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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3576-3582
The Proliferative Potential of Myeloma Plasma Cells Manifest in the
SCID-hu Host
By
Shmuel Yaccoby and
Joshua Epstein
From the Myeloma and Transplantation Research Center, Arkansas Cancer
Research Center, University of Arkansas for Medical Sciences, Little
Rock, AR.
 |
ABSTRACT |
The low proliferative activity of myeloma plasma cells prompted the
notion that the clonotypic B cells that exist in the blood and bone
marrow of all myeloma patients contain the proliferative myeloma cells
(stem cell). We have exploited our severe combined immunodeficiency
(SCID)-hu host system for primary myeloma to investigate whether
myeloma plasma cells are capable of sustained proliferation. Purified
CD38++CD45 plasma cells consistently
grew and produced myeloma and its manifestations in SCID-hu hosts (8 of
9 experiments). In contrast, the plasma cell-depleted bone marrow cells
from 6 patients did not grow or produce myeloma in SCID-hu hosts.
Similarly, whereas plasma-cell containing blood cells from 4 patients
grew and produced myeloma in hosts, neither the
PC-depleted blood cells from 3 of the patients nor a blood
specimen that did not contain plasma cells grew in SCID-hu hosts,
regardless of their CD19-expressing cell contents. Also, in hosts
injected with blood cells, although the myeloma cells were able to
disseminate through the murine host system, they were only able to grow
in the human bones within a human microenvironment and were not
detectable in the murine blood or other organs. Interestingly, the
circulating plasma cells appear to grow more avidly in the SCID-hu
hosts than their bone marrow counterparts, suggesting that they
represent a subpopulation of the plasma cells in the bone marrow.
Although our studies clearly demonstrate the proliferative potential of
myeloma plasma cells, they are suggestive, not conclusive, as to the
existence of a preplasmacytic myeloma progenitor cell.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
MULTIPLE MYELOMA is a B-cell neoplasia,
characteristically associated with monoclonal tumor cells of plasma
cells morphology and function. Disease manifestations include high
levels of monoclonal Igs, severe skeletal anomalies resulting from
activation of osteoclasts and bone resorption, and immune suppression.
Myeloma tumor cells display low proliferative activity expressed in
labeling indices of less than 0.5% in all but the most advanced stages
of the disease. This low proliferative potential, expressed also in
lack of in vitro growth and the inability to establish cell lines but
from the most advanced cases of extramedullar myeloma, has prompted the
notion that a cell earlier along the B-cell differentiation pathway is
the proliferative progenitor of the myeloma plasma cell. Indeed, B
lymphocytes and pre-B cells that carry the signature of the myeloma
clone have been identified. Initially these cells were recognized by
their idiotype,1,2 by their Ig isotype (light chain
excess), and by their abnormal phenotype.3-7 More recently,
the specific sequence of the third complementarity determining region
of the rearranged myeloma Ig gene, CDR3, was used to identify clonal B
lymphocytes and pre-B cells.8-13 Preplasmacytic cells in
the blood and bone marrow of patients with myeloma could be differentiated into monotypic plasma cells.14-16 It was
suggested by some that the circulating clonal B cells contain the
myeloma stem cells, that they are resistant to therapy, and that they are responsible for disease dissemination14 and
relapse.17,18 However, the debate on the role
of clonal B and pre-B cells in the disease process could not be tested
for lack of an adequate assay.
The SCID-hu host19 provides a hospitable host for primary
human acute leukemia cells.20 Recently, we reported that
the SCID-hu host reproducibly supports the growth of myeloma cells and
development of typical myeloma manifestations when inoculated with
freshly obtained bone marrow cells from patients with
myeloma.21 We have used the SCID-hu host system to
determine if purified myeloma plasma cells alone can grow and produce
myeloma in SCID-hu hosts or if production of myeloma in the hosts
requires preplasmacytic and accessory cells present in the bone marrow
and blood.
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MATERIALS AND METHODS |
Myeloma cells.
Heparinized bone marrow aspirates and peripheral blood samples were
obtained from patients with active myeloma during scheduled clinic
visits. Signed institutional research board-approved consent forms were
signed by all patients and are kept on record. Relevant patient
information is provided in Table 1. The
samples were separated using ficoll hypaque centrifugation (Histopaque;
Pharmacia, Uppsala, Sweden). The proportion of myeloma
cells in the light density cell preparations (specific
gravity,  1.077 g/mL) was determined using CD38/CD45 flow
cytometry.13,22 Myeloma plasma cells were identified by
their CD38++CD45negative-positive fluorescence
profile and their light scatter properties.13,22 CD38++CD45 Plasma cells were purified to
a high degree of purity using fluorescence-activated cell sorter. When
indicated, a second set of sort windows was set, excluding all plasma
cells (both CD38++CD45 and
CD38++CD45+), and the nonplasmacytic cells were
sorted and collected simultaneously; these constituted the plasma
cell-depleted cells. Aliquots of the sorted cells were used to prepare
slides for morphological and cIg analysis. When cell quantity allowed,
purity was determined by repeat flow cytometry of  20,000 cells.
Purity of the plasma cells was 99%, with an occasional
neutrophil or eosinophil visible on morphological
examination; the plasma cell-depleted specimens contained 1% cells
that fell within the plasma cell window
(Fig 1). The myeloma clone in each sample
was characterized by CDRIII- and allele-specific oligomer
(ASO)-polymerase chain reaction (PCR).13,23 BudR labeling index of light chain restricted plasma cells was determined as described by Lokhorst et al.24
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| Fig 1.
Sorting parameters and purity analysis of sorted plasma
cells and PC-depleted cells from patient no. 8. Sort windows were set
to include CD38++CD45 plasma cells (PC
window) and to exclude CD38++CD45 and
CD45+ plasma cells (PC-depleted window) (A). Sorted cells
were reanalyzed by flow cytometry for purity. The PC fraction contained
99.4% CD38++CD45 plasma cells (B) and
the PC-depleted fraction contained 98.5% cells that fell within the
parameters of the PC-depleted sort window and 1.2% cells that fell
within the PC window.
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SCID-hu hosts.
CB.17/ICr-SCID mice were obtained from Harlan Sprague
Dawley (Indianapolis, IN) and were housed and monitored in
our animal facility. The Institutional Animal Care and Use Committee
had approved all experimental procedures and protocols. SCID-hu mice were generated as reported.20 A total of 25 to 50 µL of
phosphate-buffered saline (PBS) containing 0.7 to 3.2 × 106 purified myeloma plasma cells was injected directly
into the human bone in the SCID-hu hosts. Other hosts were injected
with unsorted bone marrow cells as controls or with plasma
cell-depleted bone marrow cells. In some experiments, blood mononuclear
cells (MNC) or plasma cell-depleted blood cells were
used. In 2 experiments (patients no. 6 and 9, Table 1), cells recovered
from hosts injected with purified myeloma plasma cells were injected
into secondary hosts, and in 1 case (patient no. 9), cells recovered
from the secondary host were injected into 4 tertiary hosts. In all
experiments, increase in the levels of circulating monotypic human Ig
(hIg) of the M protein isotype was used as an indicator of myeloma cell growth.
Determination of human Ig levels.
The levels of human IgG, IgA, and  and  light chains were
determined by enzyme-linked immunosorbent assay (ELISA) as
described.21 Briefly, plates were coated with 50 µL/well
of primary antihuman and (5 µg/mL) and antihuman IgA and IgG
(10 µg/mL) and incubated overnight at 4°C. Antibodies were
purchased from The Binding Site (San Diego, CA). The
plates were washed 3 times in PBS containing 0.5% (vol/vol) of Tween
20, then once with blocking buffer (4% bovine serum albumin [BSA] in
PBS). A 50-µL sample diluted in PBS containing 1% BSA was added, and
the plates were left at room temperature for 2 hours. After washing 3 times with PBS/Tween, 50 µL biotinylated antibody (affinity-purified
antihuman and light chains at 0.5 µg/mL, antihuman IgA, and
IgG at 0.2 µg/mL) were added to each well for 1 hour. The plates were
then washed and 50 µL streptavidin-horseradish peroxidase was added
to each well and allowed to bind for 1 hour. After a final wash,
OPD solution (Dako, Carpineteria, CA) containing 3%
H2O2 was added. Absorbance at 450 nm was
determined on an Auto-Reader II ELISA reader (Ortho Diagnostic Systems,
Raritan, NJ).
Methods of analysis.
Tissues and organs recovered from SCID-hu mice were processed as
reported.21 Myeloma cells were identified morphologically, by immunohistochemical staining for cytoplasmic Ig (cIg; Dako immunoperoxidase kit), and their clonality was determined by in situ
hybridization with patient-specific probes (ASO-ISH).
Changes in bone remodeling were identified by x-radiography and
increased osteoclast activity was demonstrated by histochemical
staining for tartarate-resistant acid phosphatase (TRAP; Sigma, St
Louis, MO). Immunohistochemical staining with a
monoclonal antibody to CD34 (Cell Marque, Austin, TX) was
used to demonstrate neovascularization by human vascular endothelial
cells. Monoclonal antibody to human vitronectin receptors (Biosource
International, Camarillo, CA) was used to demonstrate the
human origin of osteoclasts in the myeloma-bearing human bones.
In situ hybridization.
Clonality of the tumor cells grown in SCID mice was demonstrated by in
situ hybridization using an adaptation of published methods,25,26 as reported.21,26 Briefly,
antisense oligonucleotide sequences (24 to 32 bp) complementary to CDR
III regions of the myeloma clone of each patient were biotinylated
during synthesis (Life Technologies, Rockville, MD).
Tissue sections were dewaxed in xylene, defatted in chloroform, and
then rehydrated. The section were treated with proteinase K (10 µg/mL
in Tris-HCl buffer at 37°C for 1 hour). Hybridization mixture
containing 50% formamide, 10% dextran, 4× SSC (SSC is 150 mmol/L NaCl, 15 mmol/L trisodium citrate, pH 7), 25 mg/mL Herring sperm
DNA, and biotinylated probe (1 to 2 µg/mL) were applied to each
section. The sections were covered with Parafilm and incubated
overnight in a humidified chamber at 37°C. After hybridization, the
slides were washed twice with 1× SSC at room temperature and
twice with 1× SSC at 37°C to 42°C (depending on the size
of the probe). Signals were visualized using an in situ hybridization
kit (Dako). Specificity was determined by using irrelevant patient
probes and sections from different patients.
Screening for Epstein-Barr virus (EBV).
Myeloma cells recovered from SCID-hu mice were analyzed for the
presence of EBV sequences by PCR.27 All samples were negative.
| |
RESULTS |
Purified myeloma plasma cells from 9 patients with myeloma were
injected directly into the human bones of SCID-hu hosts. Engraftment and growth of the myeloma cells was followed by the appearance of and
increase in the concentrations of the monotypic human Igs in the murine
sera. Purified plasma cells from 8 of the patients grew in the hosts.
Myeloma growth was detected as early as 3 weeks after the injection of
cells and was associated with the typical myeloma manifestations such
as severe bone resorption (Table 1). As we have reported previously for
SCID-hu hosts injected with myeloma bone marrow cells, myeloma growth
was restricted to the human bone, and no other human cells were
detected in the blood or other tissues of the hosts.21
Myeloma cells recovered from the SCID-hu hosts and in histological
sections of decalcified bones were of the same clone as the patients'
myeloma cells as shown by ASO-PCR or ASO-in situ hybridization
analyses. All myeloma cells in the SCID-hu hosts expressed cytoplasmic
Ig of the M protein isotype; no evidence of isotypic variance
was seen. In all cases, growth of myeloma was associated with the
expected manifestations: severe bone resorption
(Fig 2) resulting from increased activity of osteoclasts and active neoangiogenesis resulting from stimulation of
vascular endothelial cells, as we have reported for SCID-hu hosts
inoculated with unseparated myeloma bone marrow.21 The osteoclasts and vascular endothelial cells in the myelomatous human
bones were human, as attested to by their reactivity with murine
monoclonal antibodies to human Vitronectin receptor and to human CD34,
respectively (data not shown). Both antibodies reacted only with human
cells in the human bones and did not react with murine cells.
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| Fig 2.
Growth of purified myeloma plasma cells (PC) from patient
no. 1 in a SCID-hu host. SCID-hu host was inoculated with sorted PC.
(A) Human Ig concentrations. (B) Dot plot of CD38/CD45 flow cytometry
profile of the cells. The sort window used to purify plasma cells is
shown. (C and D) X-radiograms showing severe resorption of the
myelomatous implanted bone (C) compared with the nonmyelomatous bone of
the control host (D), implanted at the same time as (C).
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In the experiments in which cells recovered from the hosts were
transferred to secondary (2 experiments) and tertiary hosts (1 experiment), all hosts developed myeloma with the typical
manifestations. Within the experimental conditions and with the
limitation of the small sample size, development of myeloma and the
time to initial detection of hIg did not appear to correlate with the number of plasma cells inoculated. In contrast to hosts injected with
purified myeloma plasma cells, hosts injected with plasma cell-depleted
bone marrow cells from 6 of these patients showed no evidence of
myeloma cell growth up to 60 weeks after inoculation (Fig 3 and Table 1).
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| Fig 3.
Human Ig levels (A through C) and histology of
decalcified human bone sections (D through F) from SCID-hu
hosts inoculated with bone marrow cells (A and D), purified
plasma cells (B and E), and PC-depleted bone marrow cells
(C and F) from patient no. 2. Inserts demonstrate CD38/CD45
fluorescent profiles and sort windows of cells used for each host.
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These results indicate that CD38++CD45
myeloma plasma cells are capable of sustained proliferation and that no
other cells in the bone marrow aspirates from patients with myeloma are
required to produce myeloma in the SCID-hu hosts. To investigate
further whether other cells, specifically circulating B lymphocytes,
are able to produce myeloma in the SCID-hu hosts, we injected
light-density blood cells from 5 patients (patients no. 9 through 13, Table 1) into SCID-hu hosts. Bone marrow cells from all 4 patients produced myeloma in SCID-hu hosts. Three blood specimens contained myeloma plasma cells. These specimens produced myeloma in the hosts.
The blood from 1 patient (patient no. 11, Table 1) did not contain
plasma cells and did not produce myeloma in the SCID-hu host. In 2 experiments, blood cells depleted of plasma cells were also inoculated
into SCID-hu hosts. In both cases, the hosts did not develop myeloma
(Fig 4). Interestingly, although the blood samples contained few myeloma cells compared with the bone marrow samples from the same patients, initial detection of myeloma growth occurred earlier in hosts inoculated with blood cells than in those
inoculated with bone marrow cells. This comparison was based on the
time required for hosts inoculated with blood and bone marrow cells to
reach a landmark hIg level. The hIg levels used for this comparison
were the first reached by both hosts in each experiment (except for the
blood of patient no. 11, which did not grow at all). Hosts that
developed myeloma from blood cells showed the same manifestations as
those that were inoculated with bone marrow cells (eg, Fig 4).
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| Fig 4.
Growth of myeloma blood cells from patient no. 10 in
SCID-hu host. SCID-hu host was inoculated with blood cells (containing
0.1% plasma cells) and with PC-depleted blood cells. (A) Human Ig
levels. ( ) Blood cells; ( ) PC-depleted blood cells; ( ) host
inoculated with bone marrow cells containing 11% myeloma plasma cells.
(B) Flow cytometry dot plot of CD38CD45 profile of the blood cells. The
sort window excluding CD45 and CD45+
plasma cells is shown. (C and D) X-radiograms of host injected with
blood cells (C) and with PC-depleted blood cells (D). Note severe
decalcification of myelomatous human bone in (C).
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In some experiments, mice were implanted contralaterally with 2 human
bones each. Only 1 bone was inoculated with myeloma blood or bone
marrow cells. In all hosts, although no human hematopoietic cells were
detectable in any of the murine organs by flow cytometry, the
contralateral second bone showed radiological evidence of resorption
and massive infiltration of clonal myeloma cells by histological
examination. These results indicate that tumor cells from both the
blood and bone marrow disseminated to remote sites, and that although
tumor cells disseminate via the murine host system, they require the
human microenvironment for growth.
| |
DISCUSSION |
The phenotypic heterogeneity of clonal cells in myeloma, combined with
the low apparent proliferative activity of the recognizable myeloma
plasma cells, prompted the search for a proliferative, preplasmacytic
cell compartment with self-renewal capacity (stem cell). However, as
the data presented above clearly demonstrate, given a supportive
environment, the myeloma plasma cells, hitherto considered
nonproliferative, indeed are proliferative, and as our experiments of
sequential transfers suggest are capable of inexhaustible
proliferation. These cells can populate a nonmyelomatous human bone,
produce myeloma and its manifestations in the SCID-hu host, and
disseminate to remote sites. This proliferative potential of myeloma
plasma cells is not restricted to patients with advanced or terminal
disease, as purified myeloma cells from newly diagnosed patients grew
in the hosts equally well.
Myeloma cells from both blood and bone marrow traveled through the host
circulation to the secondary bone, but grow exclusively in the human
bones, underscoring their dependence on the bone marrow stroma. The
fact that purified plasma cells grew equally well to unseparated bone
marrow cells indicates that the nonmyelomatous bone marrow stroma and
accessory cells in the human bones of the SCID-hu hosts can support
sustained growth of myeloma cells.
Of particular interest are the data demonstrating that although fewer
in numbers, the growth of myeloma plasma cells in the blood of patients
in the SCID-hu hosts was detected earlier than those in the bone
marrow. This may suggest that circulating myeloma cells represent a
subgroup of the bone marrow myeloma plasma cells with a higher
growth potential. This notion is further supported by our observation
that the residual myeloma cells in the PC-depleted bone marrow samples
(eg, Fig 1) consistently failed to grow in the hosts. Such property of
the circulating plasma cells would be compatible with the adverse
prognostic significance of their proportion in the
circulation.28
Our results clearly demonstrate that nonplasmacytic cells in the bone
marrow are not required for purified plasma cells to produce myeloma in
SCID-hu hosts. While our results suggest that preplasmacytic cells in
the blood and bone marrow from myeloma patients cannot produce myeloma
in SCID-hu hosts, one cannot draw conclusions as to their role in
maintaining the disease process. It is possible that the SCID-hu host
does not provide a supportive environment for the preplasmacytic cells
in plasma cell-depleted bone marrow or blood specimens to produce
myeloma. Although our experiments of sequential transfer suggest that
the myeloma plasma cells are capable of inexhaustible proliferation,
this point needs to be further investigated.
| |
ACKNOWLEDGMENT |
The authors thank Cherie Johnson, Susan Mahaffey, and Jeff Woodliff for
their skillful technical assistance. We thank Dr Bart Barlogie and the
staff of the Myeloma and Transplantation Research Center for their
interest in and support of this work.
| |
FOOTNOTES |
Submitted March 8, 1999; accepted July 16, 1999.
Supported in part by Grant No. CA-55819 from the National Cancer Institute.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Joshua Epstein, DSc, MTRC, UAMS, 4301 W
Markham, Slot #776, Little Rock, AR 72205; e-mail:
jepstein{at}life.uams.edu.
| |
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TOP
ABSTRACT
INTRODUCTION