scholarly journals Mimicking Myeloma Niche Ex Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2076-2076
Author(s):  
Jana Jakubikova ◽  
Danka Cholujova ◽  
Richard W Groen ◽  
Jungnam Joo ◽  
Sun-Young Kong ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Tumor Cells ◽  
Cell Lines ◽  
Plasma Cells ◽  
Ex Vivo ◽  
3 Dimensional ◽  
Bm Niche ◽  
Hla Dr ◽  
D Model

Abstract Introduction: Recent studies have elucidated the importance of using 3-dimensional rather than 2-dimensional models in order to create an experimental system recapitulating the specialized properties of the bone marrow microenvironment. Since the neoplastic bone marrow (BM) milieu plays important roles in multiple myeloma (MM) pathogenesis, novel models to study the MM cell in its neoplastic microenvironment are needed. Methods: To mimic the neoplastic BM microenvironment of MM patients, we have established a special hydrogel-based 3-dimensional (3-D) model by ex-vivo culturing MM patient-derived mesenchymal stem cells (MM-MSCs), the predominant cellular component of the marrow niche, which promotes greater mineralization and differentiation than a 2-dimensional (2-D) system. Results: To characterize MM-MSCs in different stages of MM, we utilized an 11 multi-color flow cytometry panel. The percentage of MSCs (CD73+CD90+CD105+lin-CD45-CD34-HLA-DR-) population in BM aspirate samples of 50 MM patients (MGUS, smoldering MM, newly diagnosed MM, and relapsed or relapsed/refractory MM) was evaluated, and correlated with the distribution of (CD38+ CD138+) plasma cells. MSCs were less frequent (10x) than plasma cells, and increased with disease progression to relapsed/refractory MM. We seeded MM-MSCs (N=34) which had been expanded by adhesion methods in 2-D versus 3-D models in order to create an ex-vivo MM niche-like structure. In the hydrogel-based 3-D model, MM-MSCs formed compact clusters with active fibrous connections and meshwork-like structures at day 3 to 7. Moreover, calcium mineralization of clusters was observed, associated with the capacity for differentiation towards the osteoblastogenic or adipogenic lineage when cultured with differentiation media. Furthermore, the production of osteopontin (OPN) and angiopoietin-2 (Ang-2) was significantly higher in 3-D vs. 2-D MM-MSCs, assessed by multiplex luminex technology. Phenotypic profiling of 3-D MM-MSCs clusters revealed high expression of CD73+CD90+CD105+ and lack of expression of CD45, CD34 and HLA-DR, as in to 2-D MM-MSCs. MSC-specific markers including CD166 and HLA-ABC did not reveal any significant changes in 3-D vs. 2-D MM-MSCs; however, 3-D MM-MSCs had significantly decreased expression of CD271 and CD146 compared to 2-D cultures. We also observed significantly higher expression of extracellular matrix (ECM) molecules including fibronectin, laminin, collagen I, and collagen IV (p<0.001) in 3-D vs. 2-D MM-MSCs. Similarly, activation of integrins including VLA-2, VLA-4 and VLA-5 on the MSCs surface was also increased in 3-D MM-MSCs, as determined by confocal microscopy and flow cytometry analysis. Importantly, MM-MSCs cultured in 3-D vs. 2-D model have higher expression of N-cadherin and CXCL12 and decreased expression of nestin, reflecting the MM BM niche. Gene expression analyses of 3-D MM-MSCs revealed upregulation of BMP-2, MGP, PTGIS, COL14A1 and other genes and down-regulation of DKK1, ADAM9, OPCML genes and others compared to 2-D MM-MSCs. We also measured significantly higher production of IL-6 (p=0.002), IL-8, MCP-1(MCAF), RANTES, VEGF and HGF (p<0.001) in 3-D vs. 2-D MM-MSCs, by multiplex luminex analysis. Next, we co-cultured tumor cells from MM patients (12 MM patients) with either autologous or allogeneic MM-MSCs in 3-D vs. 2-D model. Plasma (CD38/CD138+) cells in 3-D co-culture were increased in 8/12 MM patients and equivalent to 2-D in 4/12 patients. By co-culturing MM cell lines (OPM1, RPMI-S, OCIMY5 and KMS11) labeled with CFSE fluorescent dye with various MSCs, we evaluated expression of side population (SP) cells, identified by Hoechst staining, and gating on CFSE positive MM cells, as low Hoechst stained cells. Our results showed that the SP fraction was significantly lower in 3-D compared to 2-D in co-cultures of various MM-MSCs with all 4 MM cell lines. Finally, we validated drug resistance to melphalan, bortezomib, lenalidomide, and carfilzomib in 3-D co-cultures of CFSE labeled primary tumor cells with various MM-MSCs. Conclusions: This 3-D co-culture system closely mimics the myeloma BM niche, and therefore may be useful to identify and validate novel targeted therapies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2269-2277 ◽  
Author(s):  
HM Lokhorst ◽  
T Lamme ◽  
M de Smet ◽  
S Klein ◽  
RA de Weger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Adherent Cells ◽  
Myeloma Cells ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Abstract Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion molecule 1, very late antigen 2 (VLA-2), and VLA- 5, and were positive for extracellular matrix components fibronectin, laminin, and collagen types 3 and 4. LTBMC from myeloma patients and normal donors spontaneously secreted interleukin-6 (IL-6). However, levels of IL-6 correlated with the stage of disease; highest levels of IL-6 were found in LTBMC from patients with active myeloma. To identify the origin of IL-6 production, LTBMC from MM patients and normal donors were cocultured with BM-derived myeloma cells and cells from myeloma cell lines. IL-6 was induced by plasma cell lines that adhered to LTBMC such as ARH-77 and RPMI-8226, but not by nonadhering cell lines U266 and FRAVEL. Myeloma cells strongly stimulated IL-6 secretion in cocultures with LTBMC adherent cells from normal donors and myeloma patients. When direct cellular contact between LTBMC and plasma cells was prevented by tissue-culture inserts, no IL-6 production was induced. This implies that intimate cell-cell contact is a prerequisite for IL-6 induction. Binding of purified myeloma cells to LTBMC adherent cells was partly inhibited by monoclonal antibodies against adhesion molecules VLA-4, CD44, and lymphocyte function-associated antigen 1 (LFA-1) present on the plasma cell. Antibodies against VLA-4, CD29, and LFA-1 also inhibited the induced IL-6 secretion in plasma cell-LTBMC cocultures. In situ hybridization studies performed before and after coculture with plasma cells indicated that LTBMC adherent cells produce the IL-6. These results suggest that the high levels of IL-6 found in LTBMC of MM patients with active disease are a reflection of their previous contact with tumor cells in vivo. These results provide a new perspective on tumor growth in MM and emphasize the importance of plasma cell-LTBMC interaction in the pathophysiology of MM.

The Role of B Cell-Activating Factor (BAFF) in the Biology of Multiple Myeloma (MM).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3380-3380 ◽  
Author(s):  
Noopur Raje ◽  
Shaji Kumar ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Hiroshi Yasui ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Development ◽  
Newly Diagnosed ◽  
Growth And Survival ◽  
Affymetrix U133a ◽  
Apoptotic Proteins

Abstract BAFF is a member of the tumor necrosis factor (TNF) family and is critical for the maintenance and homeostasis of normal B-cell development. Importantly, BAFF promotes the generation of rapidly dividing immunoglobulin secreting plasmablasts from activated memory B cells by enhancing their survival. Given that MM is a cancer of plasma cells and that the signaling cascades implicated in receptor ligand interactions of BAFF are crucial in MM cell biology, we hypothesized that this cytokine may play a critical role in MM cell development, survival, and proliferation. We performed gene expression profiling (GEP) on CD 138+ plasma cells isolated from 90 MM patients (45 newly diagnosed and 45 relapsed) and 11 healthy controls using the Affymetrix U133A arrays. Our data demonstrates increased expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), 2 receptors used by BAFF to exert its effects. Our data also shows an increased expression of a proliferation-inducing ligand (APRIL), another member of the TNF family with homology to BAFF. Expression levels of BAFF and BAFF-R could not be determined because of lack of these probe sets on the Affymetrix U133A arrays. GEP analysis shows increased BCMA expression (p&lt;0.0001, student T test) on newly diagnosed and relapsed MM versus normal plasma cells. Flow cytometry on MM cell lines demonstrated a differential expression of the three receptors of BAFF, with BCMA present on most cell lines but BAFF-R expressed at low levels only on LR5 cells and DOX40 MM cells. In contrast, flow cytometry performed on MM patient cells demonstrated the presence of all 3 receptors on CD 138+ cells. ELISA assays performed on 30 MM sera demonstrated a mean BAFF level of 618 pg/ml (range: 128–2126pg/ml) versus 235pg/ml (range: 158–326pg/ml) in 7 normal donor sera. Fifty six% (17/30) of MM patients had BAFF levels in excess of the highest value noted in normals. To understand the role BAFF might play in the biology of MM, we studied the effects of recombinant BAFF (rh-BAFF) on MM cells directly and in the context of its bone marrow microenvironment. (abstract # 554746) rh-BAFF conferred a survival advantage to MM cells and protected them against dexamethasone-induced cytotoxicity. Importantly, anti-apoptotic proteins Bcl2 and Mcl-1 were upregulated, as were growth and survival signals belonging to the JAK/STAT and MAPKinase pathways. Conversely, neutralizing antibody to BAFF blocked, at least in part, blocked the upregulation of anti-apoptotic proteins with associated growth and survival, confirming that these effects were due to BAFF. Importantly, all of these signals were downregulated even in the presence of bone marrow stromal cells (BMSCs). These data therefore show a role for BAFF mediating MM cell survival and provide the framework for inhibiting BAFF, either alone or in combination with dexamethasone, to improve patient outcome in MM.

scholarly journals Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2269-2277 ◽  
Author(s):  
HM Lokhorst ◽  
T Lamme ◽  
M de Smet ◽  
S Klein ◽  
RA de Weger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Adherent Cells ◽  
Myeloma Cells ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion molecule 1, very late antigen 2 (VLA-2), and VLA- 5, and were positive for extracellular matrix components fibronectin, laminin, and collagen types 3 and 4. LTBMC from myeloma patients and normal donors spontaneously secreted interleukin-6 (IL-6). However, levels of IL-6 correlated with the stage of disease; highest levels of IL-6 were found in LTBMC from patients with active myeloma. To identify the origin of IL-6 production, LTBMC from MM patients and normal donors were cocultured with BM-derived myeloma cells and cells from myeloma cell lines. IL-6 was induced by plasma cell lines that adhered to LTBMC such as ARH-77 and RPMI-8226, but not by nonadhering cell lines U266 and FRAVEL. Myeloma cells strongly stimulated IL-6 secretion in cocultures with LTBMC adherent cells from normal donors and myeloma patients. When direct cellular contact between LTBMC and plasma cells was prevented by tissue-culture inserts, no IL-6 production was induced. This implies that intimate cell-cell contact is a prerequisite for IL-6 induction. Binding of purified myeloma cells to LTBMC adherent cells was partly inhibited by monoclonal antibodies against adhesion molecules VLA-4, CD44, and lymphocyte function-associated antigen 1 (LFA-1) present on the plasma cell. Antibodies against VLA-4, CD29, and LFA-1 also inhibited the induced IL-6 secretion in plasma cell-LTBMC cocultures. In situ hybridization studies performed before and after coculture with plasma cells indicated that LTBMC adherent cells produce the IL-6. These results suggest that the high levels of IL-6 found in LTBMC of MM patients with active disease are a reflection of their previous contact with tumor cells in vivo. These results provide a new perspective on tumor growth in MM and emphasize the importance of plasma cell-LTBMC interaction in the pathophysiology of MM.

scholarly journals DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5618-5618 ◽  
Author(s):  
Priya Choudhry ◽  
Margarette C. Mariano ◽  
Arun P Wiita
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Ex Vivo ◽  
Single Agent ◽  
Cd38 Expression

Abstract Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy. Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega). Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here. Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

Junctional Adhesion Molecule-A/ F11 Receptor (JAM-A/ F11R) Expression in Multiple Myeloma (MM): a Candidate Biomarker of Aggressive Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2830-2830
Author(s):  
Bani M Azari ◽  
Marc J. Braunstein ◽  
H. Uwe Kluppelberg ◽  
Sadeaqua S Scott ◽  
Eric LP Smith ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Tumor Cells ◽  
Cell Lines ◽  
Adhesion Molecule ◽  
Mrna Levels

Abstract Abstract 2830 Poster Board II-806 Background: Multiple myeloma (MM) is an incurable disease of clonal plasma cells that accumulate in the bone marrow (BM), causing monoclonal IG production, bone marrow failure, osteolytic lesions and kidney disease. Although initially treatable, MM ultimately becomes refractory to treatment, and is invariably fatal, when tumor cells that harbor genetic mutations expand without regulation. Therefore novel treatment targets need to be identified. A key mechanism in MM pathogenesis is regulation of tumor growth by the bone marrow (BM) microenvironment, particularly by bone marrow neo-vascularization and adhesion of tumor cells to the marrow stroma. Aberrantly expressed genes that regulate angiogenesis by MM cells enhance MM progression and constitute targets in its treatment. JAM-A/F11R is an endothelial cell (EC) adhesion molecule of the immunoglobulin superfamily which is a multifunctional cell membrane protein that mediates intracellular signaling events that alter EC migration and paracellular permeability. For example, in breast cancer, attenuation of JAM-A increases tumor invasion and metastasis through a decrease in tumor adhesion (Ulas Naik Cell Adh Migr. 2008 Oct;2(4):249-51.). In this study we explored the JAM-A/F11R expression in MM tumor cells and in patients to determine the potential role of this molecule in the pathogenesis and progression of MM. Methods: The MM cell lines examined were RPMI-8266, U266, and NCI-H929. Human umbilical vein endothelial cells (HUVECs) served as controls. Informed consent was obtained from patients and control subjects. Primary BM tumor cells were enriched to > 95% CD138+ cells by positive selection using anti-CD138 MACS MicroBeads. The CD138-negative fraction was used for outgrowth of confluent EPCs (> 98% vWF/CD133/KDR+). JAM-A mRNA expression was assessed using an microarray gene expression profile, JAM-A probe based real-time PCR, and JAM-A levels in each sample were measure using a standard curve and normalized to GADPH. JAM-A protein levels in MM cell lines and primary tumor cells were measured by flow cytometry and immunofluorescence. For serum studies, peripheral blood was obtained from 25 newly diagnosed MM patients and 8 healthy, age- and sex-matched controls, and JAM-A levels were measured using an ELISA. Statistical analysis was performed using Student's t-test, two-tailed, with P ' .05 considered significant. Results: JAM-A mRNA levels were significantly increased in MM cell lines RPMI-8266, U266, and NCI-H929 compared to HUVECs (U266, P = 3×10-5; RPM1-8266, P = 1×10-6; NC1-H929, P= 5×10-4). The JAM-A mRNA levels were significantly greater in RPMI-8226; P < .04 compared to TNFα-activated HUVECs for 24 hours which is a proangiogenic switch for HUVEC gene expression. The elevated mRNA expression of the JAM-A in MM cell lines was confirmed by immunofluorescence and flow cytometry which showed the presence of both membrane and cytoplasmic JAM-A protein. Microarray analysis of gene expression profiles from 20 patients' corresponding tumor cells and microenvironmental EPCs showed that JAM-A had a higher level of expression in tumor cells versus MM EPC by 12.62 fold, (P=.0000642). Furthermore, JAM-A had a higher level of expression in MM EPC versus normal control EPC by 2.41 fold, (P=.00113) reflecting a complex regulatory role of F11 signaling in MM, similar to breast cancer (Naik, U. et al 2008). JAM-A was also found to be 12.6 fold greater in tumor cells compared to EPCS (P=.0000642). In addition, circulating levels of soluble JAM-A were found to be significantly greater in the serum of MM patients compared to controls (P < .005), with an average 2-fold increase. Serum levels of JAM-A in MM patients also decreased 71% with treatment n=5, P<.05. Conclusion: We show for the first time that JAM-A expression is highly elevated in MM tumor cells and its levels respond to treatment. In addition, MM patients have higher circulating JAM-A levels compared to healthy individuals and circulating JAM-A levels were reduced following treatment, suggesting that JAM-A may serve as a novel biomarker in MM. Current studies in the lab are aimed at correlating these levels with clinical parameters to determine whether JAM-A levels reflect disease severity and response to treatment. Results of these analyses, as well as results of ongoing experiments using JAM-A siRNA and antibody-inhibition approaches to target JAM-A in myeloma tumor and ECs will be presented. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Flow Cytometry Assessment of In Vitro Generated CD138+Human Plasma Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Rayelle Itoua Maïga ◽  
Jennifer Lemieux ◽  
Annie Roy ◽  
Carl Simard ◽  
Sonia Néron
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Human Plasma ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Peripheral Blood Mononuclear

The in vitro CD40-CD154 interaction promotes human B lymphocytes differentiation into plasma cells. Currently, CD138 is the hallmark marker enabling the detection of human plasma cells, both in vitro and in vivo; its presence can be monitored by flow cytometry using a specific antibody. We have developed a culture system allowing for the differentiation of memory B lymphocytes. In order to detect the newly formed plasma cells, we have compared their staining using five anti-CD138 monoclonal antibodies (mAbs). As a reference, we also tested human cell lines, peripheral blood mononuclear cells, and bone marrow samples. The five anti-CD138 mAbs stained RPMI-8226 cells (>98%) with variable stain index (SI). The highest SI was obtained with B-A38 mAb while the lowest SI was obtained with DL-101 and 1D4 mAbs. However, the anti-CD138 mAbs were not showing equivalent CD138+cells frequencies within the generated plasma cells. B-A38, B-B4, and MI-15 were similar (15–25%) while DL-101 mAb stained a higher proportion of CD138-positive cells (38–42%). DL-101 and B-A38 mAbs stained similar populations in bone marrow samples but differed in their capacity to bind toCD138highandCD138locell lines. In conclusion, such cellular fluctuations suggest heterogeneity in human plasma cell populations and/or in CD138 molecules.

Regulation of the New CXCR7 Receptor in Plasma Cell Dyscrasias.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3527-3527
Author(s):  
Anne-Sophie Moreau ◽  
Xavier Leleu ◽  
Xiaoying Jia ◽  
Judith Runnels ◽  
Costas Pitsillides ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Tumor Cells ◽  
Cell Lines ◽  
The Other ◽  
Pcr Analysis ◽  
Boyden Chamber ◽  
Embryonic Cells ◽  
Rt Pcr ◽  
Migration Assay

Abstract Background: Multiple Myeloma (MM) and Waldenstrom Macroglobulinemia (WM) are characterized by widespread involvement of the bone marrow (BM) as the result of successful homing, engraftment and growth of tumor cells at that site. The receptor for SDF-1, CXCR4 has already been implicated by us and others in the migration and homing of tumor cells to the bone marrow. Recently, a new receptor for the chemokine SDF-1 has been described, namely CXCR7. To date, it has been implicated in the migration of embryonic cells during neurogenesis in zebrafish. It has been reported to be expressed in cancer cells but its function remains unknown. We have previously shown that disruption of the CXCR4/SDF-1 axis interferes with homing of MM cells to the BM. To better understand the homing and migration of MM and WM tumor cells, we sought to study the expression and function of CXCR7. Methods: MM (U266, MM1.S, RPMI, OPM2, OPM1, KMS12BM) and WM (BCWM.1 and WSU-WM) cell lines were used. CD19+ and CD138+ cells were extracted from patient bone marrow samples by microbeads selection after informed consent. To determine the expression of CXCR7 on MM and WM cell lines as well as primary samples, we used flow cytometry and RT-PCR analysis. The migration of tumor cells towards SDF-1 was studied using the transwell boyden chamber migration assay. The adhesion of tumor cells to fibronectin using a fluorometric assay. The CXCR7 inhibitor CCX-754 (ChemoCentryx Inc., Mountain View, CA) was used. Results: CXCR7 was expressed in all cell lines tested except WSU-WM by flow cytometry and RT-PCR. Interestingly, U266 cell line did not express surface CXCR4 and expressed CXCR7 and therefore was used in further experiments to determine the differential role of CXCR7 in SDF-1 related function. Primary WM (n=14) and MM (n=7) cells expressed low intensity CXCR7 by flow cytometry. Use of the above cells in experiments utilizing small molecule antagonists of both CXCR4 and CXCR7 suggested that inhibiting the binding of SDF-1 to one receptor could impact the signaling functions of the other. Experiments are ongoing to clarify the nature of the interaction between these two SDF receptors. Furthermore, adhesion of U266 cells to fibronectin was increased in presence of SDF-1, and inhibited in the presence of CCX-7754. Further analysis to examine CXCR7 and CXCR4 antagonism in adhesion are ongoing. Conclusion: we showed for the first time CXCR7 expression on MM and WM tumor cells. Our results lead us to conclude that through binding of a shared ligand, CXCR7 and CXCR4 may modulate the biological activity of the other.

Disease-Associated Changes In The Repair Efficiency Of Double Strand Breaks Affect Melphalan Sensitivity Of The Bone Marrow Plasma Cells and Correlate With The Clinical Outcome Of Anti-Myeloma Therapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3723-3723
Author(s):  
Maria Gkotzamanidou ◽  
Masood A. Shammas ◽  
Evangelos Terpos ◽  
Enrique M. Ocio ◽  
Jesús F. San Miguel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Γh2ax Foci ◽  
Repair Efficiency ◽  
Bone Marrow Plasma ◽  
Rpmi 8226

Abstract Melphalan is an interstrand cross-link (ICL)-inducing agent and one of the most active chemotherapeutic drugs in the treatment of patients with multiple myeloma (MM). There is clear evidence that the formation and subsequent persistence of ICL correlates with its cytotoxicity. Previous studies have established that during ICL repair, replication forks stall at the ICL inducing the formation of a lethal form of DNA damage (DNA double-strand breaks, DSBs), which is repaired mainly by homologous recombination (HR) and non-homologous end joining (NHEJ). In this report, we investigated the molecular mechanisms of therapeutic efficiency and drug resistance to ICL-inducing agents using melphalan as a model. We studied two MM cell lines (melphalan-sensitive RPMI-8226 and melphalan-resistant RPMI-LR5) and 70 MM patients (38 males/32 females; median age 59 years) who underwent high-dose melphalan (HDM) therapy with autologous stem cells transplantation (ASCT) as first line therapy. Patient response status was assessed 100 days after ASCT according to the International Myeloma Working Group Criteria; patients were grouped into responders (≥PR, n=48) and non-responders (<PR, n=22). Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples obtained from MM patients, at diagnosis or at least 1 week prior to the treatment with any anti-myeloma drug. In addition, bone marrow plasma cells (BMPCs) were isolated from bone marrow trephine aspiration samples during diagnostic clinical assessment. Primary cells (PBMCs and BMPCs) and MM cell lines were ex vivo treated with melphalan either alone or in combination with RI-1 (selective inhibitor of HR) or NU7026 (selective inhibitor of NHEJ) and the extent of the N-ras-specific ICLs and DSBs (intermediates of ICL repair) were evaluated using a quantitative PCR assay and quantification of γH2AX foci, respectively. The γH2AX foci were viewed under a laser-scanning confocal immunofluorescence microscope and quantitated using Image J software. The induction of the apoptotic pathway by melphalan, using a photometric enzyme-immunoassay, was also studied. Following ex vivo treatment of BMPCs with melphalan, ICLs reached maximal levels within 8h of the melphalan treatment. Thereafter, ICLs levels were reduced with the repair efficiency being significantly higher in non- responders (half-time of damage removal, t1/2 23h) than in responders (t1/2 48h) (P<0.01). Moreover, γ-H2AX foci formation followed the timing of ICL formation and reached maximal levels within 8h. Thereafter, γ-H2AX foci levels declined rapidly, suggesting the resolution of the intermediate DSBs by downstream pathways (HR, NHEJ). Interestingly, the repair efficiency of DSBs in BMPCs was significantly higher in non-responders (t1/2 9h) than in responders (t1/2 12h) (P<0.02). Similar results were obtained using PBMCs. Also, in both BMPCs and PBMCs, the melphalan-induced apoptosis inversely correlated with the repair efficiencies of ICLs and DSBs, with the toxicity being higher in responders than in non-responders (P<0.01). Moreover, RPMI-LR5 cells showed higher repair efficiencies of both ICLs and DSBs and lower toxicity than RPMI-8226 cells. Interestingly, in all cell types analyzed, significant correlation between ICL and DSBs levels was observed (linear regression analysis, R2=0.67, P<0.01). To further elucidate the mechanism of drug-induced DSBs repair, MM cell lines and primary cells (BMPCs and PBMCs) were treated with melphalan in combination with nontoxic doses of RI-1 or NU7026. We found that the combined treatment of melphalan with RI-1 or NU7026 significantly increased the melphalan only-induced phosphorylation of H2AX (suggesting that both HR and NHEJ contribute to the repair of melphalan-induced DSBs), delayed the repair of ICLs and strongly enhanced the cytotoxic activity of melphalan (all P<0.01). Collectively, these results highlight that in BMPCs significant changes in the repair efficiency of DSBs occur in MM patients. These changes affect the removal of the cytotoxic ICLs, modify drug sensitivity of the malignant plasma cells ex vivo, and correlate with the clinical outcome of anti-myeloma therapy. Interestingly, these changes are also reflected in PBMCs. Specific inhibition of HR and/or NHEJ may be useful as an adjunct to melphalan therapy in MM patients. Disclosures: No relevant conflicts of interest to declare.

Ex-Vivo Drug Response Profiling for Precision Medicine Approaches in Acute Myeloid Leukemia with the Open Microwell Microfluidic Platform

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1675-1675 ◽  
Author(s):  
Laura Rocchi ◽  
Andrea Faenza ◽  
Laura Rambelli ◽  
Viviana Guadagnuolo ◽  
Giovanni Marconi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Tumor Cells ◽  
Gold Standard ◽  
Drug Response ◽  
Predictive Power ◽  
Ex Vivo ◽  
Drug Efficacy ◽  
Autologous Serum ◽  
Equity Ownership

Abstract Background: Patient stratification to match individual patients with the most effective drug treatment is still a major open challenge in cancer care. For instance, cytarabine is the main drug used for AML treatment but 30% of patients fail to respond to this agent. Laboratory developed tests determining ex-vivo cellular response to cytotoxic anticancer drugs have demonstrated good correlations with clinical response, sometimes surpassing the predictive power of molecular and genetic profiling. Standardizing sample processing to remove operator-dependent biases and maintaining live cells in a functional status that closely resembles in-vivo function are major challenges affecting these tests. Here we present the open microwell (OMW) platform, a microfluidic-based system that integrates the entire process of ex-vivo testing of anticancer drug efficacy and enables drug testing in the clinical setting prior to therapy administration. The concept was validated for the first time on 13 AML patients at Sant'Orsola hospital, Italy, showing the possibility to initiate the analysis readily after sample collection, thus minimizing drifts in cell function that typically start occurring within hours from sampling, and provide results in about 24 hours, with a fully-automated system. Methods: 13 refractory, relapsed or newly diagnosed AML patients were enrolled in the study. 2 ml of fresh bone marrow in EDTA and 1 ml of serum blood were collected from each patient. White blood cells (WBC) were separated from bone marrow by standard Ficoll-Paque, suspended in medium additioned with 2% autologous serum and loaded in microfluidic chips featuring 16 microchannels and 1200 microwells/channel (70 μm diameter), open at the bottom end (Fig. 1A-B). After settling down in microwells, cells were stained with CMAC cell tracker, anti-human-CD34/CD45 fluorescently-labeled antibodies and Propidium Iodide (PI) by injecting the reagents in the microchannels. Cytarabine or combination therapies (FLAI-3, FLAI-5, FLA, MEC-4) were then injected in the microchannels and cells were incubated for 24 hours in the system (Fig. 1C). Four channels were used per therapeutic condition, including reference (ref), high (ref x 10) and low (ref/10) dosages plus a non-treated channel as reaction control. Finally, a custom software was used to detect cells in images, classify AML blasts and analyze cell death (Fig. 1D). Drug efficacy was determined by evaluation of both cell depletion and apoptosis induction. Results: We first validated the OMW platform against gold standard (FacsAria flow cytometer) for the detection of tumor cells and measurement of cell viability and apoptosis. Count of blast frequency was carried out on 5 patient samples using anti-CD34/CD45 staining. Results (Fig. 2B) show a correlation between the gold standard and OMW (n=5, R2=0.99, p<0.0001). AML blast viability was carried out on 4 patient samples after 24h drug stimulation and in control situations (vehicle only). OMW and the gold standard (incubation in 96-well plates followed by flow cytometry analysis) showed a correlation (n=4, R2=0.86, p<0.0001, Fig. 2C). We tested the predictive power of the OMW system by comparing ex-vivo drug response analysis with clinical outcomes. The outcome was predicted correctly in 4 out of 4 (100%) patients enrolled in this arm of the study (Fig. 2D). CellPly test score was set as the ratio between the number of dead cells treated at high dosage and control. Equivalent results were obtained using the ref dosage. The test was performed as a blind prospective study for 2 patients and in a retrospective way on residual AML blasts after therapy for 2 patients. In prospective cohort, we assessed therapy response with a bone marrow biopsy performed at day +28 ± 7 from induction/re-induction course. Conclusion: The OMW platform was able to count AML blasts and analyze viability and apoptosis showing high concordance with conventional diagnostics represented by flow cytometry. The assay requires 30 μl of bone marrow sample and simple manual pre-processing. Profiling of patient response to specific drugs or combination is provided within 24h. These features make the OMW platform suitable for bedside analysis, to evaluate drug activity on tumor cells within a heterogeneous sample and promptly guide personalized treatment in hematologic malignancies, with a first proof achieved on AML patients. LRo and AF equally contributed. Disclosures Rocchi: CellPly S.r.l.: Employment. Faenza:CellPly S.r.l.: Employment. Rambelli:CellPly S.r.l.: Employment. Guadagnuolo:CellPly S.r.l.: Employment. Pecorari:CellPly S.r.l.: Employment. Giulianelli:CellPly S.r.l.: Employment. Biscarini:CellPly S.r.l.: Employment. Martinelli:Novartis: Speakers Bureau; Roche: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; MSD: Consultancy; Celgene: Consultancy, Speakers Bureau; Genentech: Consultancy. Guerrieri:CellPly S.r.l.: Equity Ownership. Bocchi:CellPly S.r.l.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Role Of Folate Receptor Targeted Therapy In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4436-4436
Author(s):  
Sonali Panchabhai ◽  
Katalin Kelemen ◽  
Sinto Sebastian Chirackal ◽  
Rafael Fonseca
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Targeted Therapy ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Newly Diagnosed ◽  
Tumoricidal Activity ◽  
Significant Difference

In multiple myeloma (MM) the interaction of plasma cells, bone marrow stromal cells and tumor associated macrophages (TAMs) plays a significant role in conferring resistance to therapy and in the maintenance of residual disease. Folate receptors (FR) are specifically expressed on metabolically active malignant cells and TAMs and their expression in normal tissues and resting macrophages is limited. FR mediates folate uptake by receptor-mediated endocytosis. This qualifies the receptor to be exploited for drug delivery of folate conjugated cancer therapeutics. Our primary goal in this study is to evaluate the expression of functional FR in MM cells and TAMs with a view to exploit this for folate conjugated targeted therapy for MM. First we evaluated the presence of TAMs in paraffin embedded bone marrow slides of newly diagnosed MM patients with CD68 (pan-Macrophage marker) and CD163 antibodies (specific marker of TAMs) and found extensive infiltration of macrophages in bone marrow from MM patients. Next to evaluate expression of FR in MM cells, we employed an FR antibody and evaluated MM cell lines with immunoblot, flow cytometry and confocal microscopy. In a panel of ten MM cell lines, we found that out of the three FR isoforms (α, β and γ), FR beta (FR-β) is expressed by all of them. Next to test whether this expressed receptor is indeed functional, we incubated the cells with folate deficient media, added different concentrations of EC17 (folate conjugated to FITC, Endocyte Inc.) to the medium and looked for the uptake by flow cytometry after washing off the drug at different time points (after 10, 20, 40 and 60 min).We observed that the uptake begins in 10 min and is saturated at 1 hour with 100nM Folate-FITC. With confocal imaging, Folate-FITC was found in the cytoplasm of MM cells suggesting internalization of Folate-FITC and localization in the cytoplasm. In addition to myeloma cell lines, we also confirmed the uptake of Folate-FITC in CD138+ plasma cells of a newly diagnosed myeloma patient by flow cytometry. This strongly suggests that MM cells express functional FR-β. To test the specificity of this FR mediated uptake, we pre-incubated cells with 0.1mM folic acid in medium for 30 min and then added EC17. This maneuver blocked the activity mediated by FR and no uptake was observed , which proves that the Folate-FITC is internalized only through the FR. To evaluate the expression of FR in-vivo samples, we stained paraffin embedded bone marrow slides of newly diagnosed MM patients with FR-β antibody and TAM specific markers. We observe that FR is expressed on both MM cells as well as TAMs. To assess the endurance of cytotoxic effect of folate conjugated chemotherapeutic agents, we treated MM cell lines with folate conjugated vinka alkaloids and compared them to unconjugated drug and found no significant difference in their action suggesting conjugation with folate does not alter its efficacy. To assess potential toxicity of folate conjugated therapeutics, we obtained CD34+ cells and looked for the uptake of Folate-FITC with flow cytometry. We found no uptake and this is in line with previous reports suggesting that CD34 positive cells express nonfunctional FR. So we propose that FR qualify as potential targets for cancer treatment. Folate targeted therapy using folate-conjugated drugs which can selectively act against both MM cells and supporting TAMs has the potential of specific anti-MM tumoricidal activity. This therapeutic approach would broaden the use of drugs that could be conjugated with folate for MM therapy. Additionally assessment of TAMs in bone marrow sections of MM patients would add another feature for grading, classifying and prognosticating MM. Disclosures: Fonseca: Cylene: Research Funding; AMGEN: Consultancy; Millennium: Consultancy; Binding Site: Consultancy; Onyx: Consultancy, Research Funding; Lilly: Consultancy; BMS: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Medtronic: Consultancy; Otsuka: Consultancy; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties.

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