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Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 630-640
Establishment of a Reproducible Model of Chronic-Phase Chronic
Myeloid Leukemia in NOD/SCID Mice Using Blood-Derived Mononuclear or
CD34+ Cells
By
Ian D. Lewis,
Louise A. McDiarmid,
Leanne M. Samels,
L. Bik To, and
Timothy P. Hughes
From the Leukaemia Research Laboratory, Division of Haematology,
Hanson Centre for Cancer Research, IMVS, Adelaide, SA, Australia.
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ABSTRACT |
An animal model of chronic myeloid leukemia (CML) will help
characterize leukemic and normal stem cells and also help evaluate experimental therapies in this disease. We have established a model of
CML in the NOD/SCID mouse. Infusion of  4 × 107
chronic-phase CML peripheral blood cells results in engraftment levels
of 1% in the bone marrow (BM) of 84% of mice. Engraftment of the
spleen was seen in 60% of mice with BM engraftment. Intraperitoneal injection of recombinant stem cell factor produced a higher level of
leukemic engraftment without increasing Philadelphia-negative engraftment. Granulocyte colony-stimulating factor and
granulocyte-macrophage colony-stimulating factor did not increase the
level of leukemic or residual normal engraftment. Assessment of
differential engraftment of normal and leukemic cells by fluorescence
in situ hybridization analysis with bcr and abl probes
showed that a median of 35% (range, 5% to 91%) of engrafted cells
present in the murine BM were leukemic. BM engraftment was multilineage
with myeloid, B-cell, and T-cell engraftment, whereas T cells were the
predominant cell type in the spleen. BM morphology showed evidence of
eosinophilia and increased megakaryocytes. We also assessed the ability
of selected CD34+ CML blood cells to engraft NOD/SCID
mice and showed engraftment with cell doses of 7 to 10 × 106 cells. CD34 cells failed to engraft at
cell doses of 1.2 to 5 × 107. CD34+ cells
produced myeloid and B-cell engraftment with high levels of
CD34+ cells detected. Thus, normal and leukemic stem
cells are present in CD34+ blood cells from CML patients
at diagnosis and lead to development of the typical features of CML in
murine BM. This model is suitable to evaluate therapy in CML.
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INTRODUCTION |
CHRONIC MYELOID LEUKEMIA (CML) is a
disorder of the hematopoietic stem cell characterized by the presence
of the Philadelphia (Ph) chromosome,1 which is produced by
a translocation between the abl proto-oncogene on chromosome
92 and the bcr gene on chromosome 22.3
Chromosomal and isoenzyme studies have shown the involvement of the
granulocytic, monocytic, erythroid, megakaryocytic, and B-lymphocytic
lineages, confirming the origin of CML in a pluripotent hematopoietic
stem cell (HSC).4 Allogeneic bone marrow transplantation
(BMT) is currently the only curative treatment for CML, but age and
donor availability restrict this therapy to approximately 25% of
patients.5 For the remaining patients, the disease is
incurable, although interferon may improve survival.6
Despite the multilineage involvement in CML, there is preferential
expansion of the myeloid and megakaryocyte compartments that results
from an increase in the number of colony-forming cells in the BM and
peripheral blood (PB).7 However, at the primitive
hematopoietic cell level, CML BM contains reduced numbers of long-term
culture-initiating cells (LTC-IC) compared with normal BM, but there is
an exponential increase in LTC-IC in the PB.8 The
overgrowth of leukemic cells in the BM and PB does not completely eradicate normal hematopoietic cells in CML. Growth of CML BM on
allogeneic stroma results in decline of Ph+ cells, and,
after 4 to 5 weeks, hematopoiesis is predominantly Ph.9 In CML, as in normal
subjects,10 the CD34+ HLA-DR
and CD34+ CD38 fractions of BM contain
cells that have properties of HSC. We have previously shown that
bcr-abl preprogenitors are present
in these fractions in both CML BM and PB.11 Furthermore,
this residual normal stem cell compartment is present in most patients
at diagnosis but as the disease progresses it gradually
disappears.12,13 Available evidence suggests that these
normal cells are extremely rare, and there is concern whether they are
clonal cells that have not yet acquired the Ph chromosome14 or do not express bcr-abl.15 There is
preliminary evidence that the CD34+
HLA-DR compartment is polyclonal in early, but not
in late, chronic-phase CML.16
The critical biologic differences between Ph+ and
Ph primitive progenitors that have been identified
can be used to develop purging strategies or novel therapies such as
selection of cells based on phenotype11,17,18 or depletion
of leukemic cells by cell culture,19 chemotherapeutic
agents,20 antisense oligonucleotides,21 or
ribozymes.22 However, it is necessary to test these
biologic differences at the stem cell level. The survival of normal and leukemic stem cells after specific manipulations can be assessed by
their ability to engraft immunodeficient mice.
Various strains of immunodeficient mice have been used to study normal
hematopoiesis. Umbilical cord blood progenitors engraft severe combined
immunodeficient (SCID) mice without any requirement for exogenous
cytokine.23 In contrast, adult BM only engrafts SCID mice
in the presence of human stroma24 or after exogenous cytokine administration.25 Cytokines are also necessary in
the mouse model of acute myeloid leukemia,26,27 but acute
lymphoblastic leukemia engrafts SCID mice without
cytokine.28 Mouse models of CML have been more difficult to
establish. Infusion of blast crisis CML cell lines, such as
K562,29 EM-2,29 BV173,30 or
KBM-5,31 causes disseminated leukemia in SCID mice.
Similarly, cells from CML patients in blast crisis engraft and
disseminate in SCID mice.29 Initial studies using
chronic-phase CML cells at doses of 1 to 5 × 107 administered intravenously (IV), administered
intraperitoneally (IP), or implanted under the kidney capsule resulted
in local recovery of cells administered IP only.29 This
failure of engraftment may be a result of the low cell dose
administered, because Sirard et al32 have shown mean
engraftment levels of 0.02% to 10% after IV infusion of 8 to 14 × 107 chronic-phase CML cells, but cells from only 5 of 10 patients engrafted at mean levels of 1% or more.
The aim of our study was to establish a model of CML in non-obese
diabetic/LtSz scid/scid mice (NOD/SCID) mice. This strain is profoundly
immunodeficient with defective T- and B-cell function and marked
impairment of macrophage, natural killer cell, and hemolytic complement
activity.33 We aimed to determine a dose of CML blood cells
that would reliably achieve engraftment and characterize the engrafting
human cells in terms of lineage and bcr-abl expression. We used
exclusively PB because it is relatively easily collected in large
amounts at diagnosis and has been shown to engraft SCID mice at
equivalent rates to BM.32 Our previous studies have
identified CML blood as an abundant source of normal preprogenitors.11 Ex vivo manipulation of CML blood is a
promising approach for autologous transplantation, so a detailed
assessment of the engraftment potential of CML blood is a necessary
precedent to studies of purging strategies.
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MATERIALS AND METHODS |
Patient Material
PB was collected by apheresis from patients with recently diagnosed CML
as approved by the Human Ethics Committee of the Royal Adelaide
Hospital (Adelaide, South Australia). Cells were diluted in Hank's
Balanced Salt Solution (HBSS; GIBCO BRL, Victoria, Australia). Light-density mononuclear cells were collected after centrifugation at
400g over a Lymphoprep density gradient (1.077g/dL; Nycomed Pharma AS, Oslo, Norway) and washed twice in HBSS. Cells were cryopreserved in 10% dimethylsulphoxide, 20% fetal calf serum (Commonwealth Serum Laboratories, Victoria, Australia) by controlled rate freezing. Before infusion, cells were rapidly thawed at 37°C and 4% sodium citrate was added. Cells were washed twice in
Ca+2 Mg+2 free phosphate-buffered saline (PBS),
4% sodium citrate, and 1% human serum albumin (CSL) and kept at room
temperature before infusion.
The phenotype of patient samples was assessed by standard
technique34 using directly conjugated monoclonal antibodies
to CD34, CD33, CD3, and CD19 (Becton Dickinson Immunocytometry Systems, San Jose, Ca). Cells stained with the appropriate directly conjugated isotype antibodies were used as controls.
Selection of CD34+ Cells
Cryopreserved cells.
Cells were thawed, washed, and resuspended at 1 to 2 × 107/mL in isolation buffer (Ca+2
Mg+2 free PBS, 0.6% sodium citrate, 2% human serum
albumin) and CD34+ cells were selected as described
previously.10 Briefly, cells were rosetted
with Dynabeads M-450 CD34 (Dynal, Oslo, Norway) at 4°C for 40 minutes with gentle rotation. Rosetted cells were selected by placing
the tube on a Dynal MPC magnet and pipetting the nonrosetted cells. The
rosetted cells were resuspended in isolation buffer and the separation
procedure was repeated 5 times. The nonrosetted cells
(CD34) were pooled for use in control mice. Beads
were detached from positively selected cells by incubating with
anti-Fab antiserum (DETACHaBEAD; Dynal) at 37°C for 90 minutes.
Released cells (CD34+) were aspirated after placing the
tube on the magnet. Released cells and nonrosetted cells were counted
and purity determined by flow cytometry.
Fresh cells.
Cells collected at diagnosis in 2 patients had CD34+ cells
selected with the Isolex 50 magnetic cell separation system (Baxter Healthcare Corp, Irvine, CA) as previously described.35
Briefly, 4 × 109 cells were incubated with 2 mg 9C5
anti-CD34 antibody (Dynal) at room temperature (RT) for 30 minutes with gentle rotation. Cells were rosetted with Dynabeads and
retained in the chamber attached to the magnetic column and nonrosetted
cells drained. Rosetted cells were washed in buffer and the separation
procedure was repeated 3 times. Rosetted cells were released by
incubating with 100pKat chymopapain (ChymoCell-R; Baxter) at RT for 15 minutes. Isolated cells had CD34 levels evaluated and were
cryopreserved as described above.
NOD/SCID Mice
A breeding colony of NOD/SCID mice was established at the University of
Adelaide from animals originally obtained from Walter and Eliza Hall
Institute, Melbourne. Experiments were performed as approved by the
Animal Ethics Committee of the University of Adelaide. Mice were kept
in microisolator cages in a laminar flow room in specific pathogen-free
conditions. Before cell infusion, mice 6 to 8 weeks old were irradiated
with a total dose of 300 cGy, delivered at 600 cGy per minute, by a
CsCl blood cell irradiator. Higher doses of irradiation (350 and 400 cGy) were attempted but resulted in high mortality between days 10 and
18. Cells were infused, in a volume of 500 to 1,000 µL, into a tail
vein 24 hours after irradiation, and mice were maintained on sterilized
food and acidified water supplemented with 60 mg trimethoprim and 300 mg sulfamethoxazole (Bactrim; Roche Products, New South Wales, Australia) per 100 mL water 3 days per week. Some mice received IP
injections of recombinant human hematopoietic growth factors (HGF),
either stem cell factor (SCF; Amgen, Thousand Oaks, CA), granulocyte
colony-stimulating factor (G-CSF; Amgen), or granulocyte-macrophage colony-stimulating factor (GM-CSF; Sandoz, New South Wales, Australia), at 5 µg 3 times per week. Mice were closely observed and killed if
they developed lethargy or weight loss. Mice were killed between days
28 and 50 by cervical dislocation. The BM from both femurs and tibias
was taken and the spleen was homogenized.
Analysis of Engraftment
Fluorescence in situ hybridization (FISH) analysis for human cell
engraftment.
Human chromosome 8 was detected using a probe for the  satellite
region at the D8Z2 locus (ATCC clone pJM128; American Type Culture
Collection, Rockville, MD), which does not cross-react with mouse chromosomes. The probe was biotin-labeled by standard nick
translation with biotin-14-dATP (BioNick Labelling System; GIBCO BRL)
to 200 to 500 bp. BM and spleen cells from transplanted mice were
washed twice in PBS and deposited on poly-l-lysine-treated glass
slides (Sigma, St Louis, MO). One drop of 75 mmol/L KCL was added to
cells that were then fixed with cold 75% methanol 25% acetic acid and
allowed to air dry.36 The in situ hybridization technique
used was a modified version of the methods described by Tkachuk et
al37 and Lichter et al.38 Cells were treated with RNAse A (Boehringer Mannheim, New South Wales, Australia), at 10 µg in 100 µL 2× SSC for 1 hour at 37°C, followed by 2 washes with 2× SSC and serial dehydration with cold 70%, 90%,
and 100% ethanol. Cells were denatured in 70% formamide, pH 7, at
70°C for 4 minutes and then immediately dehydrated again with 70%, 90%, and 100% ethanol. The hybridization mixture consisted of 5 ng
probe DNA in 10% dextran sulphate, 2× SSC, 50% formamide, and
0.1% polyoxyethylenesorbitan monolaurate (Tween 20; Sigma), which was
denatured at 96°C for 4 minutes followed by immediate cooling, and
then added to individual slides preheated to 37°C and incubated
overnight in a humid atmosphere (Omni-Slide; Integrated Sciences, New
South Wales, Australia). After hybridization, slides were washed twice
for 10 minutes each in 50% formamide at 42°C and then twice in
2× SSC and 1× SSC at RT. Hybridized probe was detected
using avidin-fluorescein isothiocyanate (FITC; Vector, Burlingame, CA),
nuclei counterstained with propidium iodide (PI), and slides mounted in
DABCO (1,4 diazabicyclo[2.2.2.]octane; Sigma). Cells were examined at
1,000× magnification under oil immersion using an Olympus BH2
microscope (Olympus, Tokyo, Japan) with fluorescence attachment. A
minimum of 300 cells were examined. Engraftment was determined by
calculating the number of cells with 2 fluorescent signals as a
percentage of the total number of PI-stained cells.
FISH analysis for leukemic engraftment.
The hybridization protocol is similar to that described for D8Z2.
Before RNAse treatment slides were treated with 0.01 pg/mL proteinase K
(Merck, Victoria, Australia) for 30 minutes at 37°C and then washed
with 2× SSC before the addition of RNAse A. LSI bcr
SpectrumGreen/abl SpectrumOrange dual-color DNA probe mixture (Vysis,
Downers Grove, IL) was prepared according to manufacturer's instructions. The denaturation, hybridization, and posthybridization washes were performed as described for D8Z2. Dual-labeled cells were
examined using a dual band-pass filter for both FITC and Texas red.
Where possible, 300 cells were examined. Scoring criteria for normal
and leukemic cells were as recommended by the manufacturer.
Immunophenotype analysis.
Mouse BM and spleen suspensions of transplanted mice were assessed for
presence of human cells by immunolabeling with conjugated anti-CD45
(Becton Dickinson) with comparison to an anti-IgG control. Red blood
cells were lysed before immunolabeling in 10× vol 0.83% ammonium
chloride at 37°C for 10 minutes. Differentiation of engrafted cells
was determined by dual-color labeling with anti-CD45 FITC and
anti-CD34, 33, 3, and 19 phycoerythrin (Becton Dickinson), respectively. Expression of these antigens was compared with control cells labeled with anti-IgG controls. We gated cells to include both
lymphoid and myeloid fractions.39
Colony-forming unit-granulocyte-macrophage (CFU-GM) assay.
Triplicate 1-mL cultures with 5 × 105 BM cells were
established in 35-mm plates in 0.9% methylcellulose (Methocel; Dow
Chemical Co, Midland, MI) in Iscove's Modified Dulbecco's Medium
(GIBCO BRL) supplemented with 30% fetal calf serum and 3 mmol/L
L-glutamine. Cultures were stimulated by 10 ng each of recombinant
human interleukin-3 (Sandoz), GM-CSF, and SCF, a combination that does
not stimulate murine progenitors. After 14 days of incubation at
37°C in 5% CO2, CFU-GM were scored as aggregates of
greater than 50 cells.
Morphological assessment.
Cytocentrifuge slides were prepared from BM and spleen, air-dried, and
then stained with Jenner-Giemsa (BDH Ltd, Poole, UK).
Conventional 4-µm histologic sections of spleen and decalcified tibia
were cut from formalin-fixed, paraffin-embedded material and stained
with hematoxylin and eosin.
Statistical Analysis
Results are expressed as the mean ± 1 standard error of the mean
(SEM) percentages, unless otherwise stated. Analysis of engraftment used regression analysis and analysis of variance.
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RESULTS |
Patient Characteristics
Cryopreserved PB from 15 patients with newly diagnosed chronic-phase
CML was infused into 163 NOD/SCID mice in different experiments. The
white blood cell count (WCC) at presentation ranged
between 39 and 376 (median, 133) × 109/L
(Table 1). Twelve patients had exclusively
100% Ph+ metaphases in the BM at diagnosis, with 1 patient
having 82% Ph+ metaphases, 1 patient having a complex
translocation, and 1 patient having an additional minor abnormality
(Table 1). Analysis of thawed material by FISH showed 37% to 51%
(median, 48%) bcr-abl+ cells in 5 samples
analyzed. The immunophenotype of thawed cells was quite variable. CD34
levels ranged between 3% and 31% (median, 18%). Myeloid cells, as
assessed by CD33 expression, ranged between 7% and 60% (median,
30%), and T lymphocytes, as assessed by CD3, ranged between 12% and
49% (median, 27%). The level of B cells was low and less than 10% in
all cases. Trypan blue estimation of cell viability before infusion
into mice showed 66% to 88% viability.
Sensitivity of FISH to Assess Engraftment
The use of human-specific probes to assess engraftment of human cells
into murine hosts allows direct visualization of human cells with
relatively simple enumeration. Only 1,000 cells are required, which is
much lower than is required for flow cytometry. All viable, nucleated
cells are seen and counted. We assessed the accuracy and sensitivity of
this technique by a mixing experiment in which human cells were mixed
with murine cells in defined proportions. Two blinded observers
analyzed 300 cells from each dilution. The results are shown in
Table 2. It can be seen that, at low levels of human cells, the concordance rate between observers and the actual
level of human cells is high; therefore, the method is suitable for
analyzing engraftment to the 1% level. This dilution experiment was
repeated for CD45 analysis and shows similar sensitivity at low levels
of engraftment (Table 2).
Engraftment Studies With Unselected CML PB Cells
In separate experiments, 115 mice were infused with 1.1 to 7.6 × 107 cells from 13 patients (all patients in Table 1 except
patients 01 and 07). Some mice received HGF. Eighteen mice (16%)
became ill between days 10 and 18, with lethargy, ruffled fur, and
wasting, suggesting radiation toxicity, and were killed. An additional 6 mice (5%) became ill between days 42 and 50, with features of lethargy and wasting, and were killed. Analysis of these mice did not
show evidence of overwhelming leukemia, and they may have succumbed to
graft-versus-host disease-type illness, as has previously been
described in NOD/SCID mice occurring at this timepoint.40
BM engraftment.
Human cells 1% were detected by FISH in 69 of the 91 mice (76%)
analyzed between days 28 and 51, with a range of engraftment of 1% to
87% (Fig
1A). The median level of engraftment
in these 69 mice was 9%. BM engraftment 10% was seen
in 32 of the 69 engrafted mice (46%). Engraftment was correlated with
total cell dose (r = .52). Eighteen mice received less than 4 × 107 cells, 8 mice showed no engraftment, and 10 mice had a median engraftment of 3% (range, 1% to 7%). In the 73 mice receiving 4 × 107 cells, 61 mice engrafted
with a level of 20% ± 3% (median, 10%; Fig 2). The effect of cell dose is further
shown in Fig 3. Different cohorts of mice
were infused with different cell doses from 4 patients. In each case,
higher engraftment was seen at the higher cell dose (r = .71).
Analysis of predictors of engraftment showed that the total cell dose,
CD34+ cell dose, and the donor (P < .01) were
independent predictors.
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| Fig 1.
Analysis of engraftment of human cells in NOD/SCID mice
by FISH. (A) Human cells detected by D8Z2 probe show two bright green fluorescent signals in contrast to murine cells with no signal (original magnification × 200). (B) Differential engraftment of normal and leukemic CML cells in NOD/SCID mice detected by dual probes
for bcr and abl. Normal cells show two red abl
signals and two green bcr signals. Leukemic cells show a single
red and green signal representing normal abl and bcr
genes and the yellow signal representing fusion of abl and
bcr genes (original magnification × 1,000).
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| Fig 4.
Morphologic analysis of murine BM and spleen engrafted
with CML cells. (A) Histologic section of tibia of NOD/SCID mouse
irradiated but not infused with human cells at day 42 (original
magnification × 400). (B) Histologic section of tibia of mouse with
28% human cells detectable at day 42. Hypercellular marrow with
proliferation of megakaryocytes and eosinophils are shown (original
magnification × 400). (C) Histologic section of spleen from control
mouse showing monomorphic lymphoid population (original magnification × 400). (D) Histologic section of spleen of mouse with 33% human
cells detectable at day 42. Infiltration with megakaryocytes is shown. (E) Cytospin preparation of spleen from control mouse (original magnification × 400). (F) Cytospin preparation of spleen of mouse with 56% human cells detected at day 42 showing prominent
magakaryocytes (original magnification × 400).
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| Fig 2.
Results of engraftment by FISH of 19 cohorts of 2 or 3 mice receiving different doses of chronic-phase CML cells collected from patients at diagnosis. Results are the mean ± SEM engraftment for each cohort of mice.
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| Fig 3.
Effect of cell dose on engraftment. The graph shows
the increased engraftment in cohorts of mice receiving different
cell doses from the same patient sample. Results are the mean BM
engraftment for each cohort. Patients no. ( ) 2, ( ) 3, ( ) 4, and ( ) 8.
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Engraftment was confirmed by analysis of human specific CD45, a pan
leukocyte marker. All mice that had engraftment 1% by FISH had
CD45+ cells detected. The level of engraftment by CD45 was
slightly lower compared with FISH. This difference may have been due to the exclusion of some human cells by the gate applied or the failure to
lyse all mouse red blood cells, resulting in a dilution of human cells.
The ability of engrafted cells to produce CFU-GM was assessed in 17 mice with engraftment levels of 1% to 87%. CFU-GM were detected in 15 of 17 cases, with a level of 35 ± 7 (range, 1 to 94) colonies per
105 BM cells plated. The total number of human CFU-GM
present in 2 femurs and 2 tibias was 7 to 2,690 (mean, 697).
Morphologic assessment showed hypercellularity of BM with increased
numbers of megakaryocytes and eosinophilia in highly engrafted animals, but there was no fibrosis (Fig 4A and B). Assessment of
the peripheral blood WCC in 6 mice showed a normal WCC.
Splenic engraftment.
In 64 mice that had BM engraftment, 42 (66%) had detectable human
cells in the spleen, with engraftment of 16% ± 3% (range, 1% to
92%). The donor was a predictor of spleen engraftment (P < .01); mice infused with cells from patients 02, 03, and 04 had higher
levels of splenic engraftment, whereas mice infused with cells from
patients 06, 11, and 12 had low splenic engraftment despite quite high
BM engraftment. There were no statistically significant differences
between these groups in terms of WCC at diagnosis, the percentage of
Ph+ cells by karyotype, or the percentage of CD3 cells in
the blood. In 16 mice the level of splenic engraftment exceeded the
level of BM engraftment. No mice had isolated splenic engraftment. Mice with high splenic engraftment had moderate splenomegaly. Histologic examination of the spleen showed infiltration by megakaryocytes in most
engrafted mice (Fig 4C through F).
Leukemic engraftment.
The differential engraftment of leukemic and normal human cells was
assessed by FISH analysis with directly conjugated probes for
bcr and abl (Fig 1B). There was no cross-reactivity of
the probes with murine cells. In 25 mice with BM engraftment 9%, the
level of bcr-abl+ cells detected was 39% ± 5%
(range, 5% to 91%) of the engrafted human cells. We also analyzed 6 spleen specimens that had 29% to 87% engraftment for the presence of
leukemic cells. Of the engrafted cells, a lower level of
bcr-abl+ cells was detected compared with
BM (11% ± 2%; range, 4% to 17%).
Use of HGF to improve engraftment.
The role of HGF in promoting engraftment of human cells was assessed in
nine experiments with 59 mice. IP injections of 5 µg of SCF, G-CSF,
or GM-CSF were administered every second day, except in 4 mice that
received SCF + G-CSF or SCF + GM-CSF. The use of HGF did not produce an
increase in engraftment compared with control mice that received no
growth factor. Mice receiving GM-CSF or SCF had levels of engraftment
that were not statistically significantly different from no growth
factor. Human engraftment with no growth factor was 22% ± 6% (n = 22), compared with 19% ± 5% for SCF (alone or in combination, n = 28), and 26% ± 8% for GM-CSF (n = 7). The use of G-CSF alone
resulted in significantly lower levels of human engraftment  6% ± 3% (n = 8; P = .01).
The influence of HGF on leukemic cell engraftment was also assessed,
and there was a trend for mice receiving SCF alone or in combination to
have higher levels of bcr-abl+ cells detected
(P = .02). In mice receiving SCF (n = 7), the level of leukemic
engraftment was 56% ± 8% compared with 34% ± 7% for mice
receiving no HGF (n = 12), 28% ± 6% for mice receiving GM-CSF
alone (n = 4), and 32% ± 18% for mice receiving G-CSF alone (n = 2; Fig 5).
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| Fig 5.
Influence of HGF on leukemic engraftment. Results are the
mean ± SEM of bcr-abl+ cells by FISH as a
proportion of total human cells (scf, stem cell factor; gm, GM-CSF; g,
G-CSF; nil, no growth factor).
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Phenotype of engrafted cells.
The immunophenotype, as assessed by dual-color immunofluorescence, of
the engrafted human cells in the BM is shown in
Table 3. The engrafted cells were
predominantly myeloid in origin, as shown by the expression of CD33
(Fig 6A). Primitive cells, defined by
expression of CD34, were detected in 10 of 24 samples analyzed at
levels of 1% to 14% of engrafted cells. CD19 cells were rare in the
BM, with 1.9% ± 1.2% of human cells being positive. Analysis of
CD3 showed predominant expression in the BM of 2 patients, with mean
levels of 54% and 58%. The samples infused into these mice contained
12% and 49% CD3 cells, respectively. These CD3+ cells
consisted of distinct populations of CD4+ and
CD8+ cells (data not shown). Analysis of sorted
CD3+ cells by FISH for bcr-abl showed only 2%
leukemic cells, which is within the background level of bcr-abl
detection, suggesting that virtually all the CD3 cells were
Ph. Expression of CD56 was assessed in the BM of 6 mice and 6% ± 3% of human cells expressed this antigen.
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| Fig 6.
(A) Immunophenotype of BM specimen at day 42 showing high
engraftment of predominantly myeloid cells with lower levels of CD3 and
CD19 cells. (B) Immunophenotype of spleen cells from the same mouse
showing virtually exclusive T-cell engraftment.
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Analysis of cell surface phenotype of human cells in the spleen showed
a different picture (Fig 6B). No CD34 cells were detected and CD33
cells were only 9.3% ± 6.3% of engrafted cells. The predominant cell type expressed CD3 with 72.8% ± 7% positive (Table 3). These CD3 cells also contained both CD4 and CD8 cells and were predominantly CD45RO+. The CD3 content of the infused cells was 6.2 to 32 × 106 (median, 12 × 106), and the
total number of T cells recovered from the spleens was 0.1 to 46 × 106 (median, 1.0 × 106). The B
lymphocytes were also more frequent in the spleen, with 6.1% ± 2.2% cells detected.
To assess the kinetics of engraftment, 6 mice were infused with 7.4 × 107 cells and 2 of these mice were killed at each
time-point; days 14, 28, and 42. The mean BM engraftment
was 38%, 13%, and 30% at each of these timepoints, respectively. At
day 28, a mean of 62% and 8% of engrafted cells expressed CD33 and
CD34, respectively. By day 42, only 7.5% and 0.5% of engrafted cells
expressed these antigens. This was associated with an increase in CD3
cells from a mean of 4% at day 28 to 56% at day 42. No engraftment of
the spleen was noted at day 14 or 28, but 33% human cells were
detected at day 42, with 94% of these being CD3+.
Engraftment Studies With Selected CML CD34 Cells
CD34+ cells were selected from 4 patient samples (05, 08, 10, and 11) using Dynal beads on thawed material. Two patient samples (01 and 07) had large scale selection of CD34+ cells by an
Isolex column. One of these samples (01) had CD34+ cells
selected at diagnosis on leukapheresis product that were then
cryopreserved before use. The mean purity of recovered cells was 93%
(range, 88% to 98%). Cell doses of between 1 × 104
and 1 × 107 were infused from these 6 patient samples
into 36 mice (Table 4). Mice were killed at
day 28 (13) or day 42 (25). There was a correlation between the number
of CD34+ cells infused and the level of engraftment
(r = .47). Engraftment was seen in 2 mice at day 28 with levels
of 2% and 63%, respectively. At day 42, 7 mice engrafted between
0.7% and 54%. Eight of 10 mice receiving 6 × 106
cells showed engraftment, but none of the 26 mice receiving lower cell
numbers did. Only 2 samples had engraftment in the spleen at levels of
1% and 4%. The 2 mice that had the highest engraftment levels (54%
and 63%) received CD34 cells selected from fresh leukapheresis material.
The engrafted cells from 3 mice were phenotyped and showed myeloid and
B-lymphoid cells in the BM. The mean level of CD34 expression was 10%.
The mean level of CD33 expression was 80% and of CD19 was 10.7%. CD3
cells were not detected (Table 5).
Assessment of leukemic cell engraftment by FISH was performed on the 3 samples with the highest engraftment. The number of bcr-abl+ cells was variable, being 23%, 35%, and
64% (Table 5).
To confirm that the engrafting cell in CML is CD34+,
parallel experiments with the infusion of CD34 cells
were performed. Cells were infused from 5 patients (05, 07, 08, 10, and
11) at doses ranging between 1.2 and 5 × 107 cells
into 12 mice. No engraftment by either FISH or CD45 analysis was seen
in any of the mice infused (Table 4).
| |
DISCUSSION |
We have established a model of CML in NOD/SCID mice by infusing large
numbers of cryopreserved CP CML PB into sublethally irradiated mice.
The major determining predictor of engraftment was the cell dose
infused with 84% of mice receiving 4 × 107 cells
showing evidence of human cells 28 to 42 days postinfusion. Of these,
46% of mice had greater than 10% human cells detected in their BM.
SCF, G-CSF, or GM-CSF alone or in combination failed to improve overall
engraftment rates. In addition, we detected human cells in the spleen
of 60% of mice that had BM engraftment. Previous studies of murine
models of CML have also shown that the cell dose infused is critical
for engraftment and that HGF do not improve engraftment levels. The
study of Sirard et al32 infused 8 to 14 × 107 fresh PB or BM mononuclear cells from
newly diagnosed CML patients into SCID mice and showed the majority of
mice achieving 0.1% to 10% engraftment. Mice receiving 2 to 6 × 107 cells achieved negligible engraftment.32
Our study shows higher engraftment in the NOD/SCID mouse using lower
cell doses. This may be the result of using cryopreserved material
because of the selective loss of the more mature, nonengraftable cells
during cryopreservation. However, the more immunosuppressed NOD/SCID mouse has been shown to improve engraftment of human cord blood compared with SCID mice,41 and this may account for the
lower doses required. We also show morphologic evidence of a CML-like disease in the BM and spleen of engrafted mice but no leukocytosis, suggesting that our model recapitulates some of the features of chronic-phase CML.
The ability to assess engraftment of leukemic and normal cells is
critical if the application of cell selection or purging techniques as
therapeutic modalities in CML are to be tested in an animal model. We
have used FISH to detect bcr-abl+ cells in
engrafted mice as a measure of leukemic engraftment. This allows direct
visualization and enumeration of cells and avoids the reliance on
proliferating cells necessary for cytogenetic analysis. Analysis of the
CML cells before infusion from 5 patients showed that 37% to 51% of
cells were bcr-abl+ by FISH, indicating that the
innoculum has equivalent numbers of leukemic and nonleukemic cells. The
high number of nonleukemic cells may be accounted for by the high
levels of T cells in some samples. We analyzed 25 specimens
posttransplantation by FISH for bcr-abl and of the human cells
a mean level of 39% were leukemic, with a range of 5% to 94%. Thus,
there is a wide range in the level of leukemic engraftment in these
mice, but the majority of cases show preferential engraftment of normal
cells.
The role of HGF is relevant, because they are used clinically to
mobilize stem cells or posttransplantation to increase the speed of
engraftment and may modulate normal or leukemic engraftment. We found
that SCF has a role in promoting the engraftment and proliferation of
leukemic cells in vivo, which is consistent with in vitro
findings,42 but G-CSF and GM-CSF did not promote leukemic engraftment.
The immunophenotype of engrafted cells shows differential engraftment
of myeloid and lymphoid cells between the BM and spleen. Myeloid cells,
defined by expression of CD33, were confined to the BM. This is not
dependent on exogenous myeloid growth promoting factors, because it was
seen in mice that received no growth factor as well. The engraftment of
primitive cells was confirmed in some mice by detection of CD34. The
levels were low but in keeping with the frequency of these cells in CML
BM and also consistent with the data of Sirard et al.32 We
were able to show B-cell lymphoid development in the spleen of some
mice, but levels of B cells within BM were negligible.
The finding of almost exclusive T-cell involvement of engrafted murine
spleen and high levels of T cells in the BM of some mice is intriguing.
The early study of Sawyers et al29 showed recovery of
predominantly CD3+ cells from the peritoneal cavity of SCID
mice receiving 1 to 5 × 107 CP CML cells IP. The
study of Sirard et al32 shows expression of CD13 and CD19
of engrafted CML BM cells. Infusion of human PB lymphocytes (PBL) can
engraft both SCID43 and NOD/SCID44 mice, but
only after IP infusion of cells. This results in engraftment of
predominantly T cells, with the spleen being the primary site of
engraftment and very few cells being detected in the BM. In contrast,
infusion of adult BM into NOD/SCID mice together with exogenous
cytokine results in myeloid and B-cell engraftment in BM and spleen,
with no erythroid or T-cell development.45
Our results show engraftment of polyclonal T cells in the spleen and BM
of some mice. These are likely to be derived from long-lived
recirculating T cells present in the innoculum, but calculation of
total T cells recovered from murine BM and spleen shows expansion of
the cells compared with input numbers in some mice. This finding is
supported by the finding of increased engraftment of T cells at day 42 compared with earlier timepoints. The studies with normal adult BM and
CML BM showing minimal T-cell engraftment from cells administered IV
and the finding that PBL only engraft after IP injection suggest that
the route of delivery of T cells is important. In this study, we show
delayed engraftment of T cells that are
bcr-abl after IV infusion of CML PB. The
mechanism of this is unclear, but it is possible that T cells in CML
are part of a preleukaemic Ph clone14
that has different engraftment characteristics than normal T cells.
Alternatively, the failure of engraftment of the CD34 fraction, which is enriched for T cells, may
indicate that CD34+ leukemic cells facilitate the
engraftment of T cells in this model.
We have shown that the infusion of 7 to 10 × 106
purified CML CD34+ cells engrafts immunodeficient mice. The
demonstration of bcr-abl+ cells by FISH indicates
the leukemic stem cell resides in the CD34 population. Experiments with
CD34 cells showed no engraftment, confirming this
finding. Splenic engraftment was only seen in 2 mice at low levels.
There was a high level of CD34+ cells detectable in the BM
6 weeks posttransplantation and high numbers of myeloid progenitors.
Furthermore, these cells differentiate into myeloid cells and B
lymphocytes, but no T cells were detected. This pattern of cell
differentiation is similar to studies using CD34+-enriched
umbilical cord blood.46 Other studies show engraftment of
purified CD34 cells from cord blood (5 × 104
cells),47 AML (5 to 7 × 106),26 and juvenile CML (JCML; 2 to 3 × 105).48 Thus, in CML higher doses of CD34 cells
are necessary for engraftment, although the studies in AML and JCML
required HGF. We have not assessed the role of HGF in engraftment of
purified CD34+ CML cells, but they may allow a lower cell
dose to be administered. Functional differences in the CD34 cell in
each of these examples may explain the requirements for differing cell
dose to achieve engraftment. In CML it is known that the majority of
primitive cells have phenotypic characteristics of proliferating
cells,49 and there is evidence to suggest cycling cells do
not efficiently engraft Balb/c mice,50 although this is in
an unirradiated model. Also, the frequency of the pluripotent HSC as
defined by the phenotypes CD34+ HLA-DR
or CD34+ CD38 is reduced in
chronic-phase CML compared with normal hematopoiesis. In normal
hematopoiesis, the frequency of DR and
CD38 cells is 3% to 8% and 3% to 7% of the
CD34+ population, respectively.10,51 In CML, we
have shown previously that these populations are rarer, comprising 1%
each of the CD34+ population.11 Another
possibility is the interaction of the CML CD34 cell and the murine BM
stroma. For successful BM engraftment, homing of infused cells to BM
stroma is critical. Homing is integrin-dependent52 and CML
cells have an integrin-mediated defect in adhesion53 that
may impair their homing ability. Hence, the rarity of engraftable leukemic stem cells and defective homing ability may both contribute to
the high CD34+ cell requirement of CML cells in the murine
model.
In summary, we have shown engraftment of mononuclear and
CD34+ selected cells from the PB of CML patients into
NOD/SCID mice as evidenced by detection of human cells in the BM and
spleen by FISH and immunophenotype. We achieved good levels of
engraftment with lower cell doses than has been reported for the SCID
mouse model of CML. Our model recapitulates the characteristic features seen in the BM with megakaryocytic and myeloid overgrowth. This model
is suitable for evaluating purging strategies and novel therapies in
the treatment of CML.
| |
FOOTNOTES |
Submitted July 2, 1997;
accepted September 12, 1997.
Supported in part by the Anti-Cancer Foundation of the Universities of
South Australia. I.D.L. is the recipient of a National Health & Medical
Research Council of Australia Postgraduate Medical Research
Scholarship.
Address reprint requests to Timothy P. Hughes, MD,
Division of Haematology, Hanson Centre for Cancer Research, IMVS, Frome Road, Adelaide, SA, Australia.
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.
| |
ACKNOWLEDGMENT |
The authors thank Dr Robert Moore for producing the tissue sections and
Dr Bill Venables for statistical advice. Dr Ken Langley (Amgen) kindly
provided SCF and Dr Glen Pater (Sandoz, Australia) kindly provided
GM-CSF.
| |
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Abstract
Introduction
Abstract