Background
The supposition of a multiple myeloma stem cell (MMSC) has been made for a few decades but identification of the exact cell or population has been difficult to accomplish. Biologically, B cells are derived from the common lymphoid progenitor cell and driven through pro-B to pre-B cell subsets by activation of transcription factors and subsequent expression of the μ chain immunoglobulin and rearrangement of the heavy chain. Development then moves to secondary lymphoid organs (i.e. spleen, lymph nodes) where exposure to antigens induces generation of germinal centers, somatic hypermutation at the Ig locus and proliferation to create clonal-specific memory and short-term and long-term antibody-secreting plasma cells (PCs) that can respond to subsequent antigen exposures. Memory and long-term PCs reside in the BM where they receive support from the BM stroma for survival and activation. The specific cell population, within the B cell/PC lineage, that contains the supposed MM CSC, however, is still unknown.
Cellular identification
The key cellular component of MM, the monoclonal (M) protein-secreting plasma cell, is a terminally differentiated cell type that arises from the B cell lineage. The pathological highlights of MM suggest, however, that malignancy is incurred in B cells and not in the plasma cell population. Early studies demonstrated that in MGUS and MM patients, a fraction of B cells, branded as clonotypic B cells, were present at differentiated states; though this population exhibited heterogeneity [
10,
11]. Additionally, Bergsagel
et al. identified these clonotypic cells in assorted numbers among patients, with steady levels observed during treatment but significantly higher levels during relapse states [
12]. These circulating B cells also had chromosomal aberrations and Ig rearrangements particular to a certain idiotype seen in the malignant PC [
13‐
15]. These cells also could differentiate into antibody secreting plasma cells suggesting that the progenitor population of myeloma is contained in the B cell fraction and has progenitor-like characteristics [
16]. Further studies of these B cells identified somatic hypermutation in the VDJ region of the genome with deficiency of intraclonal variation suggesting a post-germinal center B cell [
10,
17]. Phenotypic studies of these circulating clonotypic B cells demonstrated that they resembled memory B cells, a post-germinal center, pre-plasma cell generated to establish long-term immunity [
18]. This property along with the ability to self-renew suggested that this phenotype may be the population containing the malignant myeloma stem cell but the research by Rasmussen
et al. [
18] has been the only reported suggestion of memory B cells as the myeloma CSC population.
Biological characteristics
Biological activity of the proposed MMSCs has been variable due to the plasticity of the surface markers and the assay used to determine clonogenic activity. This has led to some confusion regarding the surface marker phenotype of the MMSC population (summarized in Table
1). The first model to understand the biology of myeloma stem cells was done by directly injecting myeloma cells from the BM of patients into a subcutaneously implanted human fetal bone chip in SCID mice (named as SCID-hu) [
19]. These mice developed clinical characteristics of MM, such as hypercalcemia and circulating M protein. In a later study, it was reported that cells from reconstituted SCID-hu mice were able to engraft secondary SCID recipients, which validates the transferable phenotype of a stem cell population [
20]. However, the engrafted population was primarily a CD38++CD45- surface phenotype and no CD19+ B cell was capable of growth [
20]. A secondary study has also identified the CD19-CD45-CD38 + CD138+ population as being the tumorigenic stem-like population for MM in another mouse model [
21].
Table 1
List of cell surface markers utilized to identify proposed multiple myeloma stem cell
CD19 + CD38-CD27+ | Rasmussen, T et al., Leukemia and Lymphoma 2004 [ 18] |
CD19 + CD138- | Pilarski, LM et al., Blood 2000 [ 22] |
Pilarski, LM et al., Exp Hematology 2002 [ 23] |
Matsui, W et al., Blood 2004 [ 24] |
CD19 + CD138-CD27+ | Matsui, W et al., Cancer Research 2008 [ 25] |
CD138-ALDH+ | Reghunathan, R et al., Oncotarget 2013 [ 26] |
Matsui, W et al., Cancer Research 2008 [ 25] |
CD19 + CD34-Lchain(λ) + ALDH+ | Boucher, K et al., Clinical Cancer Research 2012 [ 55] |
CD38 + CD45- | Yaccoby, S et al., Blood 1999 [ 20] |
CD19-CD45-CD38 + CD138+ | Kim, D et al., Leukemia 2012 [ 21] |
In stark contrast though, Pilarski
et al. isolated clonotypic circulating B cells from a progressed MM patient, with the ability to engraft immunodeficient mice and demonstrating clinical phenotypes of lytic bone disease and the presence of circulating M-protein [
22]. Furthermore, this leukemic B cell had Ig rearrangements identical to the CD138+ plasma cell, suggesting the involvement of the CD19+ B cell as the progenitor population [
14]. The ability to transfer into secondary recipients was not performed, however. Nonetheless, this study was subsequently followed to better ascertain the surface marker phenotype of the myeloma CSC. CD19+ cells lacking the plasma cell marker, CD138/syndecan1, were able to give rise to the tumor population in NOD/SCID mice generating both CD19+ and CD138+ myeloma cells [
23]. This further suggested that the myelomagenic population was contained in the B cell lineage but not in the plasma cell pool.
Another study confirmed the lack of CD138 expression in the MMSC phenotype, as primary myeloma BM samples expressing CD138+ were unable to engraft NOD/SCID mice, with the engraftment potential contained solely in the CD138- cell population, incurring plasma cell proliferation and inducing production of M protein
in vivo[
24].
In vitro colony forming assays further demonstrated the clonogenic capacity of CD138- and not CD138+ cells validating the
in vivo transplantation studies. Additional studies to identify the cell surface subset of myeloma CSCs found that the cells resembled a memory B subset in that the clonogenic population expressed CD19 + CD27 + CD138 [
25]. This population, obtained from peripheral blood of myeloma patients, engrafted NOD/SCID animals and was transferrable to secondary recipients as the CD19+ cells from the BM of the primary mice engrafted. Additionally, the potential validity of CD138- MMSC and “stemness” has been described by Reghunathan
et al. using human MM cell lines to demonstrate the CD138- MMSC neoplasticity [
26].
Though research suggests conflicting subsets in identification of the MMSC, the issue may be simply due to the source of the cells, the isolation procedure of these cells or the in vivo or in vitro assay used to determine potential clonogenicity and progenitor status. Collectively, however, the MMSC population appears to reside in the B cell lineage but not in the plasma cell pool.
Signaling pathways
CSCs utilize many of the pathways that regulate and maintain normal stem cells, adapting the ability to self-renew to maintain the malignancy. A feature identified from embryonic stem cells (ESC) to HSCs is the use of pathways established in many developmental mechanisms, including Hedgehog (Hh), Wnt and Notch pathways. Early reports demonstrated the role of these pathways in a number of cancers establishing the manipulation of self-renewal mechanisms by malignant cells to continue disease progression [
27‐
30].
Hedgehog signaling was the first to be implicated in the maintenance of MM CSCs demonstrating overexpression in the pathway both from human myeloma cell lines and primary human myeloma samples [
31]. Biologically, Hedgehog is involved in stem cell maintenance of ESCs and utilizes a ligand-receptor mechanism of Hh to Patched 1 (Ptch1) to the receptor Smoothened to induce activation of the pathway [
32]. Cyclopamine, which targets and inhibits Smoothened, was found to induce apoptosis in MM cells [
31]. The use of cyclopamine in specifically treating cancer stem cells was demonstrated in lung and prostate cancer studies, inducing apoptosis and inhibiting growth of the malignant cells [
33,
34]. One clinical trial utilizing a small molecule inhibitor of the Hedgehog pathway demonstrated significant responses in over 50% of advanced metastatic basal cell carcinoma patients [
35]. Another clinical use of this molecule was performed in a medulloblastoma patient with some success [
36]. However, the clinical use of Hedgehog inhibitors in treating MM has not been reported.
The Wnt pathway utilizes 19 conserved glycoproteins that bind to the transmembrane receptor Frizzled, activating canonical β-catenin signaling and noncanonical pathways to induce proliferation and activation [
37]. In fact, genetic manipulation of normal Wnt signaling affects the development and function of multiple organs [
38]. Aberrant activation of the Wnt pathway promotes proliferation of both MM cell lines and primary patient samples [
39]. This activation is induced by intracellular mechanisms and through crosstalk with the BM microenvironment [
40‐
43]. Small molecule inhibitors of the Wnt pathway have disrupted the maintenance of MM cells both
in vitro and
in vivo providing the possibility of developing Wnt-targeted inhibitors for clinical treatment of MM [
44,
45] gp96 is a molecular chaperone in the endoplasmic reticulum and it is regulated by the unfolded protein response pathway [
46]. It was shown recently that gp96 is a critical chaperone for Wnt co-receptor LRP6 [
47]. Targeted inhibition of gp96 genetically and pharmacologically has shown to be an effective strategy against multiple myeloma through inhibition of Wnt-LRP6-surivivin pathway [
48].
The Notch signaling pathway is involved in various cellular events from proliferation, differentiation and apoptosis to cell maintenance and survival [
49]. The expression of Notch in stem-like populations in various cancers promotes survival of the CSC and progression of disease [
50‐
54]. Activation of Notch in MM promotes proliferation and induces enhanced development of the disease [
55‐
57]. One study investigated the expression of Notch on BM clonotypic B cells from MM patients and found high expression of Notch on these cells indicating an involvement of Notch signaling in maintaining the MMSC [
58]. Inhibitors of Notch signaling have successfully prevented localization and migration of MM cells to the BM and induced apoptosis of these cells but this has not been attempted in clinical settings [
59‐
61].
These studies underlie the fact that CSCs, and more specifically, MMSCs, utilize mechanisms similar to normal stem cells to survive, maintain disease and increase progression of disease. Identifying the specific MMSC population that maintains MM would be advantageous in developing a therapy unique to targeting these cells.