Single-Cell Transcriptomics Identifies Intra-Tumor Heterogeneity in Human Myeloma Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3385-3385 ◽  
Author(s):  
Amit Kumar Mitra ◽  
Holly Stessman ◽  
Michael A. Linden ◽  
Brian Van Ness
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Lines ◽  
Tumor Heterogeneity ◽  
Myeloma Cell ◽  
Gene Model ◽  
Color Flow ◽  
Myeloma Cells ◽  
Genetic Signature ◽  
Human Myeloma Cell

Abstract Multiple myeloma (MM) is a plasma cell neoplasm with significant complexity and heterogeneity. Proteasome inhibitors (PI) including bortezomib (Velcade/Bz), carfilzomib (Kyprolis/Cz) and Ixazomib are effective chemotherapeutic agents in the treatment of MM, used alone or in combination with other anti-cancer agents. However, in spite of the recent improvements in treatment strategies, MM still remains a difficult disease to cure with median survival rate of around 7 years. In a recently published study, we have shown that the heterogeneity in response to proteasome inhibitor (PI)-based treatment in MM is governed by underlying molecular characteristics of the subclones within tumor population (Stessman et al. 2013). We confirmed the presence of residual resistant sub-population comprising up to 15% of the bulk Bz-sensitive cell population in drug-naïve MM tumors. We hypothesize that this pre-existing resistant sub-population may give rise to emerging resistance in course of treatment with PIs. In the current study, we used single cell transcriptomics analysis to identify tumor subclones within Human Myeloma Cell Lines (HMCLs) based on a 48-gene model of predictive genetic signature for baseline PI response. Automated single-cell capture and cDNA synthesis from cellular RNA were performed using Fluidigm’s C1TM Single-Cell Auto Prep System. The cDNA was then harvested and transferred to BioMark HD System for single-cell targeted high-throughput qPCR-based gene expression analysis of a 48 gene-panel using Fluidigm DELTAgene assays. Our 48-gene model combines our previously published 23 gene expression profiling (GEP) signature that could discriminate between sensitive and resistant responsiveness to Bz, and the Shaughnessy et al prognostic 17-gene GEP model along with control genes, including cell cycle genes, anti-apoptotic genes, proteasome subunit genes, house-keeping genes and internal negative controls. Based on the differential expression of these 48 genes used in the modeling, distinct subclonal populations were then identified using a combination of Fluidigm’s analysis software and the R Statistical analysis package. Further, a principal component analysis (PCA) score plot was generated as a two-dimensional grid to visualize the separate populations associated with resistant profiles. Finally, hierarchical clustering (HC) analysis was used to generate heat maps that group expression patterns associated with response. Our results demonstrated the presence of pre-existing subclones of cells within untreated myeloma cells with a characteristic genetic signature profile distinct from the pre-treatment overall (bulk) profile of myeloma cells. As an additional validation of subclonal architecture, we demonstrated the presence of subclones within HMCLs using multi-color flow cytometry. The results presented will help identify the presence and extent of intra-tumor heterogeneity in MM by single cell transcriptomics and may define residual pre-existing subclones resistant to PI therapies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 517-526 ◽  
Author(s):  
B Klein ◽  
XG Zhang ◽  
M Jourdan ◽  
J Content ◽  
F Houssiau ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Lines ◽  
Interleukin 6 ◽  
Neutralizing Antibodies ◽  
Myeloma Cell ◽  
B Cell Differentiation ◽  
Myeloma Cells ◽  
Autocrine Regulation ◽  
Spontaneous Proliferation ◽  
Human Myeloma Cell

Abstract To explore the mechanisms involved in the pathogenesis of human multiple myeloma (MM), we investigated the potential role of interleukin-6 (IL-6), a B-cell differentiation factor in humans, and a growth factor for rat/mouse heterohybridomas and murine plasmacytomas. Using a heterohybridoma assay, we found that two well-documented human myeloma cell lines, RPMI 8226 and U266, did not secrete IL-6 and did not express RNA messengers for IL-6. Neutralizing antibodies to IL-6 did not inhibit their proliferation, and recombinant IL-6 did not stimulate it. Taken together, these data show that IL-6 is not the autocrine growth factor of these human myeloma cell lines. A high production of IL-6 was found in the bone marrows of patients with fulminating MM, compared with patients with inactive or slightly active MM, or to healthy donors. This IL-6 production was assigned to adherent cells of the bone-marrow environment but not to myeloma cells. A spontaneous proliferation of myeloma cells freshly isolated from patients was observed in short-term cultures. Recombinant IL-6 was able to amplify it two- to threefold. The spontaneous proliferation of the myeloma cells was inhibited by anti-IL-6 antibodies and reinduced by recombinant IL-6. After 2 to 3 weeks of culture, the myeloma-cell proliferation progressively declined and no IL-6-dependent myeloma cell lines could be obtained despite repeated additions of fresh IL-6 and costimulation with other cytokines such as tumor necrosis factor (TNF)beta, or IL-1 beta. These data demonstrated a paracrine but not autocrine regulation of the growth and differentiation of myeloma cells by IL-6.

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

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5080-5080
Author(s):  
Shankaranarayana Paneesha ◽  
Raghu Adya ◽  
Hemali Khanji ◽  
Ed Leung ◽  
C. Vijayasekar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Mrna Expression ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Human Myeloma Cell

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

Integrative Genomic Approach Identifies Deregulated MicroRNAs in Human Myeloma Cell Lines.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1684-1684
Author(s):  
Marta Lionetti ◽  
Luca Agnelli ◽  
Laura Mosca ◽  
Katia Todoerti ◽  
Domenica Ronchetti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Mirna Expression ◽  
Mirna Gene ◽  
Myeloma Cell ◽  
Host Gene ◽  
Expression Levels ◽  
Mirna Genes ◽  
Genome Wide ◽  
Human Myeloma Cell

Abstract The recent discovery of microRNAs (miRNAs), small noncoding RNAs involved in the regulation of cell cycle, survival, and differentiation programmes, has added a further level of complexity to normal and cancer cell biology. Loss or amplification of miRNA genes by broad cytogenetic abnormalities or minute molecular aberrations has been observed in a variety of human malignancies, with the consequent altered expression of these regulatory genes. Additionally, approximately one third of miRNAs are located within the intronic regions of coding transcription units, and recent evidence indicates that the expression of these miRNAs largely coincides with the transcription of the corresponding host genes. To date, no evidence of deregulated miRNA expression has been reported in multiple myeloma (MM). To provide insights into miRNA biology in MM, we performed an integrative analysis of genome-wide, gene expression and miRNA expression profilings in a panel of 16 human myeloma cell lines (HMCLs). Global miRNA and mRNA expression data were generated on Agilent miRNA microarrays (representing 470 human mature miRNAs) and GeneChip® HG-U133A arrays, respectively, and both quantile-normalized. Genome-wide profiling data were generated on GeneChip® Human Mapping 250K NspI arrays and copy number (CN) values were inferred using the circulary binary segmentation (DNAcopy R Bioconductor package). To measure the correlation between the expression levels of each miRNA and the corresponding CN value or host gene expression, conventional non-parametric analyses were performed (Kendall’s tau and Wilcoxon rank-sum tests). As regards miRNA gene CN, the most frequent alteration identified was represented by gain/amplification (for all miRNA genes investigated, an increased CN was present in at least 3 HMCLs, with an average frequency of 58%), followed by loss (5%) and biallelic deletion (0.3%). Our analysis revealed that 14 different miRNA transcripts (miR-15a, miR-19a, miR-21, miR-22, miR-30d, miR-99b, miR-130b, miR-132, miR-140, miR-185, miR-339, miR-491, miR-503, miR-768-3p) had concordant levels with the inferred CN value of the corresponding miRNA gene. Notably, the identified miRNAs mapped to different genomic regions, some of which are involved in recurrent CN alterations in MM, such as 8q24, 19q13.33, or chromosome arms 13q, 16q, 17p, 17q, 22q, and for some of the miRNAs a role in other types of cancer has already been suggested. As regards intragenic miRNAs, 187 miRNA/host gene pairs were obtained after localizing miRNAs within the absolute 5′ and 3′ regions of genes represented on the HG-U133A arrays; 25 of these showed a significant correlation between miRNA and mRNA levels. Among the most correlated miRNA/hostgene pairs we identified miR-152/COPZ2, miR-342-3p/EVL, miR-335/MEST, miR-25 and miR-106b/MCM7. For some of the identified pairs, miRNA expression levels were validated by means of Q-RT-PCR. In conclusion, we showed that miRNA expression in HMCLs could be affected by the presence of genomic lesions or may correlate with host-gene modulation, suggesting a possible role in the molecular pathogenesis of MM. Our integrative approach represents the basis for further investigations, also in primary tumors, aimed at functionally characterizing specific miRNAs in MM.

scholarly journals Expression and function of Fas (APO-1/CD95) in patient myeloma cells and myeloma cell lines

Blood ◽  
1995 ◽  
Vol 85 (12) ◽  
pp. 3566-3576 ◽  
Author(s):  
JJ Westendorf ◽  
JM Lammert ◽  
DF Jelinek
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Cell Lines ◽  
Protective Factor ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Color Flow ◽  
Fas Antigen ◽  
Myeloma Cells ◽  
And Function

Cross-linkage of the Fas antigen induces programmed cell death in many normal and malignant lymphoid cells by a process known as apoptosis. In this study, we examined the sensitivity of myeloma cell lines and patient plasma cells to a cytolytic anti-Fas monoclonal antibody (MoAb). Eight of 10 myeloma cell lines were induced to undergo programmed cell death by anti-Fas MoAb as determined by DNA fragmentation and morphologic changes. Of the two myeloma cell lines that were resistant to anti-Fas treatment, one did not express the Fas antigen. Only the U266 cell line expressed Fas, but was not killed by the anti0Fas MoAb. To extend these studies, we have examined the expression and function of Fas in freshly isolated CD38hiCD45neg-int plasma cells from patients with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), and primary amyloidosis (AL). By three-color flow cytometry, we found Fas expression in CD38hiCD45neg-int plasma cells from all patient groups to be variable, as Fas was expressed in 15 of 28 MM, 3 of 6 MGUS, and 2 of 7 AL patients. In morphologic studies of apoptosis, Fas-positive myeloma cells in patient bone marrow mononuclear cell (MNC) cultures appeared to be resistant to anti-Fas-mediated apoptosis. By contrast, purified myeloma cells from the same patient were sensitive to anti-Fas treatment, suggesting the presence of a protective factor(s) in unseparated MNC cultures that may inhibit Fas-induced apoptosis of plasma cells. Of interest, serum from normal individuals and myeloma patients also protected myeloma cell lines from undergoing Fas-mediated apoptosis. These studies show that Fas expression in myeloma cell lines and CD38hiCD45neg-int patient plasma cells is variable and may reflect a variance in the maturation status of the various plasma cell populations. Moreover, Fas-mediated killing of patient cells and myeloma cell lines was also variable, which may be influenced, in part, by the presence of a soluble protective factor.

CD138+cell separation affects cancer gene expression in human myeloma cell lines

2013 ◽  
Vol 91 (2) ◽  
pp. 187-188
Author(s):  
Rasmus Sørrig ◽  
Niels Emil Ulrich Hermansen ◽  
Christoffer Hother ◽  
Peter Gimsing
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Cell Separation ◽  
Myeloma Cell ◽  
Cancer Gene ◽  
Human Myeloma Cell

scholarly journals Incorporation and Effect of Arachidonic Acid on the Growth of Human Myeloma Cell Lines

1999 ◽  
Vol 8 (2) ◽  
pp. 115-118 ◽  
Author(s):  
V. Desplat ◽  
C. Dulery ◽  
V. Praloran ◽  
Y. Denizot
Keyword(s):  
Arachidonic Acid ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Base Hydrolysis ◽  
Membrane Phospholipids ◽  
Myeloma Cells ◽  
Lipoxygenase Products ◽  
Kinetics Of ◽  
Phospholipid Species ◽  
Human Myeloma Cell

The objectives of this work are to investigate the incorporation of arachidonic acid (AA) in the human myeloma cell lines OPM2, U266 and IM9, and to assess the effect of AA and lipoxygenase products of AA on their growth. The kinetics of acylation of [3H]AA indicates that myeloma cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]AA is incorporated unmodified in U266, IM9 and OPM2 phospholipids, and is linked by an ester bond. Prelabeling-chase experiments indicate no trafficking of labeled AA among the various phospholipid species. Addition of AA and lipoxygenase products of AA (leukotriene B4and C4, lipoxin A4and B4, 12- and 15-hydroxyeicosatetraenoic acid) have no effect on U266, IM9 and OPM2 proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while human myeloma cells readily incorporate AA in their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.

The PPARß Activation Mechanism Which Suppresses the Constitutive NF-kB Activity in Human Myeloma Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4739-4739
Author(s):  
Ken-ichiro Otsuyama ◽  
Jakia Amin ◽  
Saeid Abroun ◽  
Abul Islam ◽  
Karim Shamsasenjan ◽  
...  
Keyword(s):  
Cell Lines ◽  
Target Genes ◽  
Myeloma Cell ◽  
Activation Mechanism ◽  
Pcr Analysis ◽  
Myeloma Cells ◽  
Ppar Agonist ◽  
Stimulation Of ◽  
Human Myeloma Cell ◽  
Human Myeloma Cells

Abstract [Purpose] It is considered that human myeloma cells have the constitutively high NF-kB activity involved in survival and proliferation. PPAR (Peroxisome proliferator-activated receptor) ß is ubiquitously expressed in all cells and considered to be involved in the lipid metabolism and regulating the inflammatory response and cell proliferation. We already have found that adrenal cortex hormones (DHEA and DHEA-S etc), dexamethasone (Dex) and baicalein augmented the expression of reporter gene. However, it remain to be clarified the role of PPARß in human myeloma cells. We focus on the mechanism of PPARß suppressed NF-kB activity. [Method] To know NF-kB activity of human myeloma cells, we performed EMSA with NF-kB consensus oligo. To investigate NF-kB and PPARß after stimulation of PPAR agonist, we did EMSA with NF-kB and PPAR consensus oligo. To confirm whether it is repression according the NF-kB activity suppression accompanying PPARß activation to the interaction of PPARß and NF-kB, we conducted immunoprecipitation - western blot analysis. To check whether the expression of NF-kB target genes (cIAP1, Bcl-xL, etc) were suppressed after stimulation of PPAR agonist, we performed RT-PCR analysis. [Result and discussion] It was suggested that human myeloma cell lines have constitutive NF-kB activity, and its activity mainly regulated p50. NF-kB activity and its target genes were repressed by stimulation of PPAR agonist. From the above, it was suggested that the activated PPARß interacted NF-kB and then its activity was suppressed.

Usp9x Silencing and Enzyme Inhibition Suppress Myeloma Cell Survival and in Vivo Tumor Growth.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2936-2936
Author(s):  
Luke F. Peterson ◽  
Hanshi Sun ◽  
Yihong Liu ◽  
Malathi Kandarpa ◽  
Diane Giannola ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Protein Expression ◽  
Tumor Growth ◽  
Cell Survival ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Protein Levels

Abstract Abstract 2936 Due to clinical success with proteasome inhibitors and E3 ligase modulators, proteins of the ubiquitin/proteasome system have emerged as novel therapeutic targets in several hematological malignancies. Deubiquitinases (DUBs) play a key role in regulating all aspects of this pathway and are aberrantly expressed or activated in several hematological malignancies. Usp9x is a high MW DUB, which has previously been shown to alter many signaling pathways, and its overexpression has been associated with drug resistance and poor prognosis in myeloma patients. Usp9x is reported to control cell survival through deubiquitination of Mcl-1 and other substrates, thereby reducing their proteosomal degradation. Since Mcl-1 plays a major role in myeloma cell survival and drug resistance, we compared Usp9x gene expression in myeloma cell lines to primary myeloma specimens. Usp9x gene expression varied among myeloma cell lines by 1.5 to 2.5-fold and endogenous Usp9x protein levels were even more variable (range 1.5 to 8-fold). Elevated Usp9x protein expression was not consistently associated with elevated Usp9x enzymatic activity as myeloma cells with the highest Usp9x gene and protein expression (KMS-11) had minimal Usp9x DUB activity. Moreover, Mcl-1 levels did not consistently correlate with Usp9x gene, protein or DUB activity, suggesting that a more complex mechanism regulates Usp9x activity and Mcl-1 stability in myeloma cells. To further examine the relationship between Usp9x and Mcl-1, we suppressed Usp9x expression (with siRNA and shRNA) and inhibited Usp9x activity with small molecule DUB inhibitors, WP1130 and VM030. Silencing Usp9x expression in H929 and MM1.S cells with shRNA vectors resulted in a reduction in Mcl-1 levels and induction of apoptosis, which approached the level of cell death achieved with direct Mcl-1 silencing. Similar results were obtained with siRNA-based Usp9x silencing in RMPI-8226 and KMS-11 cells; however, this approach led to the activation of a homologous DUB, Usp24, primarily through an increase in its protein stability. Usp24 silencing led to a decrease in myeloma cell survival, suggesting that Usp9x and Usp24 are coordinately regulated and play a role in myeloma cell survival. Measurement of Usp9x and Usp24 gene expression levels in primary myeloma samples and cell lines demonstrated that myeloma cell lines express 2 to 100-fold lower levels of these DUBs compared to primary tumors. Immuno-depletion studies illustrated that both Usp9x and Usp24 were activated in myeloma cells, and targeting both DUBs with WP1130 or VM030 led to a rapid reduction in Mcl-1 protein levels and the onset of apoptosis in both primary myeloma cells and cell lines. Further, treatment of NSG mice bearing MM1.S or RPMI-8226 tumors with VM030 resulted in Usp9x and Usp24 inhibition, reduction in Mcl-1 protein levels and suppression of myeloma tumor growth with limited toxicity. Together, these results suggest that Usp9x and Usp24 are highly expressed and activated in myeloma cells and both DUBs contribute to cell survival, but through different mechanisms. These results support the use of DUB inhibitors with specificity for Usp9x and Usp24 in the treatment of myeloma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of syndecan regulates human myeloma plasma cell adhesion to type I collagen

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 767-774 ◽  
Author(s):  
RC Ridley ◽  
H Xiao ◽  
H Hata ◽  
J Woodliff ◽  
J Epstein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
B Cells ◽  
Heparan Sulfate ◽  
Cell Lines ◽  
Type I Collagen ◽  
Myeloma Cell ◽  
Type I ◽  
Myeloma Cells ◽  
Human Myeloma Cell

The syndecans comprise a family of integral membrane proteoglycans that regulate cell behaviors by binding to extracellular matrix and binding growth factors. In mouse blood cells, syndecan expression is restricted to cells of the B-cell lineage where it is expressed by pre-B cells and plasma cells, but is absent from circulating B cells. In the present study, we examined the expression, structure, and function of syndecan on human myeloma cell lines and myeloma patient bone marrow cells. On myeloma cells, syndecan is a small (modal relative molecular mass [M(r)] = 120 Kd) heparan sulfate proteoglycan localized at the cell surface. Syndecan was detected by immunodot blotting on 7 of 10 human myeloma cell lines and by reverse transcriptase polymerase chain reaction on 10 of 14 patient samples. Cell binding assays show that myeloma cells expressing syndecan bind to type I collagen via heparan sulfate chains, while those cell lines not expressing syndecan do not bind to collagen. Furthermore, the cell lines expressing syndecan were negative for CD19 and CD45 staining, indicating that syndecan expression is restricted to tumors having a well-differentiated phenotype. We conclude that syndecan acts as a matrix receptor on human myeloma cells but is not expressed by all tumors, suggesting that syndecan may participate in regulating myeloma cell adhesion to the bone marrow stromal matrix.

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