Mature Myeloma Cells Research Articles

PF573 SINGLE CELL RNA-SEQ REVEALED A RARE ITGA2 EXPRESSING CELL SUBPOPULATION WITH DISTINCT CANCER STEM CELL MARKER SIGNATURE IN MULTIPLE MYELOMA

Background: Multiple myeloma (MM) stem cells (MMSC) are a rare cell population capable of self-renewal and differentiation into the predominating mature myeloma cells. Specific targeting this cell population could effectively eradicate the tumors in the patients. Previous studies reported CD138 non-expressing myeloma cells as putative MMSC. However, conflicting results by subsequent studies demonstrated the tumor-initiating and clonogenic cells expressed CD138 and apoptotic features in CD138 non-expressing cells. Aims: In this study, we aimed at identification of MMSC by examination of the transcriptome of myeloma cells at single cell level. Methods: Single cell RNA-Seq with flow-based SMART-Seq2 protocol was employed to determine the transcriptome of 768 myeloma cells from MM1 s and NCIH929 cell lines (384 cells each line) and 68 cells from a clinical MM bone marrow sample. The percentages of myeloma cells expressing the candidate biomarker(s) protein were determined by flow cytometry. Results: Results of quality control analyses indicated ∼400K read counts per cell with 15–20% of multiple mapping representing ribosomal RNAs and 12–15% mitochondrial RNAs and with 6700–8600 variable genes for transcriptome clustering analyses. By unsupervised analysis using t-distributed stochastic neighbor embedding (t-SNE), myeloma cells were clustered according to their cell cycle statuses. After controlling the cell cycle effects by regression, two clusters with similar cell numbers were observed in each cell line. Interestingly, the results of supervised analysis with t-SNE on 24 cancer stem cell markers expression revealed a rare subpopulation of myeloma cells (MM1 s: 2.6%; NCIH929: 3.6%) that were signified by Integrin alpha 2 (ITGA2) expression but no expression of OCT4 and NANOG stem cell markers. Flow cytometry using anti-ITGA2 antibody confirmed the presence of this rare cell population (∼0.3%) in MM cells. For the cells from the clinical sample, 3 clusters were observed by unsupervised analysis after controlling cell cycle effects. Studies of the signature genes for each cluster indicated two clusters were derived from erythroid cells and lymphocytes; while the 3rd cluster (n = 49) from myeloma cells, within which one ITGA2-expressing cell was idenitifed. ITGA2 encodes a cell surface protein CD49b integrin receptor as a marker for primitive human hematopoietic stem cells. In leiomyoma, CD49b marked the side population cells and the stemness of CD49b-expressing cells was also demonstrated. Recently, increased NOTCH1 and its downstream targets expression implying high activity of NOTCH signaling, which are involved in stem cell self-renewal and differentiation, were reported in CD49b-expressing trophoblast progenitor cells in human placenta. All these findings suggested the link of ITGA2 and stem cell properties. Functional studies of the ITGA2 positive myeloma cells are currently in progress regarding their clonogenic potentials and self-renewal capacity. Summary/Conclusion: Our study revealed a rare subpopulation of ITGA2-expressing cells with distinct cancer stem cell markers signature in MM cell lines and a clinical sample, potentially representing MMSC.

Modeling Cell–Cell Interactions in Regulating Multiple Myeloma Initiating Cell Fate

Cancer initiating cells have been documented in multiple myeloma and believed to be a key factor that initiates and drives tumor growth, differentiation,metastasis, and recurrence of the diseases. Although myeloma initiating cells (MICs) are likely to share many properties of normal stem cells, the underlying mechanisms regulating the fate of MICs are largely unknown. Studies designed to explore such communication are urgently needed to enhance our ability to predict the fate decisions of ICs (self-renewal, differentiation, and proliferation). In this study, we developed a novel system to understand the intercellular communication between MICs and their niche by seamlessly integrating experimental data and mathematical model. We first designed dynamic cell culture experiments and collected three types of cells (side population cells, progenitor cells, and mature myeloma cells) under various cultural conditions with flow cytometry. Then we developed a lineage model with ordinary differential equations by considering secreted factors, self-renewal, differentiation, and other biological functions of those cells, to model the cell–cell interactions among the three cell types. Particle swarm optimization was employed to estimate the model parameters by fitting the experimental data to the lineage model. The theoretical results show that the correlation coefficient analysis can reflect the feedback loops among the three cell types, the intercellular feedback signaling can regulate cell population dynamics, and the culture strategies can decide cell growth. This study provides a basic framework of studying cell–cell interactions in regulating MICs fate.

Bone Marrow Stromal Cells From Myeloma Patients Support the Growth of Myeloma Stem Cells

It has been well documented that bone marrow stromal cells (BMSCs) of multiple myeloma patients play a pivotal role in supporting the growth of mature myeloma cells. With evolving concepts concerning the presence of myeloma stem (initiating) cells, we aimed this investigation to specifically address the supportive role of BMSCs for myeloma stem cell growth in vitro and in vivo. BMSC lines were derived from myeloma or control patients (myeloma or control BMSCs). Myeloma stem cells of the RPMI 8226 myeloma cell line were recognized through the identification of "side populations" (SP) with Hoechst dye staining. SP cells formed more colonies when grown on myeloma BMSC than on control BMSC. Additionally, higher percentages of SP cells were observed when grown on myeloma BMSCs than on control BMSCs. In the mouse model, SP cells inoculated with myeloma BMSCs grew faster than those inoculated with control BMSCs. Of note, SP cells demonstrated an increased expression of CD184 (CXCR4) compared with non-SP cells. The expression of CD184 in SP cells was further increased when they were cultured with myeloma BMSCs. CD184(+) SP cells formed more colonies than CD184(-) SP cells. Treatment with AMD 3100, an inhibitor of CD184, reduced colony formation by CD184(+) SP cells when co-cultured with myeloma BMSCs. This was associated with the decreased activation of ERK, a downstream target of activated CD184, in myeloma cells. These findings indicate that the myeloma BMSCs create a microenvironment supportive of myeloma stem cells via, at least partially, the CXCR4 signaling pathway.

SDF-1 Is Responsible for the Constitutively High NF-kB Activity in Human Myeloma Cells.

The chemokine, stromal cell-derived factor 1 (SDF-1; CXCL12) and its receptor, CXCR4 are considered to be essentially required for plasma cell homing to the bone marrow (BM). It is well known that plasma cells in the BM (long-lived plasma cells) survive for a long time and have the constitutively high NF-kB activity. Since human myeloma cells are considered to be derived from these committed long-lived plasma cells, we investigated the role of SDF-1 on the survival of primary myeloma cells from myeloma patients and the possible relationship with NF-kB activity. First, we confirmed that all primary myeloma cells expressed CXCR4 but not CCR9 or CCR10 receptors on their surface and the levels of CXCR4 expression apparently correlated with maturity of BM plasma cells; mature myeloma cells (MPC-1+) as well as polyclonal plasma cells expressed higher levels of CXCR4 than those on immature myeloma cells (MPC-1-). The production of SDF-1 was found strongly in BM stromal cells but not in primary myeloma cells as well as myeloma cell lines. On the other hand, high DNA binding activity of NF-kB was constitutively detected in primary myeloma cells as well as myeloma cell lines, and these NF-kB activities significantly correlated with the expression levels of CD54 on their surface, for CD54 gene is one of the strict NF-kB target genes. Based on the expression levels of CD54 protein, interestingly, primary myeloma cells showed weaker NF-kB activities than those in monoclonal plasma cells from MGUS and polyclonal plasma cells from polyclonal gammopathy. Plasma concentrations of SDF-1 were also significantly correlated to the expression levels of CD54 on primary myeloma cells significantly (P<0.01). Furthermore, it was confirmed that addition of SDF-1 significantly increased the expression levels of CD54 in the in vitro culture of primary myeloma cells. Therefore, these results indicate that SDF-1 is responsible for high expression levels of CD54 and possibly the constitutively high NF-kB activity in primary myeloma cells.

Gelatin-Coated Insert-Well Culture Markedly Improved In Vitro Culture of Primary Myeloma Cells.

In order to clarify the pathogenesis of multiple myeloma (MM) and identify the molecular target for MM treatment, it is important to understand the biology and molecular mechanism of survival and proliferation of myeloma cells in vitro and in vivo. Still, it is hard to keep primary myeloma cells viable at least for 2 weeks in vitro, and these cells rapidly enter apoptosis even in the presence of IL-6. Co-culture with BM stromal cells is considered to be one of the most important factors as well as addition of cytokines for improvement of in vitro culture of primary myeloma cells. Based on our previous data, we devised a new method where bone marrow mononuclear cells (BMMNC) from BM aspirates were put inside insert-wells (8.0 um pore size, #3182, BD) coated with gelatin in the presence of the mixture of cytokines (galectin-1, SDF-1, IL-6 and IGF-1) in the serum-free synthetic medium; addition of galectin-1 and SDF-1 was essential especially at the beginning of this culture. By this method, we observed that BM stromal cells attached to gelatin and survived well with rather low proliferation; myeloma cells interacted well with these stromal cells. Thus, we could maintain viability of primary myeloma cells at least for 4 weeks and the recovery of viable myeloma cells (CD38++ cell) appeared to be about 80% in 30 cases of MM we examined. Phenotypic data also showed that ratio of immature (MPC-1−) and mature (MPC-1+) myeloma cells in the BMMNC before culture was approximately maintained after 2 or 4 weeks in this method. Therefore, these results suggest that gelatin-coated insert-well culture can control the viability of stromal cells and thus maintain the culture of primary myeloma cells in vitro. This new method would contribute to the further understanding of biology and drug sensitivity of primary myeloma cells.

An Increase in MPC-1 - and MPC-1 - CD45 + Immature Myeloma Cells in the Progressive States of Bone Marrow Plasmacytosis: The Revised Phenotypic Classification of Monoclonal Marrow Plasmacytosis (MOMP-2005)

The heterogeneity of bone marrow plasmacytosis is clearly analyzed by multicolor staining with anti-CD38 antibody. To date, at least 5 subpopulations of plasma cells have been identified in the bone marrow of multiple myeloma (MM) patients with regard to the expression of MPC-1, CD49e (VLA-5), and CD45: MPC-1(-)CD49e(-)CD45(+) proliferative immature cells, MPC-1(-)CD49e(-)CD45(-) immature myeloma cells, MPC-1(+)CD49e(-)CD45(-) and MPC-1(+)CD49e(-)CD45(+) intermediate myeloma cells, and MPC-1(+)CD49e(+)CD45(+) mature myeloma cells. We performed phenotypic analyses in 75 cases of monoclonal bone marrow plasmacytosis, including 46 cases of MM and 29 cases of monoclonal gammopathy of undetermined significance (MGUS). In 31 cases of progressive MM disease, MPC-1(-) immature and MPC-1(-)CD45(+) proliferative immature myeloma cells were significantly increased up to >25% and >10%, respectively, of the plasma cell fractions (CD38(++) cells), whereas there were no increases in MPC-1(-) or MPC-1(-)CD45(+) proliferative immature myeloma cells in 15 cases of stable disease. Interestingly, the proportions of MPC-1(-) and MPC-1(-)CD45(+) immature monoclonal plasma cells also increased in the 7 progressive cases of MGUS. Finally, we present the revised (2005) phenotypic classification of monoclonal marrow plasmacytosis (MOMP-2005).

The Plasticity of Myeloma Plasma Cells as Expressed by Dedifferentiation into an Immature, Resilient, Apoptosis-Resistant Phenotype.

Progression of myeloma (MM) is considered to be a multistage and dynamic process of cell differentiation, survival, proliferation and dissemination. We have previously demonstrated the proliferative potential of purified CD45lowCD38high mature MM cells in SCID-hu mice (Yaccoby & Epstein, Blood, 1999), the ability of CD138-selected MM cells to produce myeloma in our novel SCID-rabbit model (Yata et al., ASH, 2003), and the interdependence of MM bone disease and tumor growth whereby MM cells induce osteoclast activity and are dependent on osteoclasts in vivo (Yaccoby et al., BJH, 2002) and ex vivo (Yaccoby et al., Cancer Res., 2004). The aim of this study was to determine the osteoclast-induced phenotypic changes associated with survival of MM cells in long term co-culture. CD138-selected (>95% purity) MM cells from 16 patients were co-cultured with human osteoclasts for up to 20 weeks. The pre-cultured baseline cells were typically CD45low/inermediateCD38high, CD19−CD34−. At the end of long term co-culture (>6 weeks) MM cells had BrdU labeling index (LI) of 2.5±2.0 and their viability was 97%±1%. The phenotype of co-cultured MM cells consistently shifted to a less mature phenotype, with CD45 expression increasing from CD45low to CD45intermmediata/high and reduced expression of CD38 from CD38high to subpopulations with CD38intermediate levels, as determined by flow cytometry and confirmed by qRT-PCR. Further flow analysis revealed that co cultured MM cells also expressed low levels of CD19 and CD34, and identified a small subpopulation of CD138lowCD45high MM cells. Morphologically, the co-cultured MM cells uniformly gained plasmablastic characteristics when compared to pre-cultured cells. Previous reports suggested that IL-6 was important for maintaining subpopulation of CD45-expressing MM cells. However, blocking IL-6 activity in co-cultures with anti-IL6 and anti-IL6R neutralizing antibodies (5 μg/ml, each) did not affect the immature phenotype of MM cells. Intriguingly, long term co-culture of normal CD34+ hematopoietic stem cells (HSCs) with osteoclasts results in loss of CD34 expression, suggesting a common mechanism for osteoclast-induced MM PC and HSC plasticity. To investigate if the observed phonotypic changes are associated with apoptosis resistance, we determined the effects of 3 days exposure to the pro-apoptotic agent dexamethasone (DEX, 10−7 M) on MM cells cultured alone or in co-cultures (n=5), at initiation (baseline) and after 6 weeks of co-cultures. The percent apoptotic cells was determined by trypan blue exclusion and annexin V flow cytometry. When baseline MM cells were cultured alone, DEX significantly increased the percent of apoptotic cells over that spontaneous rate (p<0.01). In contrast, when MM cells recovered from co-cultures after 6 weeks they survived and were resistant to DEX-induced apoptosis (16%±11% and 24%±21% apoptotic cells in the absence and presence of DEX, respectively). As reported, osteoclasts supported survival of MM cells at baseline and after 6 weeks of co-culture (p<0.01), and protected MM PCs from DEX-induced apoptosis. Our data demonstrate the phenotypic plasticity of primary myeloma cells, whereby mature MM cells are reprogrammed and acquire autonomous survival properties after co-culture with osteoclasts. We hypothesize that in vivo these cells are dormant, resistant to spontaneous and drug-induced apoptosis, and could be responsible for relapse.

Growth mechanism of human myeloma cells by interleukin-6.

Human myeloma cells are heterogenous morphologically and phenotypically. Myeloma cells can be classified into at least 5 subpopulations; MPC-1-CD45+CD49e-, MPC-1-CD45-CD49e- immature myeloma cells, MPC-1+CD45-CD49e-, MPC-1+CD45+CD49e- intermediate myeloma cells and MPC-1+CD45+CD49e+ mature myeloma cells. Interleukin-6(IL-6) is a major growth factor for human myeloma cells, but only MPC-1-CD45+CD49e- immature myeloma cells can response directly to IL-6 to proliferate. In the U-266 cell lines, IL-6 can lead to the induction of CD45 expression and CD45+ U-266 cells can proliferate in response to IL-6. In primary myeloma cells, MPC-1-CD45-CD49e- immature myeloma cells sorted from bone marrow samples can be changed to CD45+ cells by addition of IL-6 in vitro. In both CD45- and CD45+ U-266 cells, STAT3 and MAPK(ERK1/2) can be activated in response to IL-6 equally between them, but src family kinases such as Lyn, Fyn can be activated only in CD45+ U-266 cells. Thus, the activation of the src family kinases associated with CD45 expression is a prerequisite for the proliferation of myeloma cells. In the bone marrow of myeloma patients, most myeloma cells do not express CD45, and CD45+ immature myeloma cells are only 1 approximately 2%. In order to clarify the difference of cellular context between CD45- and CD45+ myeloma cells, PCR-based cDNA subtraction was performed from CD45+ U-266 cells to CD45-U-266 cells. The series of this subtraction selected several genes. Furthermore, sensitivity to stress stimuli between CD45+ and CD45- U-266 cells was also compared. CD45-U-266 cells were markedly more resistant to stress conditions such as serum-free condition. Therefore, we can speculate that in the bone marrow of human myelomas IL-6 can induce proliferation of CD45+ immature cells, but the amount of IL-6 is too low to support CD45+ myeloma cells and loss of CD45 results in no direct response to IL-6 to proliferate but confers resistance to stress condition leading to the longer survival at the limited amount of IL-6.

Proliferation of Immature Myeloma Cells by Interleukin-6 Is Associated with CD45 Expression in Human Multiple Myeloma

Multiple myeloma (MM) is a hematologic malignancy of human plasma cells, and myeloma cells can be classified into several subpopulations according to phenotypic differences, such as CD38+++MPC-1+CD49e- immature, CD38+++MPC-1+CD49e- intermediate and CD38+++MPC-1+CD49e+ mature myeloma cells. The expression of the CD45 molecule on myeloma cells is quite variable, and the physiological consequence of CD45 on myeloma cells is still unknown. Recently, we have found that a few MPC-1- immature myeloma cells express CD45 antigens while most myeloma cells do not express the CD45. MPC-1-CD45+CD49e- but not MPC-1-CD45-CD49e-immature cells contain proliferating cells in response to interleukin-6 (IL-6). IL-6 can also induce expression of CD45 on the MPC-1-CD45- subpopulation of immature myeloma cells. In addition, myeloma cell lines responding to IL-6 express CD45, whereas cell lines proliferating independent of IL-6 do not express CD45. In the U266 cell line, IL-6 leads to the induction of CD45 expression and cell proliferation, indicating that IL-6-induced effects are closely linked to CD45 expression. Thus, there is a heterogeneity in human myeloma cells, and among these subpopulations immature myeloma cells expressing the CD45 molecules appear to proliferate in response to IL-6. In this review we propose the involvement of CD45 in MM pathogenesis, and the possible implications of CD45 as both a phenotypic marker and a functional molecule is discussed.

Biological significance of heterogeneity in human myeloma cells.

B cells differentiate into plasma cells which produce antibodies in the bone marrow (BM). Multiple myeloma (MM) is a hematologic malignancy in human plasma cells, and myeloma cells grow mainly in BM. According to phenotypic differences, such as expression of adhesion molecules, human myeloma cells as well as normal plasma cells can be classified into several differentiation stages. We have found that cells strongly expressing CD38 antigens (CD38++(+)) in BM are all plasma cells, and that there also are no plasma cells in either CD38- cell fraction or fraction of cells weakly expressing CD38 antigens (CD38+). Myeloma cells in BM consist of CD38++(+)MPC-1-CD49e (VLA-5)-immature and CD38++(+)MPC-1+CD49e+ mature myeloma cells. Immature myeloma cells proliferate markedly in vitro and respond to interleukin-6 (IL-6), a growth factor for myeloma cells, whereas mature myeloma cells show very low proliferative activities and show no response to IL-6. Immature myeloma cells expressing CD21 molecules on their surface seem to attach to stromal cells in BM through binding to CD23 molecules. Thus, there is a heterogeneity in human myeloma cells, and immature myeloma cells appear to proliferate in response to IL-6.

Cyclin D1 and p16INK4A are preferentially expressed in immature and mature myeloma cells, respectively.

Myeloma cells consist of immature, intermediate and mature cells with respect to expression of VLA-5 (CD49e) and MPC-1 adhesion molecules. VLA-5(-)MPC-1(-) immature myeloma cells respond to interleukin 6 (IL-6) to proliferate in vitro. but VLA-5+MPC-1+ mature myeloma cells have almost no proliferative activity with higher secretory activity of M-protein in vitro. In order to further clarify the biological differences between these immature and mature myeloma cells, we examined survival of these cells with or without IL-6 in vitro, and investigated the underlying mechanism of the proliferative or non-proliferative character of these cells by examining expression of cell cycle regulators such as cyclin D1 and inhibitors for cyclin-dependent kinase (Cdk), p16INK4A, p21CIP1 and p27KIP1 by RT-PCR and immunohistochemistry. In vitro survival of these myeloma cells was examined by flow cytometric quantification of fluorescein diacetate (FDA) and propidium iodide (PI) staining. Immature myeloma cells rapidly entered apoptosis without IL-6, but mature myeloma cells could survive without IL-6 as well as normal mature plasma cells. Immature myeloma cells as well as myeloma cell lines expressed cyclin D1 mRNA and protein, but not any Cdk inhibitors. On the other hand, mature myeloma cells did not express cyclin D1 but expressed p16, not p21 or p27, as well as normal mature plasma cells. Therefore these results show that immature myeloma cells constitutively express cyclin D1 and can proliferate, and mature myeloma cells as well as normal mature plasma cells preferentially express p16 and can survive for a long time without proliferation.

MYELOMA STEM CELL PHENOTYPE: Implications For Treatment

The phenotypic heterogeneity of myeloma cells in fact delineates a differentiation process that appears to be an integral part of the disease process. Immature myeloma cells interact with their microenvironment differently than do the more mature cells. As a result of this interaction, the immature cells display different responses to chemotherapy than do the mature cells. Addressing this issue by tailoring treatment to target immature as well as mature myeloma cells may change dramatically the outcome of treatment. The ability to define the myeloma clone by molecular genetic techniques has markedly increased the ability to detect clonal cells. This technique provides a most sensitive tool for monitoring elimination of tumor cells; however, the role of the early clonal B cells identified through the use of ASO-PCR in the disease process needs to be clarified. Currently, a great deal of effort is directed towards development of treatment protocol that will eliminate all clonal cells, and a method of purging clonal cells from harvested mobilized peripheral stem cells. Understanding the biologic significance of early clonal B cells in myeloma will allow for a more rational approach to curative treatment.

Interleukin-6 gene expression is preferentially restricted in VLA-5-MPC-1- immature but not in VLA-5+MPC-1+ mature myeloma cells.

Interleukin-6 (IL-6) is essential for the proliferation of myeloma cells, and IL-6 is considered to be produced from not only myeloma cells themselves but also microenvironments including stromal cells. To clarify which subpopulation of myeloma cells can produce IL-6, we examined IL-6 mRNA expression in immature and mature myeloma cells and normal plasma cells by RT-PCR. IL-6 mRNA expression was found in all (10/10) specimens of sorted VLA-5-MPC-1- immature myeloma cells and 27% (3/11) of VLA-5-MPC-1+ myeloma cells. On the contrary, no IL-6 mRNA was expressed in VLA-5+MPC-1+ mature myeloma cells (0/4) and CD19+CD56- normal plasma cells (0/5). IL-6R gene expression was detected in all normal and malignant plasma cells without exception. Therefore, these findings suggest that IL-6 production is preferentially restricted in immature not mature myeloma cells, and this may explain why immature myeloma cells show greater proliferative activity.

2Biological aspects of multiple myeloma

Although considerable progress has been made towards understanding many aspects of myeloma, the myeloma stem cell and the factors that drive the disease remain elusive. Recent developments in the molecular analysis of clonality have helped to confirm the presence of pre-switch B cells that are of the same clone as the myeloma plasma cells. The role of these cells in myelomagenesis has not been demonstrated, and the isotypic heterogeneity of the clonally-relevant cells suggests that the pre-switch B cells are pre-malignant progenitors of the tumour cells. Thus, the circulating clonal B cells appear to be the earliest progenitors of the mature, monoclonal plasma cells. IL-6, and possibly other cytokines, are involved in driving this process. The role of IL-6 in myeloma is complex and more involved than its proposed growth factor function. In the absence of IL-6, dependent cells become apoptotic. Increased IL-6 signalling also leads to apoptosis of myeloma cells, possibly as a result of terminal differentiation. In the presence of exogenous IL-6, the IL-6 receptor appears to be the rate-limiting factor in the pathway's activity. IL-6 may regulate the survival of myeloma cells by stimulating c-myc transcription, possibly from the P0 promoter. The high levels of c-myc transcripts and protein could regulate myeloma cell proliferation and apoptotic death by controlling p53 expression and, through it, the expression of the Rb and BAX genes. Proliferative signalling in myeloma cells is likely to be intrinsic, within the tumour cell compartment. Molecules such as CD28 and B7, both expressed by less mature myeloma cells, could represent one such self-self stimulatory mechanism, with IL-6, possibly through stimulation of c-myc expression, providing the signals for survival and differentiation.

Expression of CD21 Antigen on Myeloma Cells and its Involvement in Their Adhesion to Bone Marrow Stromal Cells

The mature myeloma cells express very late antigen 5 (VLA-5) and MPC-1 antigens on their surface and adhere to bone marrow (BM) stromal cells more tightly than the VLA-5-MPC-1- immature myeloma cells in vitro. The VLA-5 and MPC-1 antigens possibly function as two of the molecules responsible for interaction of mature myeloma cells with BM stromal cells. However, the immature myeloma cells do interact with BM stromal cells, and it is unclear which adhesion molecules mediate their interaction. In this study, we found that both immature and mature myeloma cells expressed CD21, an adhesion molecule known to bind to CD23. CD21 was also detected on normal plasma cells. To evaluate the role of CD21 expression on myeloma cells, two myeloma cell lines, NOP-2 (VLA-5-MPC-1-) and KMS-5 (VLA-5+MPC-1+), were used as representatives of immature and mature myeloma cell types, respectively, and an adhesion assay was performed between the myeloma cell lines and BM stromal cells. Antibody-blocking results showed that adhesion of the mature type KMS-5 to KM102, a human BM-derived stromal cell line, or to short-term cultured BM primary stromal cells was inhibited by monoclonal antibodies (MoAbs) against CD21, VLA-5, and MPC-1, and inhibition of adhesion of the immature type NOP-2 to KM102 by the anti-CD21 MoAb was observed as well. Furthermore, CD23 was detected on KM102. Treatment of KM102 with an anti-CD23 MoAb also inhibited adhesion of either KMS-5 or NOP-2 to KM102. Therefore, we propose that CD21 expressed on myeloma cells likely functions as a molecule responsible for the interaction of immature myeloma cells as well as mature myeloma cells with BM stromal cells, and CD23 may be the ligand on the stromal cells for the CD21-mediated adhesion.

Heterogeneous Expression of a Novel MPC-1 Antigen on Myeloma Cells: Possible Involvement of MPC-1 Antigen in the Adhesion of Mature Myeloma Cells to Bone Marrow Stromal Cells

AbstractRecent immunophenotypic analysis has shown that the heterogeneous expression of the adhesion molecule VLA-5 classifies myeloma cells into VLA-5+ mature and VLA-5− immature subpopulations. To further clarify the two myeloma subpopulations, we generated a monoclonal antibody, MPC-1, by immunizing mice with an adherent human myeloma cell line, KMS-5. The MPC-1 antibody recognized a 48-Kd surface antigen on KMS-5 but not on U-266, a nonadherent human myeloma cell line. Specificity characterization showed that MPC-1 antigen was expressed on mature myeloma cells, normal plasma cells, and mature B cells, whereas pre-B cells and germinal center B cells lacked its expression. Monocytes and a human bone marrow stromal cell line, KM102, also expressed this antigen. Two subclones of MPC-1 + VLA-5 + (KMS-5Ad) and MPC-1 −VLA-5 + (KMS-5NAd) were separated from the KMS-5 cell line. The KMS-5Ad adhered to KM 102 more tightly than did the KMS-5NAd, and the U-266 (MPC-1 −VLA-5−) displayed almost no adherence to the KM102. The adhesion of the KMS-5Ad was partially inhibited by the MPC-1 antibody. These results, taken together, suggest that the MPC-1 antigen serves as a differentiation marker for B-lineage cells, including plasma cells, and may function as an adhesion molecule involved in the interaction of mature myeloma cells with bone marrow stromal cells.

Heterogeneous expression of a novel MPC-1 antigen on myeloma cells: possible involvement of MPC-1 antigen in the adhesion of mature myeloma cells to bone marrow stromal cells

Recent immunophenotypic analysis has shown that the heterogeneous expression of the adhesion molecule VLA-5 classifies myeloma cells into VLA-5+ mature and VLA-5- immature subpopulations. To further clarify the two myeloma subpopulations, we generated a monoclonal antibody, MPC-1, by immunizing mice with an adherent human myeloma cell line, KMS-5. The MPC-1 antibody recognized a 48-Kd surface antigen on KMS-5 but not on U-266, a nonadherent human myeloma cell line. Specificity characterization showed that MPC-1 antigen was expressed on mature myeloma cells, normal plasma cells, and mature B cells, whereas pre-B cells and germinal center B cells lacked its expression. Monocytes and a human bone marrow stromal cell line, KM102, also expressed this antigen. Two subclones of MPC-1+ VLA-5+ (KMS-5Ad) and MPC-1-VLA-5+ (KMS-5NAd) were separated from the KMS-5 cell line. The KMS-5NAd adhered to KM102 more tightly than did the KMS-5NAd, and the U-266 (MPC-1-VLA-5-) displayed almost no adherence to the KM102. The adhesion of the KMS-5Ad was partially inhibited by the MPC-1 antibody. These results, taken together, suggest that the MPC-1 antigen serves as a differentiation marker for B-lineage cells, including plasma cells, and may function as an adhesion molecule involved in the interaction of mature myeloma cells with bone marrow stromal cells.

Identification of immature and mature myeloma cells in the bone marrow of human myelomas

With regard to the expression of adhesion molecules, human myeloma cells freshly isolated from bone marrow were heterogeneous. By two- color analysis with anti-VLA-5 antibody (PE staining) and FITC-labeled anti-CD38 antibody, we found all myeloma cells located at CD38-strong positive (CD38++) fraction and identified two subpopulations among these myeloma cells: CD38++ VLA-5-(VLA-5-) myeloma cells and CD38++ VLA- 5+ (VLA-5+) myeloma cells. To clarify the biologic character of these two subpopulations, the morphology, in vitro proliferative activity and in vitro M-protein secretion were examined in each fraction isolated by the purification procedure or a cell sorter. Morphologic examination showed that VLA-5- myeloma cells were mostly immature or plasmablastic and VLA-5+ cells were mature myeloma cells. Furthermore, VLA-5- myeloma cells proliferated markedly in vitro and responded to interleukin 6 (IL- 6), a growth factor for myeloma cells, while VLA-5+ myeloma cells showed very low uptakes of 3H-thymidine and no responses to IL-6 but secreted higher amounts of M-protein (immunoglobulin) in vitro significantly. Therefore, we could clarify here heterogeneity of human myeloma cells in the bone marrow with regard to the expression of VLA- 5, one of integrin adhesion molecules; VLA-5- myeloma cells were proliferative immature cells and VLA-5+ cells were mature myeloma cells.

Identification of immature and mature myeloma cells in the bone marrow of human myelomas

AbstractWith regard to the expression of adhesion molecules, human myeloma cells freshly isolated from bone marrow were heterogeneous. By two-color analysis with anti-VLA-5 antibody (PE staining) and FITC-labeled anti-CD38 antibody, we found all myeloma cells located at CD38-strong positive (CD38++) fraction and identified two subpopulations among these myeloma cells: CD38 ++ VLA-5– (VLA-5–) myeloma cells and CD38++VLA-5 + (VLA-5 +) myeloma cells. To clarify the biologic character of these two subpopulations, the morphology, in vitro proliferative activity and in vitro M-protein secretion were examined in each fraction isolated by the purification procedure or a cell sorter. Morphologic examination showed that VLA-5– myeloma cells were mostly immature or plasmablastic and VLA-5+ cells were mature myeloma cells. Furthermore, VLA-5– myeloma cells proliferated markedly in vitro and responded to interleukin 6 (IL-6), a growth factor for myeloma cells, while VLA-5 + myeloma cells showed very low uptakes of 3H-thymidine and no responses to IL-6 but secreted higher amounts of M-protein (immunoglobulin) in vitro significantly. Therefore, we could clarify here heterogeneity of human myeloma cells in the bone marrow with regard to the expression of VLA-5, one of integrin adhesion molecules; VLA-5– myeloma cells were proliferative immature cells and VLA-5+ cells were mature myeloma cells.

Phenotypic difference of normal plasma cells from mature myeloma cells

We have recently shown that two-color analysis with fluorescein isothiocyanate (FITC)-anti-CD38 antibody could clearly distinguish myeloma cells (plasma cells) from other hematopoietic cells in the bone marrow. Myeloma cells (plasma cells) alone were located at CD38strong positive (++) fractions. To further distinguish normal plasma cells from mature myeloma cells phenotypically, we examined immunophenotypes of normal plasma cells and myeloma cells by two-color flow cytometry with FITC-anti-CD38 antibody and phycoerythrin staining with antibody to VLA-4, MPC-1, CD44, CD56, CD19, CD20, CD24, or CD10. Normal plasma cells were all VLA-4+VLA-5+MPC-1+CD44+ CD19+CD56- in the bone marrows from seven healthy donors, tonsils from four patients with chronic tonsillitis, a spleen from one patient with idiopathic thrombocytopenic purpura, and lymph nodes from two patients with chronic lymphadenitis, respectively. On the other hand, mature myeloma cells (12 of 20 cases), VLA-4+VLA-5+MPC-1+, were all CD19- and most of them CD56+, and there were no myeloma cells with the CD19+CD56- phenotype in the 20 cases of myelomas we tested. Thus, as for the expression of CD19 and CD56, normal plasma cells from various tissues are all CD19+CD56-, whereas no myeloma cells have the CD19+CD56- phenotype. According to this finding, we investigated the expression of CD19 and CD56 on plasma cells (CD38++ fractions) in monoclonal gammopathy of undetermined significance (MGUS). Both CD19+CD56- and CD19-DC56+ plasma cells were found in all five cases of MGUS we tested, suggesting that MGUS consists of phenotypically normal plasma cells and myeloma cells. Therefore, it is reasoned that phenotypic analysis of plasma cells with anti-CD19 and anti-CD56 antibodies can distinguish normal plasma cells from malignant plasma cells (myeloma cells), and can detect malignant plasma cells even in MGUS or premyeloma states.