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NEOPLASIA
From the Terry Fox Laboratory, British Columbia Cancer
Agency, Vancouver, Canada.
Efflux of Hoechst 33342 from normal hematopoietic cells identifies
a "side population" (SP+) of negatively staining cells
that, in the mouse, are largely CD34 Acute myeloid leukemia (AML) arises from the clonal
expansion of a malignant transformed progenitor cell.1-3
Leukemic cells that initiate long-term hematopoiesis in stromal
co-cultures (AML LTC-IC) and suspension cultures and engraft in
nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice
(NOD/SCID leukemia-initiating cells) are rare progenitors that express
a cell surface phenotype similar to that seen on normal multipotential
long-term culture-initiating cells (LTC-IC) and lympho-myeloid NOD/SCID
mouse competitive repopulating units (CRU).2,4-9 Thus,
these assays identify candidate leukemic "stem cells" that may be
responsible for maintaining the malignant clone in patients with AML.
The immunophenotypic profile of primitive AML progenitor cells is
usually characterized by the expression of CD34 but the lack of CD38,
Thy-1, CD71, HLA-DR, and c-kit
expression.1,2,10-15 However, in some reports,
CD34 Several reports demonstrate normal hematopoietic stem cells that lack
the expression of CD34, CD38, and lineage-associated markers in murine
systems and, using the sheep in utero transplantation model, in
primitive human hematopoiesis.17-19 In the NOD/SCID mouse model CD34 Goodell et al18,21 have identified murine bone marrow
cells with stem cell characteristics by their rapid efflux of the fluorescent vital DNA binding dye, Hoechst 33342. The simultaneous display of the fluorescent staining of cells exposed to Hoechst 33342 at 2 different wavelengths identifies a small and distinct subpopulation termed the side population (SP). These SP+
cells are largely CD34 These interesting findings suggest that CD34 may not be as reliable a
marker for the identification of hematopoietic stem cells as was
previously thought. The experiments described in this report were
designed to further investigate Hoechst 33342 dye efflux as a marker
for normal and malignant human progenitors. Our goals were, first, to
identify and quantitate SP+ cells in AML samples and,
second, to demonstrate the functional properties of these cells. Third,
we investigated whether SP+ cells in AML samples were part
of the leukemic clone. We determined that
SP+CD34+CD38 Patient samples
Analysis of acute myeloid leukemia cells
Fluorescence-activated cell sorting Hoechst dye was excited at 350 nm, and its fluorescence was measured at 2 wavelengths using a 424 bandpass (BP) 44 and a 660 BP 20 optical filter (Omega Optical, Battleboro, VT). A 640-nm long-pass filter was used to separate the emission wavelengths. PI fluorescence was measured through a 715-nm long-pass filter. Hoechst blue represents the 424 BP filter, the standard analysis wavelength for DNA content. Both Hoechst blue and Hoechst red fluorescence are shown on a linear scale. PI staining was used to exclude dead cells. SP+ was defined using control cells stained with both Hoechst and verapamil to establish the SP gate (Figure 1). The sort gates for CD34+/ and CD38+/ cells
among SP+ and SP cells were established using
an isotype control. The following subpopulations were sorted:
SP+CD34+CD38,
SP+CD34CD38, and
SPCD34+CD38. Cells were sorted
into Eppendorf tubes containing serum-free media or directly onto
10-well glass slides.
Cultures of acute myeloid leukemia cells Assays for AML CFCs were performed by plating sorted cells at 150 to 10 000 cells/mL in methylcellulose medium (0.92% methylcellulose, 30% FCS, 2 mM L-glutamine, 104 M  -mercaptoethanol, 1% BSA in IMDM; Stemcell)
with 3 U/mL human erythropoietin (EPO; Stemcell), 10 ng/mL GM-CSF
(Norvartis, Basel, Switzerland), 10 ng/mL IL-3, (Norvartis) 50 ng/mL SF
(Terry Fox Laboratory) and 50 ng/mL Flt-3 ligand (Immunex, Seattle,
WA). Cultures were scored after 14 days for the presence of clusters (4-20 cells) and colonies (more than 20 cells). In patients with a
leukemia-specific clonal chromosomal abnormality, colonies were plucked
onto 10-well glass slides for fluorescence in situ hybridization (FISH)
analysis. The relative number of colonies from the sorted subpopulations that would be contained in 1 × 106
unsorted cells was calculated by multiplying the percentage of the
SP+ (or SP) sorted subpopulation in the total
cell population by the number of colonies per 106 plated
cells from that sorted subpopulation.
LTCs of AML cells were established as previously
described.23 AML cells at a concentration of 85 to 7500 cells/mL in Myelocult LTC media (Stemcell) with 10 NOD/SCID mice NOD/LtSz-scid/scid (NOD/SCID) mice24 were bred and maintained under sterile conditions in the British Columbia Cancer Research Center Joint Animal Facility according to protocols approved by the Animal Care Committee of the University of British Columbia. Eight- to 10-week-old mice were irradiated with 350 cGy from a cesium Cs 137 source 24 hours before injection of AML cells. Sorted AML cells were kept in -MEM (Stemcell) with 50% FCS and injected into mice
through the tail veins. Because of concerns that the seeding efficiency
of the AML cells would be reduced when very small numbers of cells were
injected, 2 × 106 irradiated (15 Gy) normal human bone
marrow cells were co-injected with the sorted cells. After the
injection of AML cells into mice, bone marrow aspiration from the femur
was performed every 4 weeks after anesthesia with 11 µL/g Avertin
(2,2,2-Tribromoethanol; Aldrich, Milwaukee, WI) per mouse injected
intraperitoneally. Cells were stored in -MEM with 50% FCS and were
used for FACS staining. Eight to 16 weeks after injection, the mice
were killed by CO2 inhalation. Blood cells were harvested
by cardiac puncture. Bone marrow was obtained from the 4 long bones by
flushing the bones with -MEM with 50% FCS. The spleen was removed,
and a single cell solution was prepared by flushing the cells with
-MEM with 50% FCS. Mice were checked frequently and euthanized
earlier if apparent disease or distress developed.
Analysis of mice To prepare cells from mouse tissue for FACS analysis, samples were incubated with 7% ammonium chloride (Stemcell) to lyse red blood cells, pelleted, and resuspended in Hanks balanced salt solution (Stemcell) with 2% FCS (HFN) and 5% human serum to block human Fc receptors. Cells were then incubated for 10 minutes on ice with an antimouse IgG Fc receptor monoclonal antibody (2.4G2, provided by SyStemix, Palo Alto, CA) for blocking of nonspecific binding to mouse Fc receptors. Then 105 cells were incubated for 30 minutes on ice with a mouse IgG1 isotype control (Becton Dickinson Immunocytometry Systems, San Jose, CA) to evaluate nonspecific immunofluorescence or with fluorescein anti-CD45, a human-specific pan-leukocyte marker (prepared in our center from clone HB10508, American Type Culture Collection, Rockville, MD) to detect human cells. Separate aliquots of cells were incubated for 30 minutes. at 4°C with the following combinations of antibodies against human antigens for the detection of lymphoid and myeloid engraftment: (1) antihuman CD34-FITC (8G12) plus anti-CD19-PE and anti-CD20-PE (both from Becton Dickinson) and (2) anti-CD71-PE (OKT-9) plus anti-CD45-PE, anti-CD15-FITC, anti-CD66b-FITC (all from Pharmingen). Cells were washed twice with HFN, the second wash containing 1 µg/mL PI (Sigma Chemical, St Louis, MO). FACS analysis was performed on a FACScan or a FACSort flow cytometer (Becton Dickinson). Positive cells were defined as those exhibiting a level of fluorescence that exceeded 99.98% of that obtained with isotype-matched control antibodies labeled with the same fluorochromes. Mice were considered negative if there were fewer than 5 CD34+CD19/20+ human cells or fewer than 5 CD45/71+CD15/66b+ human cells per 2 × 104 viable cells analyzed.25To allow morphologic confirmation of the presence of leukemic cells, smears of mouse bone marrow or cytospins of flow-sorted CD45+ cells were prepared on glass slides. After staining with Wright Giemsa, slides were examined by bright-field microscopy, and cells were scored to determine percentages of AML blasts. Fluorescence in situ hybridization Cytospin preparations were obtained from the AML samples after FACS sorting. Colonies from methylcellulose assay of 5-week-old LTCs were plucked onto 10-well slides and fixed in methanol-glacial acetic acid 3:1 for 10 minutes. The centromeric repeat probe used to detect the +8 abnormality (plasmid D8Z2 from American Type Culture Collection), which is specific for human chromosome 8, was labeled with digoxigenin (DIG; Boehringer Mannheim, Mannheim, Germany) by nick translation. For the inv(16) rearrangement, a yeast artificial chromosome clone containing 550 kb human DNA encompassing the breakpoint on 16p13 (CEPHy904E02 854; Max Planck Institut, Berlin, Germany) was labeled by nick translation with digoxigenin. To detect the +13 abnormality, the Quint-Essential (Oncor, Gaithersburg, MD) 13-specific DNA probe labeled with DIG was purchased and used as specified by the manufacturer. The 11q23 MLL probe labeled with DIG was purchased from Oncor. Chromosomes 8 and 16 probes and all slides were treated as previously described.23 Probes were denatured, applied to denatured cells on slides, and hybridized overnight at 37°C. For probe detection slides were incubated with a sheep anti-DIG-FITC antibody (Boehringer Mannheim) at 37°C for 1 hour in the dark, washed, and further incubated for 1 hour with rabbit-antisheep FITC (Vector Labs, Burlingame, CA) and counterstained with PI as previously described.23Analysis of the slides was performed on a Zeiss (Oberkochen, Germany) Axioplan fluorescence microscope equipped with a double bandpass filter to allow simultaneous visualization of the FITC signal and the PI counterstain. Each colony was scored as either normal or abnormal only if a minimum of 5 cells showed a clear signal and if at least 80% of the cells showed either the normal or the abnormal signal.
Patient characteristics Leukemic blast samples from 16 patients (12 men, 4 women) with newly diagnosed AML were studied. In 10 patients the bone marrow (BM) was analyzed, and in 6 patients the peripheral blood (PB) was analyzed. The mean age was 54.6 years (range, 25-77 years). The FAB subtype for 2 patients was M1, M2 for 1, M4 for 2, M4Eo for 6, and M5 (a or b) for 4 patients. Refractory anemia with excess of blasts in transformation (RAEB-IT) was diagnosed in 1 patient. Mean white blood cell count was 99 × 109/L (range, 11-370 × 109/L) with a mean blast percentage of 53.3% (range, 5-91) (Table 1).
Frequency of total side population cells and immunophenotypic profiles Figure 1 shows a typical FACS profile of AML cells stained with Hoechst 33342 with and without verapamil to identify SP+ cells, followed by an analysis of gated cells for the expression of CD34 and CD38. This strategy was used to analyze the 16 AML patient samples as shown on Table 2. Mean frequency of SP+ nucleated cells in the BM or PB was 8% (range, 0.53%-29.9%). Mean frequency of the CD34+CD38,
CD34+CD38+,
CD34CD38+, and
CD34CD38 subpopulations among the
SP+ cells was 12.3% (range, 0.4%- 49.5%), 24.9% (range,
0.5%-96%), 23.9% (range, 0%-90%), and 31.8% (range,
1.4%-76.3%), respectively, with no significant difference between PB
and BM. Staining for the expression of lineage markers on
SP+ cells revealed a mean percentage of 0.6% (range,
0%-1.7%) Thy 1+, 0.6% (range, 0%-3.5%)
c-kit+, 44% (range, 16.5%-68%)
CD33+, 66% (range, 1.7%-93.5%) HLA-DR+, 50%
(range, 11%-83%) CD15+, 1.6% (range, 0.6%-2.5%)
GlyA+, 20% (range, 7.2%-39.3%) CD61+, and
24% (range, 2.1%-68.4) CD19+ cells. Within the SP, it was
also possible to detect cells with the aberrant co-expression of
lineage markers often seen on AML blasts a mean percentage of 23.2%
(range, 0%-71.4%) cells co-expressed CD34 and CD15, 5.5% (range,
0%-16.4%) co-expressed CD34 and CD33, and 30.8% (range, 0%-61.3%)
co-expressed CD34 and HLA-DR. On average, the expression of Thy 1 was
found on 0.2% (range, 0%-0.9%) and c-kit on 0.1% (range,
0%-0.6%) of the SP+CD34+ cells.
Multidrug resistance-1 expression on SP cells To investigate one possible reason for the relatively high proportion of SP+ cells in AML samples, multidrug resistance-1 (MDR-1) expression was measured by FACS using the UIC2-PE antibody. The mean frequency of MDR+ cells in the samples was 12.8% (range, 0%-54.8%). Within the side population, a mean of 8.3% of cells was positive for UIC2 (range, 0%-43.2%). There was no correlation between the frequency of SP+ cells and the frequency of MDR+ cells. In fact, 3 samples (numbers 9, 11, 15) in which no cells stained with the UIC2 antibody had easily detectable SP+ cells, and 1 sample (number 2) with a large fraction of UIC2+ cells in the total population showed no expression of MDR among SP+ cells (Table 2).Fluorescence in situ hybridization of sorted subpopulations Initial analysis of unsorted AML cells confirmed that the FISH probes selected could detect the abnormality diagnosed by conventional bone marrow cytogenetics in all patient samples. The proportion of abnormal cells detected by FISH varied from 6% to 100% among the unsorted samples and, in most cases, was similar to the proportion of metaphase cells found abnormal by conventional cytogenetics (Tables 1, 3). AML cells were sorted using Hoechst 33342 dye efflux and expression of CD34 and CD38 to isolate 3 subpopulations of interest. SP+CD34CD38 cells were studied
because, in the mouse, SP+ cells with long-term
hematopoietic reconstituting ability lack the expression of CD34 and
lineage markers.21 Hoechst dye efflux was used to further
characterize CD34+CD38 cells into
SP+CD34+CD38 and
SPCD34+CD38 fractions given
that the CD34+CD38 phenotype is known to
identify normal and AML cells that initiate long-term cultures and
growth in NOD/SCID mice. FISH analysis of the 3 sorted
subpopulations was successful with 15 patient samples (Table 3). In 14 of 15 samples, a mosaic of normal and leukemic cells was observed in
the SPCD34+CD38 subpopulation
whereas in the remaining patient (patient 9), exclusively abnormal
cells were detected. In contrast, only normal cells in the
SP+CD34+CD38 subpopulation were
found in 9 of 15 patients. An additional 3 patients (patients 9, 10, 12) had significantly smaller proportions of leukemic cells in the
SP+CD34+CD38 subpopulation than
among SPCD34+CD38 cells; in the
remaining 3 patients (patients 2, 5, 8), a similar frequency of
abnormal cells was found in both subpopulations. In all patients
analyzed, the SP+CD34CD38
subpopulation contained leukemic blasts. In 9 of 15 patients, a mosaic
of normal and leukemic cells was seen, and in 5 patients only leukemic
cells were detected by FISH. Interestingly, the latter 5 samples from
patients 1, 3, 11, 13, and 16 contained only normal cells in the
SP+34+38 subpopulation
(Table 3).
In vitro growth characteristics of SP cells Cells from the 3 FACS-sorted subpopulations from 10 patient samples were placed in CFC and LTC-IC assays. The number of cells plated varied from 50 to 10 000 cells/mL in the CFC and 85 to 7500 cells/mL in the LTC-IC assay, depending on the subpopulation frequency in the patient sample tested. In the CFC assay, colony growth was observed for 8 of 10 samples from the SP+CD34+CD38 subpopulation and 5 of 9 samples from the
SPCD34+CD38 subpopulation,
whereas only 1 of 10 samples (patient 7) had any colony growth from the
SP+CD34CD38 fraction. The
relative number of CFC per 1 × 106 unsorted cells ranged
from 0 to 1780 for SP+CD34+CD38
cells and from 0 to 15 960 for the
SPCD34+CD38 subpopulation.
Although most CFCs were found in the
SPCD34+CD38 fraction from
patients 2, 5, 7, and 8, for patients 1, 3, and 13 the CFCs were
predominantly SP+CD34+CD38 (Table
4).
LTC-IC activity was detected in 7 of 10 5-week-old cultures
LTC-ICs of SP+CD34+CD38 FISH analysis of CFC- and 5-week LTC-IC-derived colonies FISH analysis was performed on colonies plucked from direct CFC assays and colony assays from 5-week-old LTCs of FACS-sorted AML cells (Table 5). SP+CD34+CD38 CFCs from 6 of 8 samples, in which colony growth was observed and FISH analysis was
successful, were entirely normal by FISH. Similarly, LTC-IC assays of
SP+CD34+CD38 cells from 6 of 7 analyzable samples yielded colonies that were exclusively normal by
FISH. Only SP+CD34+CD38 cells
isolated from patient 2 generated CFCs and LTC-ICs that were entirely
abnormal by FISH, consistent with the finding that 100% of
SP+CD34+CD38 cells from this
patient, analyzed directly after FACS sorting, were also
cytogenetically abnormal (Table 3).
In contrast, among the 5 samples in which CFCs were detected among
SP Engraftment of NOD/SCID mice with sorted subpopulations of cells from patients with AML After Hoechst 33342 staining and FACS sorting, the SP+CD34+CD38,
SPCD34+CD38, and
SP+CD34CD38 subpopulations from
4 patients (patients 1, 4, 7, and 16) were transplanted into cohorts of
1 to 3 NOD/SCID mice. Engraftment was observed in 1 mouse at week 8 and
in 2 mice at week 12 after injection of the cells from patient 1 (Table
6). The proportion of human cells in
mouse bone marrow at week 8 was 0.77% in the mouse injected with
1 × 105
SP+CD34+CD38 cells. Among 20 000
cells from this mouse marrow analyzed by FACS, 15 cells co-expressed
CD45/CD71 and CD15/CD66b and 20 cells expressed CD19/CD20, consistent
with engraftment with both myeloid and B-lymphoid human cells. FISH
analysis of 258 sorted CD45+ cells from this animal
revealed only cytogenetically normal cells that had lymphoid morphology
on Wright-Giemsa staining. The 2 mice that showed engraftment at week
12 were injected with 1 × 105
SP+CD34+CD38 and
1 × 106
SPCD34+CD38 cells and showed
0.21% and 0.6% CD45+ cells in mouse marrow, respectively.
FISH analysis was normal for all 67 CD45+ cells analyzed
from the SP+CD34+CD38 mouse,
whereas 26 of 44 (55%) of CD45+ cells from the
SPCD34+CD38 mouse showed the
inv(16) rearrangement.
Among mice injected with cells from patient 7, one animal that
received 1 × 105
SP+CD34+CD38
One of the challenges in devising novel therapy for AML is to identify clear differences between normal and leukemic progenitor cells that can be targeted.26,27 It seems logical that the AML progenitors, identified by their ability to maintain long-term malignant hematopoiesis in vitro and in vivo (in immunodeficient mice), would be important targets for antileukemic therapy. Although cell-sorting studies have suggested much similarity between the immunophenotype of the most primitive normal and leukemic progenitors detected in these assays, some differences have been identified. For example, though most SCID leukemia-initiating cells and AML LTC-IC are similar to normal LTC-IC and NOD/SCID CRU in their expression of CD34 and their lack of expression of CD38, they often differ from many of these primitive normal cells in their lack of Thy-1 expression.1,4-6,10,28 However, among AML samples, variability in the success with which these antigens can be used to isolate and purify progenitor cell populations is frequently seen.11,16 In addition, evidence is accumulating to suggest that some markers, such as CD34, may not be as reliable in identifying primitive normal progenitors as was previously thought.19-21,29,30 Thus, it seemed relevant to examine the value of using new characteristics attributed to normal stem cells to study AML progenitors. We chose to analyze the side population of hematopoietic cells, identified by their efflux of Hoechst 33342, because little was known about the functional properties of this population in either normal or leukemic human hematopoiesis.21 Our first observation was that cells with the characteristics of SP cells, as defined by Goodell et al18 in the mouse, were easily identified in the marrow and peripheral blood of patients with newly diagnosed AML. Although the frequency of such cells varied greatly from sample to sample, in general it was much higher (4- to 500-fold) than that seen in normal mouse or human marrow samples.21 One possible explanation for the high frequency of SP+
cells we observed might be the overexpression of the multidrug
transporter protein, Pgp or MDR-1, in the AML blasts analyzed. High
levels of Pgp expression have been well-described in AML, both at the time of diagnosis and at disease recurrence.31-36 Efflux
of Hoechst 33342 is blocked by verapamil, which is known to inhibit the
Pgp transporter function. Thus, we reasoned that SP cells would
probably express high levels of this protein. However, in this study we were unable to demonstrate a correlation between the expression of Pgp
in AML cells (as detected by FACS analysis using the UIC2 antibody) and
the presence of SP cells in the same population. In fact, for several
patient samples, none of the isolated SP cells showed detectable
staining with the UIC2 antibody (Table 2). Although it is possible that
the flow cytometry technique was insufficiently sensitive to detect low
levels of Pgp expression Our second observation was that the side population detected in AML samples is heterogeneous with regard to the expression of other antigens expressed by primitive and mature hematopoietic cells. CD34, CD38, and various markers of lineage determination were expressed at different frequencies among the SP cells. Aberrant expression of antigens associated with hematopoietic differentiation is well recognized in AML and is often used for clinical monitoring of minimal residual disease.40-43 Our finding that aberrant or discordant antigen expression could be detected on the side population from AML samples is consistent with the fact that the SP population contained leukemic cells. However, expression of Thy-1 or c-kit among SP cells was infrequent (0%-0.9%), a finding in keeping with data from SP cells in normal bone marrow and AML progenitors detected in long-term suspension culture and in immunodeficient mice, both of which seem to lack these antigens.10,12,21 When SP+ cells were sorted according to their
expression of CD34 and CD38, CD34+CD38 Although we could not demonstrate the presence of LTC-IC or
NOD/SCID-repopulating cells in the SP+CD34
We thank Brigitte Gerhard for technical assistance, Gayle Thornbury, Giovanna Cameron, and Rick Zapf for FACS operation, and Christine Kelly for help with manuscript preparation.
Submitted November 22, 2000; accepted February 8, 2001.
Supported by grants from the National Cancer Institute of Canada with funds from the Terry Fox Run (D.E.H.) and by a grant from the Deutsche Krebshilfe, Bonn, Germany (M.F.-B.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: D. E. Hogge, Terry Fox Laboratory, British Columbia Cancer Agency, 601 West 10th Ave, Vancouver, British Columbia, Canada V5Z 1L3; e-mail: dhogge{at}bccancer.bc.ca.
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B. L. Abbott, A.-M. Colapietro, Y. Barnes, F. Marini, M. Andreeff, and B. P. Sorrentino Low levels of ABCG2 expression in adult AML blast samples Blood, December 15, 2002; 100(13): 4594 - 4601. [Abstract] [Full Text] [PDF]
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M. Feuring-Buske, A. E. Frankel, R. L. Alexander, B. Gerhard, and D. E. Hogge A Diphtheria Toxin-Interleukin 3 Fusion Protein Is Cytotoxic to Primitive Acute Myeloid Leukemia Progenitors But Spares Normal Progenitors Cancer Res., March 1, 2002; 62(6): 1730 - 1736. [Abstract] [Full Text] [PDF]
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progenitor
cells from patients with acute myeloid leukemia
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Abstract
Introduction