Preclinical Development Of a Nanomedicne Approach For Multiple Myeloma Targeting The Myc Oncoprotein

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4228-4228
Author(s):  
Deepti Soodgupta ◽  
Dipanjan Pan ◽  
Grace Hu ◽  
Angana Senpan ◽  
Xiaoxia Yang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Delivery ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Free Drug ◽  
Preclinical Development ◽  
Late Antigen ◽  
Mtt Assays

Abstract Purpose This study investigated alpha 4 beta 1/ Very Late Antigen-4 (α4β1/ VLA-4)-integrin targeted nanotherapy approach to deliver a new lipase-labile prodrug. Experimental Design A phospholipid-based MYC-MAX inhibitor prodrug (MI1-PD) was synthesized, and its inherent anti-proliferate potency was compared to the lipid-free compound (MI1) using mouse multiple myeloma (MM) cell line (5TGM1). VLA-4-targeted perfluorocarbon (PFC) nanoparticles binding to 5TGM1 cells was measured and compared to biomarker expression assessed with flow cytometry using antibodies. The efficacy of MI1-PD incorporated into non-targeted and VLA-4-targeted PFC NP exposed to 5TGM1 cells was assessed with MTT assays, Annexin V and cell cycle analysis. Results MI1-PD was more potent by several orders of magnitude than its free drug counterpart in culture. Targeted NP binding correlated well with biomarker expression assessment by flow cytometry in 5TGM1 cells. Non-targeted NPs had no appreciable binding to 5TGM1 cells. High anti-MM potency of MI1-PD was noted in VLA-4-targeted NPs compared to the non-targeted NPs demonstrating that the efficacy was dependent on expression of the targeted biomarker to afford particle-to-cell drug delivery. Conclusions These results suggest the feasibility of an improved integrin VLA-4- targeted nanotherapy approach to deliver a lipase- labile prodrug construct, MI1-PD. Disclosures: No relevant conflicts of interest to declare.

Characterization of Hemangioblastic Traits within Endothelial Progenitor Cells of Multiple Myeloma Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2984-2984
Author(s):  
Sadeaqua S Scott ◽  
Marc J Braunstein ◽  
Christopher Lange ◽  
Christopher Roman ◽  
Eric LP Smith ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Progenitor Cells ◽  
Tumor Cells ◽  
Endothelial Progenitor Cells ◽  
Conflicts Of Interest ◽  
Cell Phenotype ◽  
Endothelial Progenitor ◽  
Cell Cycle Status

Abstract Abstract 2984 Background: Multiple myeloma (MM), a neoplasm of committed B-lymphocytes within the bone marrow (BM), is the second most common hematologic malignancy in the U.S. Despite prolonged median survival with anti-myeloma strategies aimed at the tumor and its BM microenvironment, MM remains invariably fatal. Endothelial progenitor cells (EPCs) are CD133+/KDR+ cells that originate in the BM and play a key role in supporting tumor growth and MM progression. Using X-chromosome inactivation and RT-PCR analyses, we previously found EPCs from MM patients to be clonally restricted and to display clonotypic IG heavy-chain gene rearrangements identical to the same patients' tumor cells (Braunstein et al., 2006). Based on the shared genetic identity that we and others have demonstrated between tumor cells and EPCs in MM patients, the present study explored the hypothesis that, similar to hemangioblasts, which are CD133-expressing precursors to adult hematopoietic and endothelial cells, EPCs may be a source of vascular and MM progenitor cells. Since hemangioblasts are known to exist predominately in the quiescent phases of the cell cycle, in this study we also examined the cell cycle status of CD133-expressing BM cells from MM patients in order to gain insight into their hemangioblastic traits. Methods: BM aspirates were acquired from MM patients under IRB approval. EPCs (>98% vWF/CD133/KDR+ and CD38-) from BM aspirates of MM patients were outgrown on laminin-coated flasks as previously described. The spleen colony assay was used to determine the stem cell capacity within BM-derived EPCs by i.v. injection into NOD/SCID mice. The spleens and BM of mice were harvested 2–4 weeks later. Cells were analyzed by immunofluorescence (IF) and flow cytometry. Hoechst 33342 (Hst) and Pyronin Y (PY) were used to measure the cell cycle status of CD133+ cells using FACS analysis. Results: Two to four weeks following i.v. injection of MM EPCs, human cell surface marker expression detected by flow cytometry within mouse BM and spleen cells shifted from CD133+/CD45-lo, a progenitor cell phenotype, to CD133−/CD45-hi, a more differentiated phenotype, suggesting the ability of MM EPCs to differentiate in vivo. Cell cycle analysis of the CD133+ population in BM cells of MM patients showed that these cells were predominantly non-cycling. On average, 60.5% of CD133+ cells were found to be in the G0/G1 phase of the cell cycle, as indicated by low levels of IF staining with Hst and PY. Conclusions: CD133+ cells are strong candidates as precursors to MM tumor and vascular progenitor cells. As quiescent cells are non-dividing, they often escape cytotoxic effects of chemotherapy, resulting in relapse, and therefore, identification of these cells is critical. Ongoing studies include the engraftment of CD133+ cells in the subcutaneous NOD/SCID gamma xenotransplant model and their growth in response to anti-myeloma strategies; results of these studies will be discussed. Disclosures: No relevant conflicts of interest to declare.

Here Is the Sub-Title:Apoptosis on Multiple Myeloma U266 Cell by Phosphorylation c-Jun of Brucine

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5112-5112
Author(s):  
Yanping Ma ◽  
Zhihua Li ◽  
Yihua Wang ◽  
Jing Feng
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Expression Change ◽  
U266 Cell ◽  
Apoptotic Effect

Abstract Abstract 5112 Objective: To investigate the Mechanism of the apoptotic effect of brucine on human multiple myeloma. Method: U266 cells (5×104) were plated in the presence or absence of the brucine (0, 0.05, 0.1, 0.2, 0.4 mg/ml) in 96 well culture plates for 24–72 h. The anti-proliferative response of brucine was assessed by MTT assay. The analysis of cell cycle of U266 cell with or without brucine was mesured by flow cytometry. The expression change of c-Jun after joining brucine, brucine and JNK specific inhibitor SP600125 was detected using RT-PCR. Results The apoptotic effect of brucine show a dose and time dependent manner. Cell cycle analysis using flow cytometry revealed accumulation of cells at sub-G0/G1 phase. The apoptosis rate separately were (4.137±0.01)%, (10.55±0.03)%, (12.31±0.04)%, (27.67±0.08)%, (29.67±0.09)% (p<0.01). Detecting c-Jun gene expression respectively after joining brucine, brucine and JNK specific inhibitor SP600125 by RT-PCR. The gray scale values were (0.7961±0.007),(0.4683±0.003). Conclusions Within the 0.4mg/ml concentration of brucine can induce apoptosis in U266 cells. Brucine by JNK signaling pathway through phosphorylation of c-Jun induced apoptosis in U266 cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inhibition of HIF-1a By PX-478 Normalizes Blood Vessels, Improves Drug Delivery and Suppresses Progression and Dissemination in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3
Author(s):  
Barbara Muz ◽  
Katherine Wasden ◽  
Kinan Alhallak ◽  
Amanda Jeske ◽  
Feda Azab ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Conflicts Of Interest ◽  
Photoacoustic Imaging ◽  
Fluorescent Microscopy ◽  
Tunel Staining

Introduction: Multiple myeloma (MM) is a lymphoplasmacytic malignancy localized in the bone marrow (BM) characterized by the continuous metastasis. Despite the introduction of novel therapies, MM patients relapse due to the development of drug resistance that is, at least in part, promoted by hypoxia (insufficient oxygen). MM cells develop a hypoxic phenotype, leading to cellular adaptations that cause metastasis, angiogenesis, stemness and resistance to drugs, such as carfilzomib, promoted by a hypoxia-inducible factor-1α (HIF-1α) transcription factor. Herein, we explored the mechanisms underlying HIF pathway inhibition using for the first time in MM a HIF-1α selective small molecule inhibitor, PX-478, both in vitro and in vivo. Methods: In vitro, to test the effect of PX-478 (0 - 50 µM) in combination with carfilzomib on MM cell survival exposed to normoxia (21% O2) or hypoxia (1% O2) was assessed using MTT assay. Cell adhesion to endothelial cells (HUVECs), and cell migration to stromal cells of prelabeled MM cells treated with PX-478 was measured by fluorescent spectrophotometer and flow cytometry, respectively. Tube-like formation of HUVECs as well as survival was tested in the presence of PX-478. For in vivo study, MM.1S-Luc-GFP cells were injected intravenously (i.v.) into 40 SCID mice; 3 weeks post injection the mice were divided into 4 groups and treated with vehicle (PBS), carfilzomib, PX-478, and a combination of PX-478 and carfilzomib. Tumor progression and weight was monitored weekly by bioluminescent imaging, and survival was monitored daily. At day 28, 3 mice from each group were randomly taken: (i) to test the number of circulating tumor cells (MM-GFP+) in the peripheral blood counted by flow cytometry; (ii) to test the MM apoptosis in the femurs by TUNEL staining; and (iii) to test extramedullar metastasis of MM in the kidney, spleen and the liver using immunohistochemistry. Additionally, tumor vasculature was demonstrated in the skull using photoacoustic imaging as well as tumor involvement using fluorescent microscopy. Moreover, we tested the drug delivery by injecting fluorescent large molecule (Dextran-AF405 Mw=70,000) i.v. in MM-bearing mice treated with and without PX-478. Lastly, we tested the effect of PX-478 on prelabeled MM cell retention in the blood and homing to the BM one hour post-MM injection in naïve mice. Results: We found that PX-478 reversed the hypoxia-induced resistance of MM cells to carfilzomib, inhibited metastasis-related cell processes such as adhesion and migration, and reduced MM-mediated tube-like formation of HUVECs in vitro. In vivo, in MM-bearing mice PX-478 decreased the number of MM circulating cells, suppressed tumor metastasis, improved vascularization of the tumor thus delivery of chemotherapy, and as a result re-sensitized MM cells to carfilzomib by increasing tumor apoptosis thus completely abrogating tumor growth and significantly extending mice survival. Conclusions: This is the first study to show the efficacy of PX-478 in MM demonstrating that PX-478 is acting as a pleiotropic molecule in which it inhibited many different hypoxia-induced biological processes - migration, angiogenesis and drug resistance. By overcoming these cancer adaptations, PX-478 has a clear advantage over using agents that carry an effect against one of these processes. This data provides a preclinical basis for future clinical trials testing efficacy of PX-478 in MM. Disclosures No relevant conflicts of interest to declare.

Comparisson of Molecular Mechanisms Altered by Treatment with Different Drugs (Doxorubicin, Melphalan, Bortezomib, Aplidin and Arsenic Trioxide) in Multiple Myeloma Patients Using Oligonucleotide Microarrays.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2457-2457
Author(s):  
Patricia Maiso ◽  
Norma C. Gutierrez ◽  
Ricardo Lopez-Perez ◽  
Gema Mateo ◽  
Isabel M. Isidro ◽  
...  
Keyword(s):  
Phase Transition ◽  
Cell Cycle ◽  
Multiple Myeloma ◽  
Arsenic Trioxide ◽  
Targeted Therapies ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Altered Expression

Abstract The development of novel targeted therapies in Multiple Myeloma (MM) has opened promising expectations for the treatment of this incurable hematological malignancy. However, the molecular mechanisms of both novel biologically based therapies and conventional treatments are still unclear. The purpose of the present study was to evaluate the changes in the gene expression profile of the human multiple myeloma cell line (MM.1S) following exposure to Doxorubicin, Melphalan, Bortezomib, Aplidin and Arsenic Trioxide. We have focused on the analysis of the early steps of activation of molecular mechanisms that lead to MM cell death. For this purpose we investigated with a time-course the onset of the apoptosis for every drug, according to the flow cytometric analysis with Annexin V- FITC Apoptosis Detection Reagent Kit. Based on these results the optimal concentration and time of exposure for each of the drugs were as follows Melphalan 50 μM, 9hours; Doxorubicin 1 μM, 17hours; Bortezomib 10 nM, 6 hours; Aplidin 50 nM, 4 hours and Arsenic Trioxide 5 mM, 3 hours. Affymetrix HG-U133A array containing around 15,000 full-length genes was used for mRNA expression profiling. All the experiments were performed in duplicate. The DNA- Chip Analyzer (DChip) was used to normalize and compare samples. Genes with expression changes greater than twofold in either direction were considered significant. A total of 269, 74, 74, 808 and 525 genes showed a significantly altered expression pattern, in response to Melphalan, Doxorubicin, Bortezomib, Aplidin and Arsenic Trioxide, respectively. Our results demonstrate that treatment with Melphalan inhibits DNA replication and transcription (underexpression of POLA, RFC1) as well as proliferation and survival (underexpression of IGF-1); it blocks the cell cycle (overexpression of CDKN1A and PA26) and induces apoptosis (overexpression of GADD45B). Doxorubicin deregulates many genes involved in cell cycle arrest (high expression of CDKN1A, PA26, GADD45A and low expression of CCND2 y CDC20) as well as up-regulation of several members of the TNF family (CD95, TRAILR2 and CD27). Bortezomib increases the expression of many “heat shock proteins” (HSP 110, HSP 70B, HSP 70B′) and decreases the level expression of IGF-1. Aplidin triggers early induction of many genes involved in apoptosis (overexpression of MAP4K3 and EGR2) and down-regulation of genes that play and important role in G2/M phase transition (NEK2, CENPF, BUB1). Arsenic Trioxide induces underexpression of essential genes for G1/S phase transition and cell cycle progression (CDC7L1, CDC25A) as well as underexpression of genes that mediate spindle formation and cromosome segregation (STK6, PRC1, HCAP-G, ZWINT, KIF4A, BUB1). Moreover, Arsenic Trioxide alike Bortezomib treatment up-regulates several HSP (HSP 40, HSP70B, HSP27). TNFSF9 which inhibits proliferation was the only gene up-regulated by all the drugs. Microarray technology demonstrates that treatment with novel targeted therapies (Bortezomib, Aplidin and Arsenic Trioxide) induces deregulation of molecular mechanisms which are not involved in anti-myeloma activity of conventional treatments (Melphalan, Doxorubicin). In addition it is an efficient tool to understand the differences in mechanisms of action of novel drugs.

ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1589-1589
Author(s):  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
John A. Lust ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Significant Activity ◽  
Rpmi 8226 ◽  
Dose Dependent

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

Aberrant Bone Marrow Perivascular Niches are Involved in Multiple Myeloma Progression: Role of Notch–Delta Pathway.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2800-2800
Author(s):  
Sara Lamorte ◽  
Marta Costa ◽  
Giovanni Camussi ◽  
Sergio Dias
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Pcr Analysis ◽  
Adherent Cells ◽  
Feeder Layer ◽  
Hematopoietic Stem ◽  
Lower Chamber

Abstract Abstract 2800 Poster Board II-776 Bone marrow (BM) angiogenesis is implicated in Multiple Myeloma (MM) progression. In this study, we tested the hypothesis that MM progression occurs when aberrant BM perivascular niches are established. We isolated BM endothelial cells derived from MM patients (MM-BMECs) from BM aspirates using anti-CD31Ab coupled to magnetic beads. FACS analysis showed that of all the cell lines isolated were endothelial: more than 95% expressed Ulex Europaeus Agglutinin-1 and Factor VIII and were negative for monocyte-macrophage (CD14) and plasma cell markers (CD38). To test the hypothesis that in MM patients BM perivascular niches are aberrant we analyzed how MM-BMECs modulate hematopoietic stem cells (HSCs) properties using a BM microvascular endothelial cell line isolated from a healthy donor (BMECs) as control. We co-cultured cord blood cells CD34+ HSCs in the presence of MM-BMECs or BMECs feeder layer and we analyzed the ability of MM-BMECs compared with BMECs to modulate HSCs adhesion, chemotaxis and apoptosis. The results show that MM-BMECs promote CD34+ HSCs adhesion, recruitment and protect them from apoptosis. In detail, we showed that after 24h of co-culture there was a significant increase in the number of adherent HSCs on MM-BMECs than on BMECs: 43±9% versus 25±6%. Moreover, when HSCs were cultured for 48 hours in 1% of serum in the presence of MM-BMECs they were less sensitive to apoptosis (9±11% of Annexin V+ cells) than HSCs cultured in the presence of BMECs (14±1% of Annexin V+ cells) or without a feeder layer, as control (17±3% of Annexin V+ cells). For the migration assay a transwell chamber system, in which the upper and the lower chambers were separated by 5-μm pore-size filter, was used. BMECs, MM-BMECs or nothing was plated in the lower chamber, while HSCs were seeded into the upper chamber. Both chambers were loaded with unsupplemented EBM-2 plus 2% of serum. Cell migration was studied over a 6-8 hours period and evaluated as number of cells migrated into the lower chamber. The results showed a significantly greater migration of HSCs in the presence of MM-BMECs than BMECs: 12±2% versus 5±1% of migrated cells. Taken together, these data showed that MM-BMECs promoted HSCs migration, adhesion and survival. Next we evaluated how MM-BMECs modulate the hemopoiesis recovery after irradiation in a NOD-SCID mouse model. When injected into sub-lethally irradiated (3 Grey) NOD-SCID mice MM-BMECs were detected in the BM integrated within the murine BM vessels and promoted hematopoietic recovery. In detail, MM-BMECs provided signals favoring the commitment towards lymphoid lineage. In fact, 7 days after injection, the BM of mice injected with MM-BMECs showed an increase in the percentage of lymphoblast (2.7%), compared with mice injected with BMECs or PBS, as control (respectively, 1.5% and 1.4%); followed, 14 days after injection, by a significant increase in the percentage of peripheral blood lymphocytes in mice injected with MM-BMECs (75±6%) versus mice injected with BMECS and PBS (respectively 60±0.5% and 47±7%). Since MM is a plasma cells disorder and the Notch-Delta pathway has been shown to play a central role in regulating HSCs properties, including the decisions of HSCs to undergo T- or B-cell differentiation, we investigated the involvement of this pathway in MM-BMECs and HSCs interaction. As determined by FACS and RT-PCR analysis, MM-BMECs, compared to BMECs, over expressed Delta-like Notch ligand 4 (DII4). Thus, we investigated the role of DII4 in the MM-BMECs/BMECs-HSCs adhesion. The first results showed that the expression of DII4 by MM-BMECs is necessary to promote HSCs adhesion. In fact, using a blocking antibody against DII4 (AbαDII4) at 50ug/ml there was an impairment in HSCs adhesion to MM-BMECs (43±9% versus 24±2% of adherent cells without and with AbαDII4 treatment), but not on BMECs (25±6% versus 26±1.4% of adherent cells without and with AbαDII4 treatment). Ongoing experiments are focusing on the role of DII4 in the modulation of HSCs proliferation, protection against apoptosis and in vitro-in vivo B commitment by MM-BMECs. Taken together, all these data suggest that BMECs in MM may function as “aberrant perivascular niches”, modulating HSCs properties. This aberrant phenotype could be due to an alteration of the Notch-Delta pathway in BMECs that favors malignant clonal growth by protecting it from apoptosis, favoring migration, adhesion and providing self-renewing and/or proliferative cues. Disclosures: No relevant conflicts of interest to declare.

Lactadherin as a Probe for Phosphatidylserine Exposure and as An Anticoagulant for the Procoagulant Activity in the Study of Stored Platelets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3150-3150
Author(s):  
Jin Zhou ◽  
Jinxiao Hou ◽  
Wen Li ◽  
Xiaoqian Zhang ◽  
Yueyue Fu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Progressive Increase ◽  
Procoagulant Activity ◽  
Platelet Concentrates ◽  
Time Analysis ◽  
Coagulation Time ◽  
Enzymatic Assays

Abstract Abstract 3150 Poster Board III-87 Background Phosphatidylserine (PS) can support coagulant reactions. However, it is uncertain how the location and extent of PS exposure to the membranes of stored platelets affect such reactions. We compared annexin V with lactadherin as a way of detecting how of PS exposure influences the procoagulant properties of stored platelets in platelet concentrates (PCs). Method PS exposure and the relevant procoagulant activity (PCA) of platelets in 5 different PCs were investigated by flow cytometry, confocal microscopy, coagulation time analysis and enzymatic assays. PS exposure was separately measured using annexin V and lactadherin, respectively. Results Exposure of PS to stored platelets promoted thrombin formation. A progressive increase in PS exposure was detected by flow cytometry. Moreover, using lactadherin, we identifed higher levels of PS exposure on the platelets and platelet-derived microparticles (PMPs) compared to detection using annexin V. The percentage of PS-positive cells was 0.02 % by annexin V versus 0.3 % by lactadherin on day 0, 7.5 % by annexin V versus 12.3 % by lactadherin on day 5, and 29 % by annexin V versus 44.3 % by lactadherin on day 9. Rare microparticles (MPs) were released from fresh platelets, and, the number of PMPs increased approximately 2-fold on day 5 and further progressively increased. Using lactadherin and platelets in the earlier stage of storage, confocal microscopy revealed earlier and localized PS exposure based on plasma membrane staining. For later storage platelets, increased levels of PS-positive platelets and PMPs were clearly detected by both annexin V and lactadherin. Thirty-two nM lactadherin or annexin V prolonged coagulation time 2.4 fold versus 2 fold. The productions of thrombin and intrinsic/extrinsic factor Xase were approximately inhibited 85 % and 60 % by lactadherin and annexin V, respectively. Conclusion PS exposure was localized to the cellular rims, blebbing vesicles and thin elongated filopodia-like areas on banked platelets. Furthermore, lactadherin provides a more accurate measurement of PS exposure and the relevant with PCA, which is an important factor to consider for transfusion medicine. Our findings of elevated PS-positive platelets and PMPs indicate that platelets should not be stored for extended periods of time. Disclosures No relevant conflicts of interest to declare.

Utility of Multiparameter Flow Cytometry Immunophenotypic Studies In Patients with Systemic Light Chain (AL) Amyloidosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4051-4051
Author(s):  
Bruno Paiva ◽  
María-Belén Vidriales ◽  
Jose J. Perez ◽  
Maria-Consuelo López-Berges ◽  
Ramón García-Sanz ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Differential Diagnosis ◽  
Plasma Cell ◽  
Light Chain ◽  
Cell Cycle Analysis ◽  
Disease Assessment ◽  
Multiparameter Flow Cytometry ◽  
Al Amyloidosis

Abstract Abstract 4051 Multiparameter flow cytometry (MFC) immunophenotyping has shown to be of value for differential diagnosis and minimal residual disease assessment in multiple myeloma. However, the clinical value of MFC immunophenotyping in other plasma cell disorders (PCD) remains largely unexplored. Systemic light chain (AL) amyloidosis is a rare PCD characterized by the accumulation of monoclonal light chain fragments leading to end-organ damage and short survival. Bone marrow (BM) plasma cell (PC) infiltration in AL is usually low and thus the identification of clonal PC can be often difficult by immunohistochemistry and/or immunofluorescence. In the present study we focused on 34 BM samples sent to our institution with a suspected diagnosis of AL. MFC immunophenotypic studies were performed using the following 4-color combinations of MoAbs (FITC/PE/PerCP-Cy5.5/APC): CD38/CD56/CD19/CD45 (n=34); in addition cy-Kappa/cy-Lambda/CD19/CD38 staining was add to confirm the clonal or polyclonal nature of BMPC in equivocal cases. Ploidy and cell cycle analysis were additionally performed in a subset of cases (n=12/34). From the total 34 cases included in the present study, 28 had a confirmed diagnosis of AL. The remaining 6 cases were finally diagnosed with localized - amyloidoma - (n=2) and familial (n=1) forms of amyloidosis, multiple myeloma-associated amyloid (n=2) and congestive pericarditis (n=1). Interestingly, the presence of clonal PC was detected by MFC in 27 of the 28 (96%) patients with AL; in turn, clonal PC were undetectable in the BM of all cases with localized and familial forms of amyloidosis. The median overall level of PC (M-PC plus N-PC) seen in MFC immunophenotypic analyses of BM samples of the 28 patients with AL was 1.9% (range: 0.1% - 15%), with a significant positive correlation between PC enumerated by MFC and conventional morphology (r=0.5; p=.01). Within the BMPC compartment, the median proportion of clonal PC was of 94% (mean 81% ± 29%); in 6 cases all BMPC were clonal while in the remaining 22 patients residual normal PC persisted (median of normal PC/BMPC 13% ± 31%). The most common aberrant phenotypes were down-regulation of CD19 (92%) and CD45 (83%), followed by overexpression of CD56 (56%) and infra-expression of CD38 (42%). Aneuploidy was only found in 18% of cases, all of them hyperdiploid. Cell cycle analysis showed a median % of S-phase and G2-Mitosis PC of 0.7% and 3.5%, respectively. Concerning patients' outcome, cases with undetectable normal PC (6/28, 21%) had a significantly decreased overall survival (OS) compared to patients with persistent BM normal PC at diagnosis (22/28, 79%) with 3-year OS rates of 0% vs. 59%, respectively (p=.001). In summary, these preliminary data suggests that MFC immunophenotyping investigations may be clinically relevant in patients with suspected amyloidosis for i) differential diagnosis between AL and other forms of amyloidosis and, ii) prognostication of patients with AL according to the presence or absence of baseline persistent normal PC. Disclosures: No relevant conflicts of interest to declare.

Skeletal-Targeted, Osteolytic-Responsive Drug Delivery System Improves Anti-Tumor Efficacy in Multiple Myeloma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3155-3155
Author(s):  
Xuli Wang ◽  
Ye Yang ◽  
Scott Miller ◽  
Fenghuang Zhan
Keyword(s):  
Drug Delivery ◽  
Multiple Myeloma ◽  
Polymeric Micelles ◽  
Conflicts Of Interest ◽  
Drug Loading ◽  
Bone Matrix ◽  
Cathepsin K ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Tumor Inhibition

Abstract Abstract 3155 Background: Multiple myeloma (MM) cells often directly or indirectly induce the formation of osteoclasts, which enhance bone resorption by increasing secretion of a key protease (cathepsin K) to degrade bone matrix, leading to osteolytic lesions and serious skeletal complications. Hence, bone-targeted, osteolytic-responsive therapeutic modalities are greatly needed to improve clinical outcomes for MM. Methods and Results: A tri-block copolymer of peptide, poly(ethylene glycol) and poly(trimethylene carbonate) (Pep-b-PEG-b-PTMC) has been synthesized as a nanocarrier to improve treatment for MM. The functional peptide with the sequence of CKGHPGGPQAsp8 was designed to possess a bone tropism octapeptide (Asp8), a cathepsin K (CTSK)-cleavable substrate (HPGGPQ), multiple cationic residues and a terminal cysteine for site-specific conjugation. Maleimide-terminated diblock copolymer of PEG-b-PTMC was readily functionalized with the peptide to obtain Pep-b-PEG-b-PTMC that can spontaneously form micelles with the size of 75 nm in diameter. Sixty-six % of polymeric micelles were able to bind to hydroxyl apatite, showing high bone binding capability. The nanoparticles exhibited a negative-to-positive charge conversional profile upon exposure to cathepsin K, an overexpressed enzyme in osteolytic microenvironments. By using doxorubicin as a model drug, Pep-b-PEG-b-PTM showed 7.5 ± 0.5 % and 22.7 ± 1.5% for drug loading content and drug loading efficiency, respectively. More importantly, a unique characteristic of on-demand charge-conversional behaviour in a cathepsin K-rich condition led to enhanced cellular uptake of the nanotherapeutics, as demonstrated by confocal laser scanning microscopy. Enhanced tumor inhibition was observed in 5TGM1 MM cells when nanoparticles were pre-treated with 150 nM cathepsin K, demonstrating enzyme-triggered improved therapy. Efficacy of free DOX or DOX-loaded NPs in 5TGM1 mice bearing myeloma was further preliminarily tested. 5TGM1 mice bearing myeloma were established through injection of 5TGM1 cells (1 × 106 cells in 100 μL PBS) via tail vein, and tumor was allowed to grow for a week before initiating treatment study. Mice (n=3) were injected twice weekly with different therapeutic formulations at equivalent DOX dose (0.75 mg/Kg) or PBS. Tumor burden in the mice was monitored by ELISA measurements of serum IgG2b. Drug-loaded nanoparticles from Pep-b-PEG-b-PTMC were more efficacious in terms of mice survival rate and tumor inhibition when compared to the groups with non-targeted nanoparticles from mPEG-PTMC, free DOX or PBS controls. This improved drug efficacy may be attributed to more selective delivery of DOX to bone metastatic tissues and/or responsiveness of the nanoparticles to cathepsin K, thus improving tumor uptake of DOX, enhancing therapeutic efficacy in terms of tumor reduction as well as MM mouse survival. Conclusions: The promising results from this study may prompt the development of bone-targeted, enzyme-triggered drug delivery systems to improve their affinity to skeletal tissues, enhance selectivity for osteolytic regions and improve efficacy of anti-cancer agents, thus facilitating the development of effective nanotherapeutic modalities for multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

The Expression Of CD200 As a Prognostic Factor In Newly Diagnosed Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3082-3082
Author(s):  
Yi Li ◽  
Wenjun Wu ◽  
Jingsong He ◽  
Xiaoyan Han ◽  
Gaofeng Zheng ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Progression Free Survival ◽  
Staging System ◽  
Newly Diagnosed ◽  
New Approach ◽  
Myeloma Cells ◽  
Targeting Therapy ◽  
Molecular Therapeutic

Abstract Introduction multiple myeloma (MM) is currently an incurable hematological malignancy. Discovering molecular therapeutic targets is a new approach to improve the outcome in the treatment of the malignant disease. As CD200 is a type Ⅰmembrane glycoprotein expressed on myeloma cells, we asked if the expression of CD200 could serve as a prognostic marker for MM patients. Our data indicated that the expression level of CD200 is indeed correlated with the prognosis of the MM patients. Methods bone marrow samples from 96 newly diagnosed MM patients from April 2011 to July 2013 were evaluated by flow cytometry, using PE-conjugated anti-CD200 mAb, FITC-conjugated anti-CD138 mAb, and PE-Cy7-conjugated anti-CD45 mAb. PE-or FITC-conjugated normal mouse IgG was used as isotype-matched controls. Results 96 MM patients were investigated in the present study, including 60 men and 36 women, with a median age of 63 years (range 34–86 years). 81/96(84%) MM patients were CD200 positive with a median Mean Fluorescence Intensity (MFI) of 127 as analyzed by flow cytometry, which was consistent with the previous studies. While in 15 of 96 patients, CD200 expression was undetectable. Among the CD200 positive ones 7.40% patients were classified as stage Ⅰ, 12.35% were stage Ⅱ, and 80.25% were stage Ⅲ according to Durie–Salmon staging criteria. 40.74% patients were stageⅠ, 22.22% were stage Ⅱ, and 37.04% were stage Ⅲ, according to the International Staging System (ISS). Analysis of the CD200 positive patients revealed the MFI<127 group had a better progression free survival (PFS) (p=0.046) (Fig 1A) and overall survival (OS) (p=0.069) compared to those with MFI≥127. In the patients with age ≥65 years old, PFS (p=0.023) (Fig 1B) and OS (p=0.044) (Fig 1C) were much shorter in the MFI≥127 group, compared to the MFI<127 ones. Conclusions Our study demonstrated that the expression and MFI of CD200 on primary multiple myeloma cells is correlated with the prognosis of the MM patients. The better PFS and OS were observed in the MFI<127 group compared to the patients with MFI≥127, especially in the patients with age≥65 years old. Improved PFS in CD200 positive ones is likely due to the immune suppression mediated by CD200. Our study suggests that targeting therapy against CD200 may become a new approach to the treatment of MM in clinical practice. Disclosures: No relevant conflicts of interest to declare.

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