Human Plasma Extracellular Vesicle Isolation and Proteomic Characterization for the Optimization of Liquid Biopsy in Multiple Myeloma.

Cancer cells secrete membranous extracellular vesicles (EVs) which contain specific oncogenic molecular cargo (including oncoproteins, oncopeptides, and RNA) into their microenvironment and the circulation. As such, EVs including exosomes (small EVs) and microvesicles (large EVs) represent important circulating biomarkers for various diseases, including cancer and its progression. These circulating biomarkers offer a potentially minimally invasive and repeatable targets for analysis (liquid biopsy) that could aid in the diagnosis, risk stratification, and monitoring of cancer. Although their potential as cancer biomarkers has been promising, the identification and quantification of EVs in clinical samples remain challenging. Like EVs, other types of circulating biomarkers (including cell-free nucleic acids, cf-NAs; or circulating tumor cells, CTCs) may represent a complementary or alternative approach to cancer diagnosis. In the context of multiple myeloma (MM), a systemic cancer type that causes cancer cells to accumulate in the bone marrow, the specific role for EVs as biomarkers for diagnosis and monitoring remains undefined. Tumor heterogeneity along with the various subtypes of MM (such as non-secretory MM) that cannot be monitored using conventional testing (e.g. sequential serological testing and bone marrow biopsies) render liquid biopsy and circulating tumor-derived EVs a promising approach. In this protocol, we describe the isolation and purification of EVs from peripheral blood plasma (PBPL) collected from healthy donors and patients with MM for a biomarker discovery strategy. Our results demonstrate detection of circulating EVs from as little as 1 mL of MM patients' PBPL. High-resolution mass spectrometry (MS)-based proteomics promises to provide new avenues in identifying novel markers for detection, monitoring, and therapeutic intervention of disease. We describe biophysical characterization and quantitative proteomic profiling of disease-specific circulating EVs which may provide important implications for the development of cancer diagnostics in MM.

The Acute Phase Protein Hepcidin Is Cytotoxic to Human and Mouse Myeloma Cells.

BACKGROUND/AIM: Hepcidin is a cationic acute phase reactant synthesized by the liver. It has bactericidal properties and is a major regulator of iron homeostasis. Cationic antimicrobial peptides represent an innate antimicrobial defense system. We hypothesized that, like other cationic antimicrobial peptides, hepcidin is cytotoxic to cancer cells. MATERIALS AND METHODS: The cytotoxicity of human hepcidin against myeloma cells was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and DNA fragmentation assays. Plasma membrane damage was quantified by propidium iodide (PI) staining. Cell membrane changes were visualized by scanning electron microscopy. RESULTS: Hepcidin impaired myeloma cell survival and induced DNA fragmentation. PI staining and scanning electron microscopy revealed hepcidin-induced disruption of the plasma membrane. CONCLUSION: Human hepcidin is an anti-cancer peptide that induces myeloma cell lysis, and therefore may play a role in innate anticancer immunity. To our knowledge, this is the first biological function ascribed to human hepcidin that is not related to its antimicrobial and iron-regulatory properties.

Monoclonal glomerulopathy with features of cryoglobulinemic glomerulopathy in murine multiple myeloma model.

In vivo and animal models of monoclonal light chain-associated renal diseases are limited. The Vk*MYC transgenic model with multiple myeloma in 50-70 weeks old mice with renal involvement has been reported before, but detailed renal pathologic changes have not been well documented. This study fully investigated pathologic changes in the kidneys of Vk*MYC transgenic model using light microscopy, immunofluorescence stains for kappa and lambda light chains, and electron microscopy. Compared to the kidneys of wild-type mice, the kidneys of transgenic mice showed either mesangial segmental expansion, some with associated hypercellularity, and/or thrombotic obstruction of glomerular capillaries. The glomeruli revealed stronger lambda staining than kappa light chain staining. Six out of 12 kidneys from transgenic mice showed abundant electron-dense deposits when examined ultrastructurally. The deposits were located in glomerular capillary lumina in three cases. Large luminal and subendothelial deposits were characterized by randomly disposed microtubular structures measuring up to 16 nm in diameter, with overall features most consistent with cryoglobulins. In summary, about 50% of kidneys from the Vk*MYC mice with myeloma had features most consistent with monoclonal cryoglobulinemic glomerulopathy.

SR18292 exerts potent antitumor effects in multiple myeloma via inhibition of oxidative phosphorylation.

AIMS: Multiple myeloma (MM) was recently reported to rely on increased oxidative phosphorylation (OXPHOS) for survival, providing a potential opportunity for MM therapy. Herein, we aimed to propose a novel targeted drug for MM treatment, followed by the exploration of reason for OXPHOS enhancement in MM cells. MATERIALS AND METHODS: The expression of OXPHOS genes and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) was analyzed using bioinformatics analyses, followed by verification in MM cell lines. The effects of SR18292 on OXPHOS were measured by qRT-PCR, Western blot, transmission electron microscopy, oxygen consumption rate and so on. The proliferation and apoptosis were evaluated by CCK-8, flow cytometry and Western blot. The efficiency and safety of SR18292 were assessed in a mouse model of MM. KEY FINDINGS: The OXPHOS genes were generally overexpressed in MM cells, which was associated with poorer prognosis of MM patients. PGC-1α, a transcriptional coactivator, was upregulated in MM cells, and MM patients with higher PGC-1α expression exhibited increased enrichment of the OXPHOS gene set. Treatment with SR18292 (an inhibitor of PGC-1α) significantly impaired the proliferation and survival of MM cells due to OXPHOS metabolism dysfunction, which leads to energy exhaustion and oxidative damage. Besides, SR18292 potently inhibited tumor growth at a well-tolerated dose in MM model mice. SIGNIFICANCE: The overexpression of OXPHOS gene set mediated by upregulated PGC-1α provides a structural basis for enhanced OXPHOS in MM cells, and SR18292 (a PGC-1α inhibitor) exerts potent antimyeloma effects, offering a potential tangible avenue for MM therapy.

Ultrastructural identification of a proximal tubulopathy without crystals in a relapsed multiple myeloma patient.

A multiple myeloma patient, who had been treated with a hematopoietic stem cell transplant, underwent a renal biopsy for investigation of a possible relapse of disease as indicated by increased serum creatinine and positive urinary Bence-Jones protein containing increased kappa light chain. Paraprotein-related renal disease was not evident by light microscopy or immunofluorescence microscopy however electron microscopy demonstrated a proximal tubulopathy with intracytoplasmic non-crystalline inclusions. The ultrastructural findings suggested possible end-organ involvement by the disease and follow-up studies subsequently revealed a relapsed multiple myeloma in the patient. The case exemplifies the usefulness of electron microscopy in detecting paraproteins that, in some instances, may be difficult to demonstrate by other techniques.

Inhibition of autophagy enhances the antitumour activity of tigecycline in multiple myeloma.

Accumulating evidence shows that tigecycline, a first-in-class glycylcycline, has potential antitumour properties. Here, we found that tigecycline dramatically inhibited the proliferation of multiple myeloma (MM) cell lines RPMI-8226, NCI-H929 and U266 in a dose and time-dependent manner. Meanwhile, tigecycline also potently impaired the colony formation of these three cell lines. Mechanism analysis found that tigecycline led to cell cycle arrest at G0/G1 with down-regulation of p21, CDK2 and cyclin D1, rather than induced apoptosis, in MM cells. Importantly, we found that tigecycline induced autophagy and an autophagy inhibitor bafilomycin A1 further amplified the tigecycline-induced cytotoxicity, suggesting that autophagy plays a cytoprotective role in tigecycline-treated MM cells. Mechanisms modulating autophagy found that tigecycline enhanced the phosphorylation of AMPK, but did not decrease the phosphorylation of Akt, to inhibit the phosphorylation of mTOR and its two downstream effectors p70S6K1 and 4E-BP1. Tigecycline effectively inhibited tumour growth in the xenograft tumour model of RPMI-8226 cells. Autophagy also occurred in tigecycline-treated tumour xenograft, and autophagy inhibitor chloroquine and tigecycline had a synergistic effect against MM cells in vivo. Thus, our results suggest that tigecycline may be a promising candidate in the treatment of MM.

Hypoxia inducible factor-1 alpha as a therapeutic target in multiple myeloma.

The increasing importance of hypoxia-inducible factor-1α (HIF-1α) in tumorigenesis raises the possibility that agents which specifically inhibit this transcription factor, would provide significant therapeutic benefit. The constitutive expression of HIF-1α in about 35% of Multiple Myeloma (MM) patients suggests HIF-1α suppression might be part of a therapeutic strategy. Accordingly, we explored the effect of EZN-2968, a small 3rd generation antisense oligonucleotide against HIF-1α, in a panel of MM cell lines and primary patients samples. Here, we demonstrated that EZN-2968 is highly specific for HIF-1α mRNA and that exposure of MM cells to EZN-2968 resulted in an efficient and homogeneous loading of the cells showing a long lasting low HIF-1α protein level. In MM cells, HIF-1α suppression induced a permanent cell cycle arrest by prolonging S-phase through cyclin A modulation and in addition it induced a mild apoptotic cell death. Moreover, HIF-1α suppression caused a metabolic shift that leaded to increased production of ATP by oxidative phosphorylation (i.e. Warburg effect reversion), that was confirmed by the observed mitochondrial membrane potential decrease. These results show that HIF-1α is an important player in MM homeostasis and that its inhibition by small antisense oligonucleotides provides a rationale for novel therapeutic strategy to improving MM treatment.

Characterization of multiple myeloma vesicles by label-free relative quantitation.

Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. The role that extracellular vesicles (EVs), microvesicles and exosomes, released by MM cells have in cell-to-cell communication and signaling in the bone marrow is currently unknown. This paper describes the proteomic content of EVs derived from MM.1S and U266 MM cell lines. First, we compared the protein identifications between the vesicles and cellular lysates of each cell line finding a large overlap in protein identifications. Next, we applied label-free spectral count quantitation to determine proteins with differential abundance between the groups. Finally, we used bioinformatics to categorize proteins with significantly different abundances into functional groups. The results illustrate the first use of label-free spectral counting applied to determine relative protein abundances in EVs.

Knockdown of DEPTOR induces apoptosis, increases chemosensitivity to doxorubicin and suppresses autophagy in RPMI-8226 human multiple myeloma cells in vitro.

DEP domain containing mammalian target of rapamycin (mTOR)-interacting protein (DEPTOR) is an mTOR binding protein that is overexpressed in RPMI-8226 human multiple myeloma cells, and plays an important role in maintaining cell survival. However, knowledge on the effects of DEPTOR knockdown on the biological functions of RPMI­8226 human multiple myeloma cells, is limited. This study aimed to determine the role of DEPTOR in the proliferation, apoptosis and autophagy in these cells and to elucidate the mechanisms by which DEPTOR contributes to the chemosensitivity of myeloma cells. RNA interference was used to reduce the expression of DEPTOR. Cytotoxicity was evaluated by MTT assay. Apoptosis was examined by flow cytometry. DEPTOR mRNA and protein expression in RPMI­8226 cells treated with DEPTOR-specific short hairpin RNA (shRNA) was evaluated by RT-PCR, quantitative PCR and western blot analysis. The expression of apoptosis­associated proteins, autophagy­associated proteins, and the activation of the phosphoinositide 3­kinase (PI3K)/Akt signaling pathway were detected by western blot analysis. Autophagy was also measured by transmission electron microscopy and monodansylcadaverine (MDC). In this study, RPMI-8226 cells were transfected with the DEPTOR-specific shRNA, which resulted in the significant inhibition of the transcription and expression of DEPTOR. The downregulation of DEPTOR inhibited proliferation, enhanced the doxorubicin­induced growth inhibitory effects on RPMI-8226 cells, and increased the expression of cleaved caspase­3 and cleaved poly(ADP-ribose) polymerase (PARP). Moreover, the downregulation of DEPTOR suppressed autophagy and inhibited the activation of the PI3K/Akt signaling in RPMI­8226 cells. In conclusion, our data demonstrated that the downregulation of DEPTOR induces apoptosis, increases chemosensitivity to doxorubicin, and suppresses autophagy and the activation of the PI3K/Akt signaling pathway in RPMI­8226 human multiple myeloma cells.

In multiple myeloma, bone-marrow lymphocytes harboring the same chromosomal abnormalities as autologous plasma cells predict poor survival.

Chromosomal abnormalities in plasma cells (PCs) from multiple myeloma (MM) provide a clonal signature to identify malignant cells. BM-lymphocytes from MM aspirates, defined by stringent criteria, were screened for the same chromosomal abnormalities as autologous PCs, including translocations, deletions, and amplifications. For 200 MM patients, we evaluated BM mononuclear cells to identify lymphocytes and autologous PCs on the same slide, followed by interphase fluorescence in situ hybridization to characterize their chromosomal abnormalities. Of all patients having a given chromosomal abnormality(s) in PCs, 45% showed that same abnormality(s) in 2-37% (median = 5%) of BM-lymphocytes. Most translocations, amplifications, and deletions found in MM PCs were also detected in lymphocytes, above the healthy-donor "cut-off." In patients having chromosomally abnormal CD20(-) PCs, chromosomally abnormal lymphocytes were found among CD20+ cells confirming them as B cells. Exceptions were amplification of 1q21 or p53 deletion, which characterize PCs but were undetectable in BM-lymphocytes, suggesting that processes leading to these abnormalities may be exclusive to PCs. For a set of 75 patients whose BM-lymphocytes and PCs were analyzed by all six probe sets, 58% of those with abnormal PC also had abnormal BM-lymphocytes harboring from one to five different abnormalities. Confirming the clinical significance of chromosomally abnormal BM-lymphocytes, MM patients having abnormalities in both lymphocytes and PC had significantly worse survival than those with abnormalities only in PC (HR = 2.68). The presence of at least one chromosomal abnormality in BM-lymphocytes appears to have greater clinical significance than particular abnormalities. Chromosomally abnormal BM-lymphocytes correlate with poor outcome and by extrapolation with more aggressive disease.

Visualization of numerical centrosomal abnormalities by immunofluorescent staining.

The presence of multiple centrosomes in tumor cells is associated with the formation of multipolar mitotic spindles and results in aneuploidy of both daughter cells. Centrosome amplification is a feature of all cancer cells. We have previously described centrosome amplification in abnormal B cells. Further studies of centrosome amplification in different stages of B lineage development could provide important information about multiple myeloma pathogenesis.

Shear flow-induced formation of tubular cell protrusions in multiple myeloma cells.

Exposure of live cells to shear flow induces major changes in cell shape, adhesion to the extracellular matrix, and migration. In the present study, we show that exposure of cultured multiple myeloma (MM) cells to shear flow of 4-36 dynes/cm(2) triggers the extension of long tubular protrusions (denoted flow-induced protrusions, or FLIPs) in the direction of the flow. These FLIPs were found to be rich in actin, contain few or no microtubules and, apart from endoplasmic reticulum (ER)-like membranal structures, are devoid of organelles. Studying the dynamics of this process revealed that FLIPs elongate at their tips in a shear force-dependent manner, and retract at their bases. Examination of this force dependence revealed considerable heterogeneity in the mechanosensitivity of individual cells, most likely reflecting the diversity of the malignant B cell population. The mechanisms underlying FLIP formation following mechanical perturbation, and their relevance to the cellular trafficking of MM cells, are discussed.

Dual inhibition of akt/mammalian target of rapamycin pathway by nanoparticle albumin-bound-rapamycin and perifosine induces antitumor activity in multiple myeloma.

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway mediates multiple myeloma (MM) cell proliferation, survival, and development of drug resistance, underscoring the role of mTOR inhibitors, such as rapamycin, with potential anti-MM activity. However, recent data show a positive feedback loop from mTOR/S6K1 to Akt, whereby Akt activation confers resistance to mTOR inhibitors. We confirmed that suppression of mTOR signaling in MM cells by rapamycin was associated with upregulation of Akt phosphorylation. We hypothesized that inhibiting this positive feedback by a potent Akt inhibitor perifosine would augment rapamycin-induced cytotoxicity in MM cells. Perifosine inhibited rapamycin-induced phosphorylated Akt, resulting in enhanced cytotoxicity in MM.1S cells even in the presence of interleukin-6, insulin-like growth factor-I, or bone marrow stromal cells. Moreover, rapamycin-induced autophagy in MM.1S MM cells, as evidenced by electron microscopy and immunocytochemistry, was augmented by perifosine. Combination therapy increased apoptosis detected by Annexin V/propidium iodide analysis and caspase/poly(ADP-ribose) polymerase cleavage. Importantly, in vivo antitumor activity and prolongation of survival in a MM mouse xenograft model after treatment was enhanced with combination of nanoparticle albumin-bound-rapamycin and perifosine. Utilizing the in silico predictive analysis, we confirmed our experimental findings of this drug combination on PI3K, Akt, mTOR kinases, and the caspases. Our data suggest that mutual suppression of the PI3K/Akt/mTOR pathway by rapamycin and perifosine combination induces synergistic MM cell cytotoxicity, providing the rationale for clinical trials in patients with relapsed/refractory MM. Mol Cancer Ther; 9(4); 963-75. (c)2010 AACR.

A physical mechanism for coupling bone resorption and formation in adult human bone.

During skeletal remodeling, pre-osteoclasts and pre-osteoblasts are targeted to critical sites of the bone to resorb and reconstruct bone matrix, respectively. Coordination of site-specific recruitment of these two cell types is a prerequisite to maintain the specific architecture of each bone within strict limits throughout adult life. Here, we determined that the bone marrow microanatomy adjacent to remodeling areas is a central player in this process. By using histomorphometry and multiple immunostainings, we demonstrated in biopsies exhibiting coupled bone resorption and formation that osteoclasts and osteoblasts on the bone surface were always covered by a canopy of flat cells expressing osteoblast markers. In contrast, in biopsies in which this canopy was disrupted, bone formation was deficient. Three-dimensional visualizations revealed that this canopy covered the entire remodeling site and was associated with capillaries, thereby forming a previously unrecognized microanatomical entity. Furthermore, pre-osteoclasts were positioned along these capillaries. These findings led to a model that implicates vasculature in the site-specific recruitment of osteoclasts and osteoblasts and embraces the current knowledge on the molecular mechanism of bone remodeling.