downregulated IDO activity (9.3 mM) close to that of control, demonstrating the involvement of MM-CD28 in these interactions. We also demonstrated a significant up-regulation of the pro-myeloma survival cytokine IL-6 when immature DCs were co-cultured with CD28+ MM1S (90–300 pg/ml), a 4–9 fold increase over that of DC only control (25 – 35 pg/ml). This was further enhanced when immature DCs cultured with IL-10 (+ GM-CSF + IL-4) was used in co-cultures with MM-1S (800 – 1300 pg/ml), or with primary CD138+ myeloma cells from patient bone marrow aspirates (128–1142 pg/ml). In conclusion, our data demonstrates that blocking myeloma-CD28 - myeloma-CD86 “autocrine” interaction can enhance drug cytotoxicity, while interactions with DCs produce the essential growth cytokines IL-6 and immunosuppressive enzyme IDO with potential implications in MM survival and immune escape. Use of clinically approved agents (e.g. Abatacept®) to block myeloma-CD28 binding to its B7 ligands (increase chemotherapeutic efficacy), 1-MT to inhibit IDO and targeting DCs in the microenvironment to disrupt the tumor microenvironment could be viable therapeutic strategies for the future treatment of multiple myeloma.

Download Full-text

A Characteristic Cytokine/Chemokine Milieu Is Present in the Bone Marrow Environment of Multiple Myeloma Patients Before and After Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v114.22.2795.2795 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2795-2795

Author(s):

Yanran Cao ◽

Tim Luetkens ◽

Sebastian Kobold ◽

York Hildebrandt ◽

Maja Gordic ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Cell Lines ◽

Allogeneic Stem Cell Transplantation ◽

Myeloma Cell ◽

Luminex Technology ◽

Before And After

Abstract Abstract 2795 Poster Board II-771 Background: The interaction of Multiple Myeloma (MM) with its bone marrow (BM) microenvironment is important for the homing, survival, and proliferation of the malignant plasma cells. In this study, we aimed at answering the question which cytokines, chemokines, and growth factors are typically found in the BM of untreated MM patients as well as in MM patients after allogeneic stem cell transplantation (alloSCT) and might be involved in the pathogenesis of MM and/or Graft-versus-Host Disease (GvHD). Design and Methods: Applying Multiplex Bead-based Luminex technology and Quantikine ELISAs, we determined the concentrations of 34 cytokines/chemokines in the supernatants of 10 myeloma cell lines as well as in the plasma derived from BM and peripheral blood (PB) samples of 10 newly diagnosed MM patients, 20 MM patients post alloSCT, and 20 healthy donors. Results: Besides factors known to be secreted by myeloma cell lines (IL-1RA, IL-8, MCP-1, MIP-1α, MIP1β, MIP-3α) we observed secretion of other cytokines/chemokines such as EGF, HGF, IL2R, IL-12p40/p70, IL-22, IP-10, MIG, and RANTES. In the BM plasma samples of MM patients, we detected elevated levels of HGF, IL-2R, IL-16, EGF, IL-1RA, IP-10, MCP-1, and MIG (p<0.01 or p<0.05). Most of these factors were significantly increased in the BM compared to PB. In addition to increased levels of the factors mentioned above, the BM plasma of MM patients post alloSCT showed selectively elevated concentrations of IL-4, IL-6, IL-8, IL-12p40/p70, and eotaxin (p<0.01 or p<0.05). Eotaxin levels were particularly high in patients with chronic GvHD. Correlating the BM cytokine/chemokine levels with the clinical characteristics of MM patients, we observed that BM levels of IL-16 (r=0.829, p=0.003) and HGF (r=0.805, p=0.005) correlated positively with myeloma cell infiltration within the BM. In addition, IP-10 (r=0.770, p=0.009), HGF (r=0.645, p=0.044), and IL-6 (r=0.664, p=0.036) correlated significantly with the initial stage of disease. Conclusions: Our study demonstrates that myeloma cells themselves generate a significantly higher number of cytokines/chemokines than previously described. We show that characteristic cytokine/chemokine patterns exact in the BM environment of MM patients before and after alloSCT. Certain factors, such as MIP-1α, MCP-1, HGF, IL-16, IP-10, and eotaxin might not only be developed into diagnostic instruments and/or predictive biomarkers, but might also represent potential targets for future myeloma- or GvHD-specific therapies. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

A Novel Mouse Model of Multiple Myeloma Representative of Human Disease and Its Use in Preclinical Therapeutic Assessment

Blood ◽

10.1182/blood.v118.21.2907.2907 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2907-2907

Author(s):

Rosemary A Fryer ◽

Timothy J Graham ◽

Emma M Smith ◽

Brian A Walker ◽

Gareth J Morgan ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Cell Lines ◽

Myeloma Cell ◽

Human Condition ◽

Myeloma Cells ◽

Treatment Groups ◽

Hyperintense Signal

Abstract Abstract 2907 In order to aid the pre-clinical development of novel therapeutics for multiple myeloma, an in vivo model which recapitulates the human condition in particular tumor growth patterns and response to treatment is required. An important feature of such a model is the interaction of the myeloma cells with the bone marrow microenvironment as this is known to modulate tumor activity and protect against drug-induced apoptosis. We have developed a model with myeloma restricted to the bone marrow, which proceeds rapidly from initial inoculation to disease progression, and possesses a range of chemo-sensitive markers with which to monitor anti-tumor response. Female NOD/SCID γcnull mice were injected inta-osseously with luciferase-tagged myeloma cell lines. Disease progression was monitored weekly by bioluminescent imaging (BLI) and measurement of paraprotein levels (ELISA). These methods were compared to histological assessment of tumor infiltration and MRI which provided a quantitative measurement of progression. On T2-weighted images tumor was identified as a hyperintense signal enclosed within cortical bone. Tumor burden was quantified from regions of interest drawn on the periphery of the hyperintense signal. Luciferase-tagged cells engrafted by 3 weeks at the injection site and progressed to the femurs, spine and pelvis from week 4. BLI showed a significant increase in radiance from 5.6×105 to 43.0×105p/s/cm2/sr between weeks 5 and 7 (p<0.05). Quantification of tumor volume by MRI showed a significant increase from 6.4mm3 to 27.6mm3 between weeks 4 and 8 (p<0.05) and μCT demonstrated lytic disease. Serum levels of Igλ increased from 860ng/ml to 4325ng/ml during this period (p<0.05), which mirrored the changes seen with BLI and MRI. Flow cytometry and histology confirmed the confinement of CD138 positive myeloma cells within the bone. These results indicate successful engraftment of human myeloma cell lines with induction of myeloma in a pattern similar to the human condition. We have adapted this model to study primary patient material. 10 mice were implanted with samples from 3 cases of plasma cell leukemia with complex cytogenetics. 5 of these developed myeloma confined to the bone marrow, 2 with additional plasmacytoma localized at the injection site, over a period of 1–5months. We have characterized the original patient cells with gene expression, SNP based gene mapping and have characterized the nature of the engrafted cells using similar technology. We have also shown the model is suitable for preclinical assessment of anti-myeloma agents using bortezomib and a novel aminopeptidase inhibitor, tosedostat (CHR-2797). Non-treated mice displayed a significant increase in radiance from 16.13×105 to 69.00×105p/s/cm2/sr (p<0.01). In comparison, in the bortezomib and tosedostat treated groups no significant increase in radiance was seen (bortezomib: 5.22×105 to 1.12×105 p/s/cm2/sr; tosedostat: 9.92×105 to 13.78×105p/s/cm2/sr). Paraprotein levels mimicked these changes in BLI. At the end of treatment Igλ levels in control, bortezomib and tosedostat treated mice were 2473.7, 132.5 and 923.0ng/ml, respectively. Igλ levels in both treatment groups were significantly different from control (p<0.001). Average tumor volumes derived from MRI were significantly different in bortezomib (14.7mm3) and tosedostat treated (23.4mm3) groups compared to non-treatment (33.0mm3). The volumes for the bortezomib treated group showed no significant difference from control mice. In addition, there was a decrease in CD138 expression by flow cytometry in bone aspirates from treatment groups compared to control which was mirrored in histological samples. In conclusion using both myeloma cell lines and primary patient cells, we have developed a model which recapitulates human myeloma with secretion of paraprotein, disease confined to the bone marrow, lytic bone lesions and spinal compression. In addition, this model is suitable for assessing the efficacy of novel therapeutics in vivo, using a number of non-invasive tumor markers such as BLI and MRI. Disclosures: Morgan: J&J: Honoraria, Speakers Bureau. Davies:J&J: Honoraria, Speakers Bureau.

Download Full-text