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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2901-2910
By
From the Department of Medical Biophysics, University of Toronto and
the Ontario Cancer Institute, Toronto, Ontario, Canada; the Department
of Oncologic Pathology, Princess Margaret Hospital, Toronto, Ontario,
Canada; the Autologous Blood and Marrow Transplantation Program,
University of Toronto and the Toronto Hospital, Toronto, Ontario,
Canada; the Samuel Lunenfeld Research Institute, Mount Sinai Hospital
(Toronto) and the Department of Pathology, Women's College Hospital,
Toronto, Ontario, Canada; and the Department of Oncology, University of
Alberta and the Cross Cancer Institute, Edmonton, Alberta,
Canada.
The ribosome-inactivating protein, Shiga-like toxin-1 (SLT-1),
targets cells that express the glycolipid globotriaosylceramide (CD77) on their surface. CD77 and/or SLT-1 binding was detected by flow
cytometry and immunocytochemistry on lymphoma and breast cancer cells
recovered from biopsies of primary human cancers as well as on B cells
or plasma cells present in blood/bone marrow samples of multiple
myeloma patients. Breast cancer cell lines also expressed receptors for
the toxin and were sensitive to SLT-1. Treatment of primary B lymphoma,
B-cell chronic lymphocytic leukemia, and myeloma B or
plasma cells with SLT-1-depleted malignant B cells by 3- to 28-fold,
as measured by flow cytometry. Depletion of myeloma plasma cells was
confirmed using a cellular limiting dilution assay followed by reverse
transcriptase-polymerase chain reaction analysis of
clonotypic IgH transcripts, which showed a greater than 3 log reduction
in clonotypic myeloma cells after SLT-1 treatment. Receptors for the
toxin were not detected on human CD34+ hematopoietic
progenitor cells (HPC). HPC were pretreated with a concentration of
SLT-1 known to purge primary malignant B cells and cultured for 6 days.
The number of HPC was comparable in toxin-treated and untreated
cultures. HPC were functionally intact as well. Colony-forming units
(CFU) were present at an identical frequency in untreated and SLT-1
pretreated cultures, confirming that CFU escape SLT-1 toxicity. The
results suggest the ex vivo use of SLT-1 in purging SLT-1
receptor-expressing malignant cells from autologous stem cell grafts of
breast cancer, lymphoma, and myeloma patients.
GENE MARKING STUDIES suggest that relapse
observed in cancer patients treated with high-dose chemotherapy and
autologous stem cell transplantation (ASCT) may reflect, at least in
part, the reinfusion of contaminating tumor cells in the stem cell
graft.1,2 In myeloma, recent experiments have demonstrated
that malignant progenitors from granulocyte colony-stimulating factor
(G-CSF)-mobilized blood transferred myeloma to immunodeficient mice as
measured by the presence of lytic bone lesions and polymerase chain
reaction (PCR)-detectable disease (Pilarski et al, manuscript
submitted). Significant improvements in disease-free
survival have been demonstrated for follicle center cell lymphoma
patients transplanted with autologous stem cell grafts purged free of
PCR-detectable tumor cells with anti-B-cell antibodies and rabbit
complement.3,4
A limited number of strategies for purging tumor cells from stem cell
grafts have been developed to date. Most approaches focus on the design
of protein fusion molecules or chemically derived toxin conjugates
incorporating a cytotoxic component derived from a protein toxin
covalently linked to a growth factor, cytokine, or antibody domain,
enabling the resulting constructs to target specific cancer cells. Such
constructs were initially created to cope with the broad receptor
specificity of native protein toxins. The bacterial toxin, Shiga-like
toxin 1 (SLT-1), is internalized by cells that express the glycolipid
receptor globotriaosylceramide (Gb3, CD77) on their
surface.5,6 Our group has recently shown that SLT-1 can
successfully purge a human Burkitt's lymphoma cell line from a murine
bone marrow (BM) graft ex vivo before transplantation in severe
combined immunodeficiency (SCID) mice.7 The potential use
of SLT-1, a ribosome-inactivating protein, is presently limited to an
ex vivo application, because its toxicity includes endothelial cell
damage and hemolytic uremic syndrome observed in patients after
gastrointestinal infections with SLT-1-producing strains of
Escherichia coli or Shiga toxin-producing strains of
Shigella dysenteriae.5,8-14 We now report the
expression of SLT-1 receptors on clinical tumor specimens and cell
lines of cancers that may benefit from autologous stem cell
transplantation and have addressed the survival of CD34+
cells after exposure to SLT-1. Our findings suggest that SLT-1 could be
used as an ex vivo purging agent for autologous grafts from patients
with 3 malignancies commonly treated with high-dose chemotherapy and
autologous stem cell transplantation, namely breast cancer, lymphoma,
and multiple myeloma.15,16
Cell lines, tissue sources, and culture conditions.
Human breast cancer cell lines were obtained from the American Type
Culture Collection (Rockville, MD) and from the laboratories of Dr Ron
Buick (Ontario Cancer Institute, Toronto, Ontario, Canada) and Dr Irene
Andrulis (Lunenfeld Research Institute, Toronto, Ontario, Canada). The
MDA-MB series of cells were cultured at 37°C in Leibovitz L-15
medium in the absence of CO2, whereas the other breast
cancer cell lines were cultured in Monoclonal antibodies (MoAbs).
Antibodies used for immunocytochemistry were bought from the following
sources: anti-CD10 (Dako, Glostrup, Denmark), biotinylated mouse
antirat IgM, unconjugated rat IgM anti-CD7718 and
anti-B-B4 antibodies (Serotec, Oxford, UK),19 anti-CD13
(Coulter, Burlington, Ontario, Canada), AE1/AE3 anti-cytokeratin
(Zymed, South San Francisco, CA), anti-low molecular weight keratins
(Cam 5.2; Becton Dickinson, Franklin Lakes, NJ). The antiepithelial
cancer antibody Ber-EP420 was purchased from Dako. ONC-M38,
a hybridoma producing a murine antimucin antibody,21 was
from Dr Peter Linsley (Bristol-Myers Squibb, Seattle, WA). M38 antibody
was purified from culture supernatants by protein G affinity
chromatography. MoAb 17G10-phycoerythrin (PE) was used to
detect CD45.22 Anti-CD19 MoAb (FMC63) was coupled to PE or
fluorescein isothiocyanate (FITC) and used to detect B
cells in lymphoma biopsies and myeloma MNC as previously
described.23,24 Plasma cells in myeloma BM were identified
using an anti-CD38-PE conjugate (Leu17; Becton Dickinson) and based on
their light scattering properties.25 Hematopoietic
progenitor cells (HPC) were detected by staining the cell population
with a combination of anti-CD34-PE (8G12-PE; Becton Dickinson) and
anti-CD45-Quantum red (Sigma) conjugates and gating for the
CD34+45lo subset, as previously
described.26
Toxin purification and labeling.
SLT-1 was initially purified to greater than 95% purity by column
chromatography from a transformed E coli strain, as described elsewhere.27 The binding subunit, SLT-B, was purified to
homogeneity, as described previously.28 It was labeled with
FITC, as stated elsewhere.7
SLT-1 cytotoxicity assays for breast cancer cell lines.
The toxicity of SLT-1 towards adherent cell lines was measured using
the sulforhodamine B (SRB) dye binding assay.29 Briefly, cells cultured in 96-well plates were exposed to a range of toxin concentrations for a 1-hour period in phosphate-buffered saline (PBS). The toxin-containing solution was subsequently
diluted with the appropriate medium containing FCS and the treated
cells were cultured for another 48 hours. The medium was removed and the remaining adherent cells were fixed with ice-cold 10%
trichloroacetic acid, air-dried, and stained with 0.4% SRB (Molecular
Probes, Eugene, OR) dissolved in 1% acetic acid. The excess dye was
washed away, and the remaining bound SRB dye was extracted from the
cells with 10 mmol/L Tris base. The absorbance of the dye was read at 540 nm using a plate reader.
Flow cytometry and immunocytochemistry.
The expression of SLT-1 receptors was detected by flow cytometry using
FITC-labeled SLT-B subunit (FITC-SLT-B) and by immunocytochemistry with
an unconjugated anti-CD77 MoAb. Flow cytometry of human lymphoma samples was performed as previously described.7 Flow
cytometric data were acquired from human breast cancer aspirates or
cells recovered from minced tumor samples stained with antibodies to surface antigens (CD77, Ber-EP4, and M38) and viability dyes. A
cocktail of both Ber-EP4 and M38 antibodies was used for flow cytometry
of breast cancer biopsies because of the limited recovery of cells.
Cytospins of lymphoma and breast cancer cells were immunostained with
anticytokeratin antibodies (cam 5.2 [Becton Dickinson] and AE1/AE3
[Zymed]). All immunocytochemistry was accomplished on paraformaldehyde-fixed samples.
Purging of ex vivo malignant B cells.
MNC derived from lymphoma biopsies or from blood/BM samples of myeloma
patients were treated with 5 µg/mL SLT-1 for 60 minutes at 37°C,
washed to remove unbound toxin, and cultured for 6 to 10 days in RPMI
supplemented with 10% FCS. Cultured cells were harvested, and the
total number of viable cells was counted and stained with FITC-labeled
anti-CD19 as well as with propidium iodide (PI) to identify viable
cells. The absolute number of viable CD19+ B cells
remaining in the cultures was calculated as the percentage of viable
CD19+ cells (CD19+PIlow) multiplied
by the total number of viable cells. Plasma cells were stained with
FITC-labeled anti-CD38 MoAb as well as PI.
Quantitation of clonotypic cells in myeloma.
To confirm the purging of malignant plasma cells from myeloma BM cells,
a cellular limiting dilution reverse transcriptase-PCR (RT-PCR) assay24 was performed to detect the
presence of clonotypic IgH VDJ rearrangements using patient-specific
primers. Briefly, graded numbers of untreated or SLT-1-pretreated BM
cells were deposited into PCR tubes containing lysis buffer. RNA from
the lysed cells was reverse transcribed and the resulting cDNA was amplified in the same tube. The patient-specific IgH VDJ sequence was
identified from single plasma cells shown to be specific for this
patient and expressed by greater than 80% of individual plasma cells
recovered for this patient, confirming that the myeloma-specific Ig
sequence had been identified.24
Short-term culture of HPC.
Mononuclear cells from G-CSF-mobilized blood were pretreated with 5 µg/mL of SLT-1 for 60 minutes at 37°C, washed, and cultured in
commercial long-term culture medium (Myelocult plus 10% Hemostim; Stem
Cell Technologies, Vancouver, British Columbia, Canada) for 7 to 10 days. Harvested cells from paired untreated and SLT-1-pretreated cultures were stained with anti-CD34-PE and anti-CD45-QR conjugates. The percentage of HPC
(CD34+45loScatterlow/med) was
determined as previously described.26
Assay for colony-forming units (CFU).
MNC from G-CSF-mobilized blood samples were enriched for HPC by
negative selection according to instructions from the manufacturer (StemSep; Stem Cell Technologies). Enriched HPC (40% to 60% HPC) were
then treated or not with 5 µg/mL of SLT-1 for 60 minutes at 37°C,
washed once, and cultured to monitor the number of CFU. Increasing
numbers of toxin-treated or untreated cells (25 to 200 cells per well)
were added to a mixture of growth factors and methyl cellulose
(Methylcult; Stem Cell Technologies) according to the instructions from
the manufacturer and deposited into culture wells. The number of
colonies was counted under a microscope after 20 days, as recommended
by the manufacturer. CFU values reported represent the average of
colony numbers observed in 6 to 12 replicate wells.
Expression of SLT-1 receptor on breast cancer cell lines and SLT-1
cytotoxicity.
The expression of SLT-1 receptors on 18 breast cancer cell lines was
analyzed by flow cytometry using FITC-SLT-B. Two antibodies were used
to label breast cells: Ber-EP4, which recognizes an unknown antigen on
epithelial-derived carcinomas,20 and M38,21 which detects the epitope MUC1 on breast-derived mucin.30
Of the 18 breast cancer cell lines studied, 5 had less than 15%
positivity for FITC-SLT-B, whereas 13 (72%) expressed surface SLT-1
receptors (Table 1). Sixteen of 18 (89%)
breast cancer cell lines were positive for M38 (MUC1) and 12 of 18 (67%) were positive for Ber-EP4. The results demonstrate the frequent
expression of SLT-1 receptors on the surface of breast cancer cell
lines. The level of expression of SLT-1 receptors on breast cancer cell
lines correlates with their sensitivity to killing by SLT-1
(Fig 1 and Table 1, CD50 values).
Expression of SLT-1 receptors on breast cancer biopsies.
The expression of SLT-1 receptors on biopsies of primary human breast
cancers obtained from 10 patients was analyzed by flow cytometry and
immunocytochemistry. Cell suspensions were prepared from fine-needle
aspirates or by mechanical disaggregation of solid tumors. Eight of 10 samples (80%) showed greater than 15% positive staining for
FITC-SLT-B (Table 2) and the intensity of
fluorescence staining was high (Fig
2C). In contrast to the established breast cancer cell
lines, Ber-EP4/M38 was expressed on 4 of 10 samples, all 4 of which
were positive for SLT-1 receptors (SLT-R). Four of 8 SLT-R+
samples were negative for the breast cancer markers (Ber-EP4 and M38).
SLT-1 receptor-positive cells were confirmed as breast cancer cells by
morphological examination and immunocytochemistry with anti-low
molecular weight cytokeratin and anti-CD77 antibodies (Fig 3A and B).
Expression of SLT-1 receptor on lymphoma and myeloma.
Clinical samples from patients with hematological cancers were
routinely screened with FITC-SLT-B to monitor the expression of SLT-1
receptors. Flow cytometry data were collected on 134 sequential samples
(Fig 4) classified according to the REAL
system.31 On average, 3% cells (±4%) recovered from
nonmalignant samples stained with the fluorescent probe (results not
shown). Clinical samples were scored as positive when at least 15% of
cells (3 SD above the mean background) bound FITC-SLT-B.7
SLT-1 receptors were frequently expressed on follicle center cell
lymphoma grades I, II, and III (Fig 4), with 31 of 43 (72%)
patient samples positive. These results agree with the expression of
CD77 on normal follicle center cells.32-37 Thirty-three
percent (15 of 46 samples) of small lymphocytic lymphomas with or
without chronic lymphocytic leukemia were positive for FITC-SLT-B
staining, as were 42% (5 of 12 samples) of large B-cell lymphomas.
FITC-SLT-B staining was not observed on mantle cell lymphomas, which
are thought to arise from the normally CD77
Hematopoietic progenitor cells do not bind SLT.
HPC are phenotypically defined as CD34+45lo
Scatterlow/medium cells.26,40,41 Hematopoietic
progenitor cells from G-CSF-mobilized blood MNC were stained in
multicolor immunofluorescence to determine SLT-1 binding. A
representative flow cytometry plot is shown in Fig 5 for unfractionated
G-CSF-mobilized MNC (Fig 5C) and the same mobilized blood
MNC gated for the HPC subset, showing that normal HPC (indicated by
arrows in Fig 5C and D) do not bind SLT-1. Similar results were
obtained for HPC from 4 different mobilized blood samples.
SLT-1 purging depletes malignant cells from lymphoma, B-CLL, and
myeloma tissues.
MNC from lymphoma node biopsies or blood/BM from myeloma patients were
treated in vitro with SLT-1 to evaluate its ability to purge malignant
cells recovered from these tissues. Unlike cell lines that, after
exposure to 1 µg/mL or less of toxin, die within 2 to 3 days,7 the toxic effects of SLT-1 on freshly cultured cells
required relatively high concentrations of toxin (5 µg/mL) and were
detectable only after 6 to 10 days in culture (not shown). For 2 lymphoma biopsies, MNC were pretreated with 5 µg/mL SLT-1 and
cultured, and the number of phenotypically defined B cells was
enumerated using flow cytometry. Figure 6
(top panel) shows that the absolute number of lymphoma B cells was
reduced by 3- to 6-fold after pretreatment with SLT-1. For 1 patient
with B-CLL, B cells were reduced 28× after pretreatment with
SLT-1 (Fig 6, top panel). For B cells from myeloma blood, a similar reduction in total B cells was detected after SLT-1 pretreatment for 2 patients with the high numbers of B cells characteristic of myeloma
patients (Fig 6, bottom panel). However, for 1 myeloma patient who had
recovered from autologous transplant and had no detectable circulating
clonotypic cells (not shown; MMB 3, Fig 6, bottom panel), there was
only a marginal reduction in B cells after SLT-1 pretreatment,
suggesting that only malignant B cells are targeted by this strategy.
For myeloma BM, pretreatment with SLT-1 efficiently depleted the
majority of plasma cells by 10- to 23-fold as measured phenotypically.
Molecular analysis confirms a greater than 3 log reduction in
clonotypic myeloma cells after SLT-1 pretreatment.
One complicating factor in evaluating the extent of purging in the
experiments cited above is the presence of residual normal B lineage
cells that may resist the action of the toxin. Because phenotypic
analysis does not reliably distinguish between normal and malignant B
or plasma cells, we evaluated the extent of purging using a novel
molecular assay for clonotypic transcripts. Myeloma cells are uniquely
identified by the IgH VDJ rearrangement that characterizes the
malignant clone in each patient. Untreated or SLT-1 pretreated BM cells
from MM patient no. 4 were deposited at limiting dilution into lysis
buffer in PCR tubes, followed by RT-PCR using patient-specific primers.
Figure 7 shows that, for the untreated
cultures, by day 6, essentially all cells were clonotypic (3 of 3 tubes
were positive at the 1 cell/tube concentration). However, for the
SLT-1-treated cultures, clonotypic transcripts were undetectable even
in the tubes containing 1,000 cells per tube. The frequency of
clonotypic cells remaining after SLT-1 pretreatment was less than
1/3,000 cells in light of the fact that all 3 tube replicates were
negative from the presence of transcripts (for a total of 3,000 cells
analyzed).
HPC and CFU from G-CSF-mobilized blood escape SLT-1 toxicity.
To be effective, a purging agent must spare normal hematopoietic
progenitor cells. To evaluate the effects of SLT-1 treatment on
progenitor cells and their functional activity, we analyzed the
survival of HPC in toxin-pretreated cultures and of cells able to give
rise to hematopoietic colonies in vitro (CFU). These 2 approaches
represent surrogate assays for engraftment potential. Table 3A shows that, when MNC or
HPC-enriched MNC are pretreated with 5 µg/mL of SLT-1, a
concentration shown above to effectively purge malignant cells,
phenotypically identified HPC are present in equivalent numbers in
untreated or SLT-1-treated cultures at days 6 to 7 posttreatment. For
the HPC-enriched MNC population derived from mobilized blood, HPC were
more frequent in SLT-1-treated than in untreated cultures (Table 3,
analysis of HPC-enriched CD34+45lo
populations), probably reflecting a further enrichment of the toxin-resistant HPC as SLT-1-sensitive cells died. These experiments indicate that the HPC population as a whole survives SLT-1
pretreatment, but does not address the functional capability of these
HPC.
Shiga-like toxin 1, a ribosomal-inactivating protein produced by
pathogenic strains of E coli (eg, O157:H7), intoxicates cells expressing globotriaosylceramide (CD77, Gb3) on their
surface. It has recently been demonstrated that ex vivo treatment of
murine BM with SLT-1 can effectively purge SCID mice of a B-cell
lymphoma xenograft while sparing normal murine hematopoietic precursor cells, suggesting its potential use as an ex vivo purging
agent.7 This study demonstrates the expression of
Shiga-like toxin 1 receptors on 3 of the human cancers commonly treated
by autologous stem cell transplant (breast cancer, lymphoma, and
multiple myeloma) and proves the absence of SLT-1 receptor on human
CD34+ stem cells. Taken together, this evidence supports
the clinical use of SLT-1 as an ex vivo purging agent to deplete
malignant cells expressing receptors for SLT-1 from autologous stem
cell grafts.
The authors thank Papar Laraya, Rose Hurren, Marijke Koekebakker,
Lori-Ann Webster, James Ho, Juliet Sheldon, and Denis Bouchard for
their technical help; Dr Hans Messner and Nazir Jamal for providing
peripheral blood stem cells; and Drs Brent Zanke and Heather Lochnan
for their helpful comments. In Edmonton, Eva Pruski provided
outstanding technical assistance, Dr Robert Coupland provided biopsies
of B-lymphoma nodes, and the Red Cross Apheresis Unit provided aliquots
of mobilized blood. We thank Agnieszka J. Szczepek for sequencing the
RT-PCR products shown in Fig 8. This work is dedicated to the memory of
Dr Ron Buick (1948-1996) for all his encouragement, advice, and support
over the years.
Submitted September 30, 1998; accepted May 30, 1999.
Supported by a translational research grant to J.G. from the Leukemia
Society of America and by a grant from the Medical Research Council of
Canada to L.M.P. and A.R.B. E.C.L. was the recipient of a fellowship
from the Medical Research Council of Canada.
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 Jean Gariépy, PhD, Department of
Medical Biophysics, Room 7-117, Ontario Cancer Institute/Princess
Margaret Hospital, 610 University Ave, Toronto, ON, Canada, M5G 2M9.
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TOP
ABSTRACT
INTRODUCTION
-minimal essential medium
(
),
JS-1 (
), MCF-7 (
), MDA-MB-231 (
), MDA-MB-468 (
), MDA-MB-469
(
), SKBR3 (
), and T47D (
).
B cells
in the follicular mantle zone. Marginal zone lymphomas were also
CD77
