Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19+ clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19+ B cells. We found that in vitro MM colony-forming cells were enriched in CD138−CD19−CD38++ plasma cells, while CD19+ B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138−CD19−CD38++ plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19+ B cells was detected. In 4 out of 9 cases, CD138+ plasma cells propagated MM, although more slowly than CD138− cells. Finally, we transplanted CD19+ B cells from 13 MM patients into NOD/SCID IL2Rγc−/− mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138− and CD138+ cells have the potential to propagate MM clones in vivo in the absence of CD19+ B cells.
Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells.1, 2 The immunoglobulin gene sequences in MM plasma cells are somatically hyper-mutated and remain constant throughout the clinical course, suggesting that the disease arises from a post-germinal center B cell or a more differentiated cell.3, 4, 5 Previous studies have found that MM patients harbor phenotypic B cells expressing the immunoglobulin gene sequence and the idiotype unique to the individual myeloma clone.6, 7, 8, 9 These findings imply that clonotypic B cells may be involved in the disease process but offer no definitive proof that B cells in fact correspond to the proliferating tumor compartment.
Clonogenic MM cells are thought to be responsible for disease progression10, 11 so that it is important to identify and target them. The first successful in vitro system capable of growing human MM colonies was described by Hamburger and Salmon.10 They showed that the clonogenic frequency of clinical myeloma specimens ranged from 0.001 to 0.1% of BM cells from MM patients. In a later study utilizing methylcellulose media supplemented with lymphocyte conditioned media as growth factors, clonogenic MM progenitor cells were found in BM cells lacking expression of the plasma cell marker CD138.11, 12, 13, 14, 15 It was further reported that rituximab inhibited MM colony formation11 and that CD20+ B cells from some MM patients could produce MM plasma cells in a 3-D culture in vitro,16 which suggests that CD138− clonogenic MM cells might be B cells. However, it is still unclear whether clonogeic MM cells are B cells or plasma cells, because some CD38++ MM plasma cells lack CD138 expression.17
CD19+ B cells isolated from MM patients could reportedly generate MM disease upon transplantation into NOD/SCID mice,11, 12, 18, 19 indicating that clonotypic CD19+ B cells served as MM progenitor cells in these MM patients. However, B cell depletion by means of rituximab in MM patients was not clinically effective in most cases, at least for short periods, in which plasma cells did not express CD20.20 It is therefore still unclear whether CD19+ or CD20+ clonogenic MM progenitor cells are responsible for disease progression and maintenance. Studies using the SCID-hu or rab models, which are SCID mice implanted with human fetal or rabbit bone fragments, respectively, to create a humanized or human-like microenvironment, suggested that malignant plasma cells have tumorigenic properties.21, 22, 23
For the development of effective treatment, it is important to know whether clonogenic cells in MM are B cells or plasma cells. In the study presented here, we aimed to clarify whether CD138− clonogenic MM cells are B cells or plasma cells by using an in vitro colony assay and two types of xeno-transplant assays.
Materials and methods
BM cells from MM patients were collected from iliac bone after the patients’ informed consent had been obtained. Mononuclear cells were purified using Ficoll Paque (GE Healthcare, Piscataway, NJ, USA) and analyzed. Cord blood cells were obtained from the Keihan Cord Blood Bank (Osaka, Japan). The research was approved by the institutional review boards of Osaka University School of Medicine and the Keihan Cord Blood Bank.
A total of 50 patients diagnosed with multiple myeloma were used in this study, 16 of whom were subjected to in vitro clonogenic assay. Twelve patients were used for the SCID-rab experiments with un-fractionated BM cells, and the samples of nine patients were subjected to SCID-rab experiments with fractionated BM cells. Finally, Samples from 13 patients were used for transplants into NOG mice.
Flow cytometry and cell sorting
Single cell suspensions from BM were treated with ACK solution (150 mM NH4Cl, 10 mM KHCO3) for 3 min on ice to lyse erythrocytes, then washed with PBS containing 2% FCS, incubated with 10% human AB serum for 20 min to prevent nonspecific antibody binding, and finally stained with fluorochrome-conjugated CD138 (MI15), CD38 (HB7), CD34 (8G12) and CD19 (H1B19) mAbs (BD Pharmingen, San Jose, CA, USA) on ice for 30 min. After washing, the cells were re-suspended in 1 μg/ml propidium iodide. Analysis and cell sorting were performed on FACS Aria (BD Biosciences, San Jose, CA, USA). The BD Cytofix/Cytoperm kit (BD Pharmingen) and phycoerythrin-conjugated anti-IgLκ(G20-193) or IgLλ(JDC-12) (BD Pharmingen) for staining cytoplasmic immunoglobulin were used according to the manufacturer’s instructions.
Colony forming assay
Methylcellulose culture assays were performed in Methocult M3223 (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 10% lymphocyte conditioned media, which was generated by culturing human peripheral blood mononuclear cells in RPMI1640 supplemented with 10%fetal bovine serum and 2.5 μg/ml PHA. Colonies were counted and scored on culture days 14–21.
SCID-rab mice were constructed as previously described.23 The Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine approved the experimental procedures and protocols. Six- to eight-week-old CB.17/Icr-SCID mice were obtained from Crea Japan (Kanagawa, Japan) and 4-week-old New Zealand rabbits from Kitayama Rabesu (Nagano, Japan). The femurs and tibias from the rabbits were cut into two pieces with the proximal and distal ends kept closed. One piece of bone was inserted subcutaneously through a small (5–mm) incision, which was then closed with sterile surgical staples. Four to eight weeks after, BM cells from MM patients were injected directly into the implanted rabbit bone. The mice were periodically bled from the tail vein and changes in levels of circulating human immunoglobulin light chain (hIgL) of the M-protein isotype were used as an indicator of MM growth. Serum human immunoglobulin was measured by means of ELISA using the human kappa/lambda ELISA kit (Bethyl Laboratories, Montgomery, TX, USA). To enrich CD138−CD34− or CD138+ cells, CD138-microbeads and CD34-microbeads kits (Miltenyi Biotech, Auburn, CA, USA) were used according to the manufacture’s instruction.
Transplantation into NOG mice
Intra-BM transplantation was performed as previously reported.24 Seven-week-old female NOD/Shi-scid, IL-2 Rγnull (NOG) mice (Central Institute for Experimental Animals, Kawasaki, Japan) irradiated with 200 cGy 4-24 h before transplantation were injected into the tibia with FACS-sorted PB or BM cells from the MM patients. Transplantation into newborn NOG mice was performed within 72 h after birth. The mice were irradiated with 100 cGy 4-24 h before transplantation and injected with sorted cells via the anterior facial vein.25 Development of MM was monitored by measuring human immunoglobulin light chain (IgL)κ or λ by means of ELISA. Twelve or more weeks after transplantation, the mice were sacrificed and BM cells were collected from the tibias and femurs and analyzed by means of FACS. Cells were stained with human CD45 (HI30)-APC, CD138-PE, CD38-FITC, CD19-Cy7APC (BD Pharmingen) and mouse CD45 (30-F11)-Cy7PE (eBioscience, San Diego, CA, USA), followed by analysis using FACS.
Clonogenic cells are enriched in CD19−CD38++CD138− plasma cells of some MM patients
MM plasma cells expressing a monotypic immunoglobulin light chain, could be identified as CD34−CD19−CD38++ cells by FACS analysis in the BM samples of most MM patients (Figure 1a). CD34−CD19−CD38++ MM plasma cells were separated into CD138− and CD138+ cells (Figure 1a). CD34−CD19+ B cells, CD34−CD19−CD38++CD138− plasma cells, or CD34−CD19−CD38++CD138+ plasma cells were FACS-sorted and subjected to colony assay. An in vitro colony assay was performed in methylcellulose medium supplemented with lymphocyte conditioned media, as previously reported.11 Formation of MM colonies consisting of plasma cells (Figure 1b) was detected in 3 out of 13 MM samples. In those 3 MM samples, the frequencies of colony formation in the CD34−CD19−CD38++CD138− plasma cells were 700, 35, and 9053 colonies per 105 cells, while those in CD34−CD19−CD38++CD138+ plasma cells were much lower (Figure 1c) and CD34−CD19+ B cells did not form any MM colonies. These results indicate that clonogenic cells were found in BM cells lacking the expression of a plasma cell marker CD138, but only in CD138−CD19−CD38++ plasma cells, not in CD19+ B cells.
We further investigated whether colony-forming cells could be detected in CD19+CD27+ B cells, which are reportedly enriched with clonogenic cells.12 Samples from 3 patients were examined, and MM colony-forming cells were detected in one. In that sample, MM colonies were formed not from CD19+CD27+ B cells, but from CD19−CD138− cells (Figure 1d).
In the SCID-rab model, only CD38++ MM plasma cells engrafted and expanded in vivo without engraftment of CD19+ B cells
SCID-rab mice were constructed as previously reported.23 A rabbit bone fragment was inserted under the skin of a SCID mouse more than 4 weeks before transplantation of MM cells. First, whole BM cells from MM patients were transplanted and engraftment of MM cells was monitored by measuring human IgLκ and λ in serum of the recipient mice. Engraftment and expansion of MM cells were observed in 5 out of 12 cases (Table 1). Rabbit BM was analyzed 12 weeks or more after transplant to determine whether engraftment of not only MM plasma cells but also CD19+ B cells had taken place. Robust engraftment of human CD38++ MM plasma cells expressing the monotypic immunoglobulin light chain and containing both CD138− and CD138+ cells was detected in the rabbit BM, but no human CD19+ B cells were detected (Figure 2a). These results indicate that CD19−CD38++ plasma cells could engraft and expand at least for several months without engraftment of CD19+ B cells.
Both CD138− and CD138+ plasma cells could engraft and propagate MM clones in SCID-rab model
Next, we transplanted purified CD138− or CD138+ BM cells into SCID-rab mice to test whether proliferating cell compartments were present in the CD138− population. Transplants with 9 MM samples were performed and in 3 of the samples, rapid increase of either human IgLκ or λ was observed in serum of the mice transplanted with CD138− cells (Table 2). In the rabbit BM, CD38++ MM plasma cells including CD138− and CD138+ cells, but not CD19+ cells, were detected (Figure 2b). In 4 of 8 cases CD138+ plasma cells also engrafted and expanded, although more slowly than CD138− BM cells (Table 2, Figure 2b). In addition, CD138− cells from the SCID-rab mice that had initially been engrafted with CD138− BM cells could be secondarily transplanted to another SCID-rab recipient and propagate MM disease more rapidly than CD138+ cells (Table 2, exp. 2-2). These results indicate that CD138− plasma cells of some MM patients have the potential to propagate MM disease rapidly in the SCID-rab model, while CD138+ plasma cells can also engraft and propagate MM slowly.
CD19+ B cells from MM patients did not engraft to NOD/Shi-scid,IL-2Rγnull (NOG) mice
To examine whether CD19+ B cell fractions contain MM-initiating cells upon transplantation to immune-deficient mice, CD19+ B cells were FACS-sorted from PB of 5 MM patients and BM cells from 4 MM patients including one patient whose CD19+ B cells exclusively expressed IgL λ (Figure 3a), and transplanted directly into BM of NOD/Shi-scid,IL-2Rγnull (NOG) mice or intravenously to new born pups of NOG mice (Table 3, exp. 1-9). Engraftment of human MM cells was monitored by measuring human immunoglobulin light chain (IgL) κ and λ in serum of the recipient mice, but no human IgL was detected at any time. We also analyzed BM of the recipient mice 12–20 weeks after transplant, but no human CD19+ or CD38++ cells were detected (Table 3, Figure 3a). CD19−CD38++ plasma cells from the MM BM samples were also transplanted into NOG mice, but did not engraft (Table 3, exp. 6–9). In contrast, robust engraftment of human cells was observed upon transplantation of cord blood-derived CD34+ cells (Table 3, exp. CB, Figure 3a). In three experiments (Table 3, exp. 10–12), CD3−CD34−CD138− cells were transplanted into BM of NOG mice, but did not engraft. Finally, CD3-depleted BM cells from an MM patient whose CD19+ B cells exclusively expressed IgLκ (Figure 3b) were transplanted intravenously into a newborn NOG mouse (Table 3, exp. 13). When the BM cells were analyzed 12 weeks after transplant, significant engraftment of human CD45+ cells was observed because CD3− BM cells contained many CD34+ hematopoietic stem and progenitor cells. Small numbers of human CD38++CD138+ plasma cells were also detected, but analysis of their IgL κ and λ expression showed that they were not clonal MM plasma cells (Figure 3b). This result suggests that normal human hematopoietic cells, but not MM cells, engrafted in the recipient mice.
In this study, we showed that in vitro clonogenic cells that were detected in some MM patients lacked expression of the plasma cell marker CD138,11, 13, 14 and that they were CD19−CD38++CD138− plasma cells, not CD19+ B cells. Consistent with our results, it has been recently reported that CD138−CD38++plasma cells contain more cycling cells compared to CD138+ plasma cells.17 An in vitro colony assay of CD138−CD19−CD34− cells showed colony formation only in 4/16 (25%) patients which is a lower ratio than the one previously reported (88%) by Matsui et al.11 even though our method for in vitro colony assay was the same. Only cells that proliferate extensively in response to stimulation by lymphocyte-conditioned medium (LCM) can be detected with the clonogenic assay used in our study. When the survival of clonogenic MM cells depends on factors other than the soluble factors contained in LCM, for example attachment to stromal cells, clonogenic MM cells cannot be detected with the assay used in our study. On the other hand, cells from aggressive or advanced MM cases may be more independent of several cell extrinsic factors and efficiently produce colonies. Heterogeneity of MM patients should thus be the reason for the differences in the frequencies of MM colony formation between our study and the one by Matsui et al.11
The SCID-rab experiments showed that highly proliferative myeloma cells were present in the CD138-negative fraction in some patients, but those cells were MM plasma cells, not B cells. Consistent with the findings of previous studies,22, 23 we also found that MM developed in the SCID-hu/rab mice transplanted with CD138+ plasma cells. MM plasma cells thus have the potential to propagate and maintain MM clones, at least for several months, in the absence of clonotypic B cells. This may explain why B cell depletion by rituximab was not clinically effective, at least in the short run, for most MM patients.
Interestingly, CD138−CD38++ plasma cells were detected in the SCID-rab mice transplanted with CD138+ plasma cells. This suggests that CD138 expression on MM plasma cells may be reversible, although we cannot exclude the possibility of minor contamination of CD138− cells in the purified CD138+ cells. The significance of CD138 expression on clonogenic MM cells thus needs to be carefully interpreted. It was reported that interaction with bone marrow stromal cells induced expression of CD138 in MM plasma cells,26 suggesting that changes in CD138 expression depend on the microenvironment. In addition, Jakubikova et al. recently reported that clonogenic side populations in MM cell lines were not enriched in the CD138low/+ but not in the CD138− population, although it is not clear whether clonogeic side populations in primary MM cells are also enriched in CD138low/+ cells.27
CD19+ B cells in some MM patients generated MM disease upon transplantation into NOD/SCID mice,11, 12, 18, 19 indicating that CD19+ B cells of some MM patients definitely contain MM progenitor cells. In our experiments, however, we could not find any MM patients whose CD19+ B cells induced MM disease upon transplantation into NOG mice. However, this does not necessarily mean that CD19+ clonotypic B cells cannot be MM progenitor cells in MM patients. It should be noted that there are many difficulties involved in the engraftment of human cells in xeno-graft models. For example, mouse IL6, which is one of the major growth factors for plasma cells, does not transduce its signals through human IL6 receptors, and probably other factors lack inter-species cross reactivity between human and mice. This means that MM progenitor cells can be detected in xeno-graft assays only when they can survive independently of human IL6 or other human factors. Thus, CD19+ B cells from advanced MM patients may be independent of several survival factors and effectively engraft and propagate MM disease upon transplant into immuno-deficient mice. In addition, signals from B cell receptors (BCRs) on clonotypic CD19+ B cells need to be taken into consideration. When BCRs of clonotypic CD19+ B cells show very high affinity to xeno-antigen in mice, they may be depleted and cannot survive in xeno-graft models. Thus, assuming that CD19+ MM progenitor cells are present, they can be detected in xeno-graft assays only when their BCRs are suitable for survival in mice. We can therefore not deny the possibility that CD19+ B cells have potential as MM progenitor cells, even if CD19+ B cells do not engraft in immunodeficient mice.
Taken together, our findings show that CD138− clonogeic cells are plasma cells and not B cells in some MM patients. Furthermore, we suggest that MM plasma cells, which include CD138− and CD138+ cells, have potential to propagate and maintain MM clones for at least several months without the need for CD19+ clonotypic B cells. Our findings also indicate that clonogenic MM plasma cells should be considered important therapeutic targets. The Hedgehog signaling pathway was reported to be a promising candidate as a therapeutic target against clonogenic MM cells.15 In addition, since clonogenic MM plasma cells mainly reside in the BM microenvironment, it is also important to understand the mechanisms involved in how the BM microenvironment supports clonogenic MM plasma cells and targets them. The Notch signaling pathway may be a good candidate for such a target.28
We wish to thank Manabu Kawakami, Masashi Nakagawa (Nissei Hospital, Osaka, Japan), Tamotsu Yamagami, Masaki Murakami, Shigeo Fuji, Eui Ho Kim (NTT West Hospital, Osaka, Japan), Shinichiro Kawamoto, Noboru Yonetani, Takayuki Takubo (Osaka Medical University), Hiroya Tamaki, Hiroyasu Ogawa (Hyogo Medical College) for collecting patient samples, and the Keihan Cord Blood Bank (Osaka, Japan) for supplying cord blood samples. This work was supported by the Knowledge Cluster Initiative (Stage II) established by the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the Senri Life Science Foundation, by the Astellas Foundation for Research on Metabolic Disorders and by the Uehara Memorial Foundation (to N.H.).
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European Journal of Cancer Care (2017)