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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 746-755
Proliferation and Survival of Mammary Carcinoma Cells Are Influenced
by Culture Conditions Used for Ex Vivo Expansion of CD34+
Blood Progenitor Cells
By
A. Spyridonidis,
W. Bernhardt,
D. Behringer,
G. Köhler,
M. Azemar,
A. Pflug, and
R. Henschler
From the Experimental Hematology Group, the Department of Hematology,
and the Department of Pathology, Freiburg University Medical Center,
Freiburg, Germany; and the Institute for Experimental Cancer Research,
Tumor Biology Center, Freiburg, Germany.
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ABSTRACT |
Malignant cell contamination in autologous transplants is a
potential origin of tumor relapse. Ex vivo expansion of
CD34+ blood progenitor cells (BPC) has been proposed as a
tool to eliminate tumor cells from autografts. To characterize the
influence of culture conditions on survival, growth, and clonogenicity
of malignant cells, we isolated primary mammary carcinoma cells from
pleural effusions and ascites of patients with metastatic breast cancer and cultured them in the presence of stem cell factor (SCF),
interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (EPO), ie,
conditions previously shown to allow efficient ex vivo expansion of
CD34+ BPC. In the presence of serum, tumor cells
proliferated during a 7-day culture period and no significant
growth-modulatory effect was attributable to the presence of
hematopoietic growth factors. When transforming growth factor-1
(TGF-1) was added to these cultures, proliferation of
breast cancer cells was reduced. Expansion of clonogenic tumor cells
was seen in the presence of SCF + IL-1 + IL-3 + IL-6 + EPO,
but was suppressed by TGF-1. Cocultures of tumor cells in direct
cellular contact with hematopoietic cells showed that tumor cell growth
could be stimulated by ex vivo expanded hematopoietic cells at high
cell densities (5 × 105/mL). In contrast, culture under
serum-free conditions resulted in death of greater than 90% of breast
cancer cells within 7 days and a further decrease in tumor cell numbers
thereafter. In the serum-free cultures, hematopoietic cytokines and
cellular contact with CD34+ BPC could not protect the
tumor cells from death. Therefore, ex vivo expansion of
CD34+ BPC in serum-free medium provides an environment
for efficient purging of contaminating mammary carcinoma cells. These
results have clinical significance for future protocols in autologous progenitor cell transplantation in cancer patients.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IN RECENT YEARS, high-dose chemotherapy
followed by autologous hematopoietic stem cell transplantation has been
increasingly used for the treatment of breast cancer.1
Initial results from clinical trials using high-dose chemotherapy
protocols to treat breast cancer patients have suggested an increased
therapeutic benefit compared with conventional chemotherapy regimens,
including improved response rates and better survival.2,3
Because contamination of the autologous grafts with tumor cells does
occur,4,5 and may be of clinical
significance,6-11 attempts have been made to deplete the
tumor cells within autologous grafts by manipulation of the harvests ex
vivo.
One possibility to purge autologous grafts is antibody-mediated
selection of hematopoietic progenitor cells, eg, enrichment for
CD34+ progenitor cells. Yet, the reported clinical trials
using currently available CD34+ selection technology have
shown that it is difficult to achieve CD34+ cell purities
of greater than 90%, which are equivalent to an expected 2 log
depletion of breast cancer cells within the graft.12 Contaminating breast cancer cells have indeed been detected in positively selected CD34+ fractions from both bone
marrow12 and mobilized peripheral blood13 (and
own unpublished results). Ex vivo expansion of blood
progenitor cells has been proposed as an alternative or additional
tumor cell purging strategy, if it selectively favors hematopoietic
cell survival and expansion in culture conditions adverse for malignant
cells. A number of studies have defined optimal culture conditions to
generate increased numbers of progenitor cells from CD34+
enriched cells.14,15 Hematopoietic progenitor cells
expanded ex vivo in liquid cultures in the presence of cytokines have
so far been used to initiate long-term bone marrow
cultures,15,16 to repopulate the bone marrow of lethally
irradiated mice,17 and to accelerate hematopoietic
reconstitution in humans after myelosuppressive
chemotherapy.18
Ex vivo expansion of CD34+ hematopoietic cells could be
used as a means of tumor cell purging only if contaminating tumor cells do not coexpand in the ex vivo expansion systems. Although some studies
have suggested the absence of detectable tumor cells in the ex vivo
expanded hematopoietic cell collections, the question of whether tumor
cells survive or proliferate in the cultures could not be formally
answered in some previous studies, because the CD34+
starting cell populations were not screened19 or were
negative20 for tumor cell contamination. In contrast, Purdy
et al21 reported the possibility that malignant cells may
persist after ex vivo culture. Also, breast cancer cells contained in
harvests of bone marrow or mobilized peripheral blood have been found
to be viable and to possess the capacity for clonogenic growth in
vitro.4,22,23 We hypothesized that culture conditions may
alter the fate of tumor cells in ex vivo expansion cultures and we
therefore analyzed the influence of ex vivo expansion parameters (ie,
cytokines, culture medium, and hematopoietic cells) on the survival,
growth, and clonogenicity of primary breast cancer cells ex vivo.
Breast cancer cells were found not to be susceptible to growth
stimulation by hematopoietic cytokines used to ex vivo expand
CD34+ blood progenitor cells, but a tumor cell-suppressive
effect was obtained when using serum-free medium preparations. These
data have implications for future ex vivo expansion protocols used in
patients with mammary carcinoma.
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MATERIALS AND METHODS |
Primary breast cancer cells.
Tumor cells were derived from pleural effusions or ascites from
patients with disseminated stage of metastatic breast cancer ( 3 sites
involved). Primary tumors were histologically classified as
infiltrating ductal carcinomas and nuclear grading was II-III in all
patients. All patients had received previous chemotherapy, but no
chemotherapy had been administered throughout the last 3 months before
the specimens were obtained. Cells from the malignant effusions were
sedimented by centrifugation at 400g for 6 minutes. An aliquot
was processed for immunocytological analyses (specified below) to
assess the malignant cell content of the patient samples. Cells were
seeded into T175 flasks (Falcon; Becton Dickinson, Heidelberg,
Germany) in  -Minimal Essential Medium (-MEM; GIBCO BRL, Paisley, Scotland) supplemented with 10% fetal calf serum (FCS;
Pan Systems, Passau, Germany) at a density of 1 to 5 × 106/mL. In later experiments (shown in Fig 5), cells were
seeded directly after sedimentation into the respective assays at a
density of 0.1 to 1 × 106/mL. When reaching confluency,
cells were detached from the culture surface with
trypsin-ethylenediaminetetraacetic acid (EDTA) solution (Sigma, Munich,
Germany), washed twice with phosphate-buffered saline (PBS;
BioWhittaker, Brussels, Belgium), counted, and either reseeded in
culture medium or resuspended in HEPES (hydroxyethyl-piperazine-ethane sulfonic acid)-buffered saline (HBS) for subsequent immunocytologic staining with anticytokeratin, antiepithelial-specific, and
antimesothelial-specific monoclonal antibodies (MoAbs; see below). In
some experiments, both adherent and nonadherent cells were passaged as
indicated in the respective figure legends. In all other experiments,
only the adherent cell populations were subcultured. The tumor cell content of the successfully established cultures (termed samples 1 through 3) was documented serially by immunocytochemistry. Initially, the content of CK+/Ber-Ep4+ cells was
heterogeneous (content of CK+ cells for samples 1 through
6: 10.6%, 9.6%, 79.2%, 0.24%, 11.3%, and 10.2%, respectively;
contents of BER-Ep4+ cells for samples 1 through 6: 9.4%,
8.5%, and 80.7%), respectively. Stock cultures of
greater than 95% tumor cells (CK+/Ber-Ep4+
cells) were used as a source of tumor cells for proliferation and
clonogenic assays. Population doubling times were calculated from the
cell numbers determined at a given time point and a second time point
between 4 and 7 days thereafter before the cultures reached confluency.
Media and growth factors.
Serum-free culture medium (SFM) containing Iscove's Modified
Dulbecco's Medium (IMDM; BioWhittaker), bovine serum albumin (Boehringer Mannheim, Mannheim, Germany; 2%), cholesterol
(Sigma; 2 × 10 6 mol/L), -mercaptoethanol (Serva
Heidelberg, Germany; 5 × 105 mol/L),
and insulin (Sigma; 0.01 mg/mL) was prepared using a protocol
originally developed for clonal hematopoietic culture assays.24,25 Suppliers of recombinant growth factors and
concentrations used were: stem cell factor (SCF; Genzyme, Cambridge,
MA; 10 ng/mL), interleukin-1 (IL-1; Genzyme; 3 ng/mL), IL-3
(Genzyme; 100 ng/mL), IL-6 (Genzyme; 100 ng/mL), erythropoietin (EPO;
Erypo 4000; Cilag, Sulzbach, Germany; 1 U/mL); transforming growth
factor-1 (TGF-1; R&D Systems, Wiesbaden, Germany; 30 ng/mL), epidermal growth factor (EGF; Genzyme; 1 µg/mL), and
insulin-like growth factor (IGF; Genzyme; 0.1 µg/mL). Long-term bone
marrow culture (LTBMC) medium consisted of IMDM, 10% horse serum
(Sigma), 10% fetal calf serum (Pan Systems), and 5 × 107 mol/L hydrocortisone (Sigma).
Tumor cell proliferation assays.
Tumor cells from the stock cultures were trypsinized and seeded into
24-well plates (Falcon) in FCS-supplemented -MEM medium at 10,000 cells/cm2 and various concentrations of added growth
factors. After 7 days, cells were trypsinized for 10 minutes at 37°C
and cell counts were determined in a hemocytometer. Cell viability was
greater than 95% by trypan blue exclusion. Aliquots were also
processed for immunocytological analysis to document the tumor cell
origin of the cells (see below).
Tumor cell colony assay.
Tumor cells from stock cultures were trypsinized, washed twice, and
plated in duplicates in 35-mm Petri dishes
(1 × 104/dish; Nunc, Wiesbadem, Germany)
in a semisolid medium consisting of 55% LTBMC medium, 45% of a 2.1%
methylcellulose solution in IMDM (WAK-Chemie, Bad Homburg,
Germany), 1 µg/mL human recombinant EGF, and 0.1 µg/mL human
recombinant IGF. All cultures were evaluated light-microscopically and
contained single-cell suspensions after plating. Initial seeding of
tumor cell aggregates was never recorded. Dishes were incubated at
37°C in 5% CO2 and colonies consisting of more than 30 cells were scored under a light microscope on days 17 to 20 after
plating. Cultures without supplemental growth factors (EGF and IGF) did
not give rise to tumor cell colonies and were used as negative
controls. Colonies from each dish were picked with a Pasteur pipette,
diluted in 100 µL of PBS, and attached onto glass slides by
cytocentrifugation (Cytospin 3; Shandon, Runcorn, UK). Slides were
immunostained with fluorescein isothiocyanate (FITC)-anticytokeratin
MoAbs (see below) to determine the epithelial cell origin of the
colonies. Absolute numbers of clonogenic tumor cells per culture were
calculated by multiplying the clonogenicity of tumor cells with the
number of total cells in the cultures.
Immunocytological detection of tumor cells.
A mixture of two IgG1 anticytokeratin antibodies (AE1/AE3 [Boehringer
Mannheim] and KL1 [Dianova, Hamburg, Germany]), an anticytokeratin antibody (MNF116; DAKO, Hamburg, Germany) that specifically recognizes members of the acidic as well of the basic subfamily of cytokeratins, the antiepithelial-specific Ber-Ep4 antibody (DAKO, Glastrup, Denmark) that recognizes two glycoproteins present on
epithelial cells but not mesothelial cells,26 and a
mesothelial-specific anticalretinin antibody (Zymed Laboratories, San
Francisco, CA) were used to detect mammary carcinoma cells and
distinguish them from mesothelial or hematopoietic cell populations.
For staining with AE1/AE3 (1:50 dilution) + KL-1 (1:200 dilution) and
with Ber-Ep4 (1:40 dilution) antibodies, cells were washed twice in HBS, attached in duplicate to poly-L-lysine-coated adhesion slides (Bio-Rad, Munich, Germany) consisting of 12 spots of 5-mm diameter, and
dried on the spots according to the manufacturer's recommendations. Slides were stored in sealed plastic bags at  20°C until use. Cells
were fixed and permeabilized in serial dilutions of acetone in ethanol
for 5 minutes at 4°C, incubated with the MoAbs, and immunostained in
an alkaline phosphatase-antialkaline phosphatase assay using the APAAP
assay (DAKO). MCF-7 mammary carcinoma cells obtained from the American
Type Culture Collection (ATCC; Rockville, MD) were used
as a positive control in each assay, and an IgG1 isotype antibody
served as a negative control. Staining with these antibodies of
peripheral blood mononuclear cells, of bone marrow cells, or of
CD34+ blood progenitor cell populations from patients with
hematologic malignancies never gave positive results. Regularly, a
total of 2,000 cells per spot was examined microscopically (at least
1,700), and the incidence of cells staining bright red was determined. Absolute numbers of tumor cells per culture were calculated from the
incidence of immunocytologically detected tumor cells and the total
cell counts within a culture at the end of the culture period. For
immunostaining with the MNF116 anticytokeratin (1:700 dilution) and the
anticalretinin (1:1 dilution) antibodies, cells were formalin-fixed and
paraffin-embedded. Cytokeratin staining was performed after digestion
for 10 minutes with 0.5% proteinase K (Sigma-Aldrich GmbH, Munich,
Germany) using the avidin-biotin-complex method
(Vectorstain-Kit; Vector, Burlingame, CA). Calretinin staining was
performed after heat-induced epitope retrieval according to the
manufacturer's instructions. For immunfluorescence staining of
colonies in the tumor colony assay, several colonies per dish were
randomly selected, aspirated through a 100-µL pipette tip, resuspended in 100 µL PBS, spun onto glass slides using a
cytocentrifuge, and fixed in acetone for 5 minutes. Immunostaining was
performed for 1 hour at 4°C in a moist chamber using an
FITC-conjugate of an anticytokeratin IgG1 (Cytokeratin-FITC, clone CAM
5.2; Becton Dickinson). After washing in PBS for 5 minutes, the slides
were covered with a mixture of propidium iodide (Oncor, Gaithersburg, MD) and mounting medium (Vectashild; Vector) and examined under a
fluorescence microscope (Axiophot; Zeiss, Stuttgart,
Germany). The nuclei of both cytokeratin-positive and
-negative cells stained red, and only cells with a bright green
cytoplasm were scored as cytokeratin-positive. No staining with
anticytokeratin MoAbs was observed in hematopoietic colonies derived
from CD34+ blood progenitor clonogenic assays.
Detection of ErbB2-receptor expression by immunoblotting.
Western blot analyses of erbB2-protein expression were performed as
previously described.27 Briefly, extracts from 2.5 to 5 × 105 patient cells (except sample 3, 2.5 × 106 cells) and from 5 to 7 × 105
SKBR3 or A431 cells (ATCC) were subjected to a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were
electroblotted onto nylon membranes. The p185ErbB2 proteins were
detected using the 21N antisera and an enhanced chemiluminescence detection kit (Amersham, Braunschweig, Germany) as described.
Coculture of CD34+ blood progenitor cells and
primary mammary carcinoma cells.
CD34+ hematopoietic cells were obtained from patients with
solid tumors or hematologic malignancies who underwent blood progenitor cell (BPC) mobilization by leukapheresis after informed consent. For
the experiments using previously expanded hematopoietic cells, ex vivo
expansion of CD34+ cells was performed in 24-well plates
and 1 mL culture medium at 37°C and 5% CO2 in a
humidified atmosphere using IMDM (BioWhittaker) containing FCS or SFM
and 10 ng/mL SCF, 3 ng/mL IL-1, 100 ng/mL IL-3, 100 ng/mL IL-6, and
1 U/mL EPO as described.24,25 Cells proliferated
exponentially and reached a 8.8- to 10.2-fold increase in cell numbers
by day 7 and a 20- to 80-fold increase by day 14. After 14 days,
differentiated granulocytic and monocytic/macrophage cells predominated
in the cultures. Fresh or 14-day expanded CD34+
hematopoietic cells were cocultured for 3 days with 10 fluorescence-labeled tumor cells (see below) in 96-well flat-bottom
plates (Becton Dickinson) or for 7 days with unlabeled mammary
carcinoma cells at a ratio 1:10 in 3-mL slide flasks (Nunc, Wiesbaden,
Germany). Cocultures were preformed in the presence of
SCF, IL-1, IL-3, IL-6, and EPO. Cell viability under these
conditions remained greater than 80%. During the first 72 hours, no
significant increase of cell numbers was recorded. For tumor cell
labeling, the fluorescent dye PKH-26 (Sigma) was used according to the
manufacturer's instructions. Briefly, 105 tumor cells were
washed two times in IMDM (BioWhittaker), and the pellet was suspended
in 600 µL diluent buffer. One microliter of PKH-26 solution
(103 mol/L) prediluted into 100 µL diluent buffer was
added, and the mixture was left at room temperature for 3 minutes with
gentle agitation for several times. The labeling reaction was stopped by the addition of 1 mL heat-inactivated FCS (Pan Systems) for 1 minute. This mixture was then carefully underlayered with 1 mL
heat-inactivated FCS. Cells were cytocentrifuged for 5 minutes at
400g, the supernatant was removed, and the pellet was washed three times in serum-containing medium (IMDM/10% FCS) in a fresh tube
for every washing step. Finally, cells were taken up in culture medium,
counted, and used for the assays. The whole cultures of CD34+ cells and tumor cells were examined under a
fluorescence microscope, and cells displaying a red fluorescence were
scored positive. In the experiments performed for 7 days, both adherent
and nonadherent cells were collected, fixed on adhesion slides, and
stained with anticytokeratin and Ber-Ep4 antibodies as described above
to determine the numbers of tumor cells.
Statistical analyses.
For statistical analysis, data for the control groups were normalized
to 1, and the groups were compared using a two-tailed Student's
t-test. A P value less than .05 was considered
significant.
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RESULTS |
Isolation and enrichment of primary breast cancer cells.
Cells isolated from serous fluids (ascites or pleural effusions) of
patients with metastatic breast cancer were cultured in FCS-supplemented -MEM. In 6 of 11 specimens obtained from different patients, an outgrowth of breast carcinoma cells as identified by
positive immunostaining with anticytokeratin (CK) and Ber-Ep4 antibodies and negative immunostaining with anticalretinin antibodies was observed. The 6 samples that yielded growing mammary carcinoma cells initially presented with a very heterogenous content of CK-positive cells (0.2% to 79%). CK-negative cells consisted mostly of erythrocytes, macrophages, and lymphocytes, as evaluated through Wright-Giemsa-stained cytospin preparations. Growth kinetics of both
CK-positive and CK-negative cells were monitored by staining aliquots
with anti-CK antibodies. The absolute numbers of CK-positive cells
decreased during the first few weeks of culture, indicating that a
proportion of tumor cells did not survive in culture (Fig 1). At later time points, numbers of
CK-positive cells started to increase and, concomitantly, CK-negative
cells completely disappeared from the cultures (Fig 1). Continuous
subcultivation of the adherent cell populations for more than 10 to 30 days resulted in purities of CK-positive cells of greater than 95%. At
these and later time points, cells in the cultures were also stained
with the epithelial-specific Ber-Ep4 MoAb with all samples staining
positive in greater than 95% of cells, indicating that the cells were
of tumor origin and not derived from mesothelium which does not express
this antigen.28,29 In addition, the cells stained negative
with the mesothelial-specific anticalretinin antibody30
(Table 1). Cells from 4 of the 5 analyzed
cultured samples were found to express the erbB2-protein at levels
comparable to SKBR3 or higher than A431 cell lines, as detected by
Western blot analyses (Table 1). These levels of erbB2 expression
reflect an overexpression of this protein according to the quantitative
estimates for normal breast tissues, breast cancer tissues, and various
cell lines.31-33 Enriched primary breast cancer cells
proliferated ex vivo until the cultures terminated spontaneously within
60 to 130 days (Table 1). Cell aliquots taken during this growth period
were used for subsequent experiments.
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| Fig 1.
Primary breast cancer cell cultures. (A) Cells contained
in an ascites (upper panel) and a pleural effusion sample (lower panel)
from 2 representative breast cancer patients were cultured in
-MEM/10% FCS. At the indicated time points, numbers of CK-positive
cells ( ) and CK-negative cells ( ) were determined by
immunocytochemical analysis as described in Materials and Methods. At
the time points indicated with an asterisk (*), detection of Ber-Ep4
was also performed yielding nearly identical cell numbers as
CK-positive cells. Cultures were maintained by subcultivating total
cells, ie, adherent and nonadherent fractions. (B) Immunostaining with
anti-CK MoAbs of the cultures shown in the upper panel of (A) at the
indicated time points (original magnification, ×100).
Fig. 3.
(A) Appearance of a tumor cell colony grown for 20 days
in methylcellulose in LTBMC medium supplemented with EGF and IGF
(original magnification, ×100). (B) Immunofluorescence staining of
cells picked from tumor cell colonies shown in (A) after staining with
FITC-labeled anticytokeratin antibodies (original magnification,
×40).
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Effect of cytokines on breast cancer cells.
To address the question whether the cytokines used for ex vivo
expansion of CD34+ blood progenitor cells may influence the
growth of breast cancer cells in culture, we studied tumor cell
proliferation in the presence and absence of SCF + IL-1 + IL-3 + IL-6 + EPO, a cytokine combination previously shown to efficiently
mediate ex vivo expansion of CD34+ blood progenitor
cells.34 Enriched primary mammary carcinoma cells used
after various precultivation periods and derived from different
patients showed a 2.4- to 6.6-fold increase in cell numbers over a
7-day period in control cultures (Fig 2).
Overall, the addition of cytokines SCF + IL-1 + IL-3 + IL-6 + EPO
did not result in a significant inhibitory or stimulatory effect on tumor cell growth compared with cultures without cytokines (Fig 2A).
TGF-1, a pleiotropic cytokine that has been shown to suppress the in
vitro growth of epithelial-derived tumor cells,35 was recently found to allow reduced, but still significant expansion of
colony-forming cells without loss of primitive LTBMC-initiating cells
in cytokine-supported CD34+ BPC ex vivo
expansion.36 To determine if TGF-1 could be of use to
purge CD34+ autografts from breast cancer cells during ex
vivo expansion, we investigated its potential to influence the survival
and proliferation of breast cancer cells. As outlined in Fig 2B,
enriched primary tumor cells proliferated in control cultures, yet
their growth was inhibited in the presence of TGF-1. In all cases,
cell numbers fell below the cell numbers initially seeded into the
cultures, irrespective of the presence of cytokines, SCF + IL-1 + IL-3 + IL-6 + EPO.
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| Fig 2.
Influence of hematopoietic stimulatory cytokines (A) or
TGF-1 (B) on proliferation of primary mammary carcinoma cells.
Primary mammary carcinoma cells were precultured in -MEM/10% FCS
for the indicated periods. Enriched tumor cells (purity >95% of
CK-positive and Ber-Ep4-positive cells) were cultured for 7 days in
-MEM/10% FCS alone or with 10 ng/mL SCF, 3 ng/mL IL-1, 100 ng/mL
IL-3, 100 ng/mL IL-6, and 1 U/mL EPO (S136E) and/or 30 ng/mL of
TGF-1. ( ) Controls; ( ) S136E; ( ) TGF-1; ( ) TGF-1 + S136E. Results represent the mean values ± SD of duplicate
determinations. A P value greater than .05 was obtained when
comparing the mean values of both groups in a two-tailed Student's
t-test in (A). Statistical significant differences
(P < .05) were recorded when comparing the control group
(no cytokines) with either the TGF-1 or the S136E plus TGF-1
group in (B) using a two-tailed Student's t-test.
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Because induction of cell proliferation and differentiation by
hematopoietic growth factors has been described in nonhematopoietic malignant cells,37 we investigated whether the treatment of breast cancer cells with cytokines influences subpopulations of tumor
cells with in vitro clonogenic potential. Tumor cell colonies were
grown in EGF- and IGF-supported semisolid assays and were of irregular
shape, consisting of aggregates of closely attached cells (Fig
3A). The epithelial origin of the clonogenic cells was
confirmed by immunofluorescence staining of the cells picked from the
colonies with anticytokeratin MoAbs (Fig 3B). As outlined in Table
2, tumor cells from 3 of 5 patient samples
scored positive in the tumor colony assay. When tumor cells had been
pretreated with hematopoietic cytokines SCF + IL-1 + IL-3 + IL-6 + EPO for 7 days, tumor cells formed colonies with a comparable cloning efficiency as untreated controls (Table 2). Absolute numbers of
clonogenic tumor cells per culture were also not significantly different in control and SCF + IL-1 + IL-3 + IL-6 + EPO-treated cultures. These results suggest that a cytokine combination used to ex
vivo expand CD34+ hematopoietic cells, SCF + IL-1 + IL-3 + IL-6 + EPO, does not influence the growth of breast cancer cell
subpopulations with in vitro clonogenic potential. In contrast,
TGF-1 suppressed clonogenic tumor cell populations as determined in
tumor colony assays, resulting in a reduction of numbers of clonogenic
progenitors compared to the respective control cultures (Table 2). The
combined treatment of tumor cells with TGF-1 and SCF + IL-1 + IL-3 + IL-6 + EPO resulted in a similar inhibitory effect as
TGF-1 alone, with reduced numbers of clonogenic cells compared with
untreated control cultures (Table 2).
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Table 2.
Influence of Cytokines on Numbers of Clonogenic Mammary
Carcinoma Cells Detected in Methylcellulose Cultures
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Influence of CD34+ cells on tumor cells during ex
vivo expansion.
To analyze the potential of CD34+ cells or their progeny to
influence the growth of tumor cells during ex vivo expansion, we performed mixing experiments of CD34+-enriched BPC with
primary mammary carcinoma cells. During a 7-day culture period in the
presence of SCF + IL-1 + IL-3 + IL-6 + EPO, which led to an overall
9.5- ± 0.7-fold expansion in total numbers of hematopoietic cells,
CK+/Ber-Ep4+ cells coexpanded by a factor of
5.2 ± 1.4 (Fig 4A). Comparison with the
results of the analogous experiments in the absence of CD34+ cells shown in Fig 2 demonstrates that
CD34+ cells, under these conditions, did not detectably
influence tumor cell growth. Interestingly, the addition of TGF-1
still led to suppressed tumor cell growth, but in contrast to the
previous experiments with TGF-1 in the absence of CD34+
cells, absolute numbers of tumor cells increased. Therefore, the
presence of hematopoietic cells interferes with the potential of
exogenously added TGF-1 to inhibit tumor cell growth and survival. In another assay, highly enriched CD34+ hematopoietic cells
or late-stage hematopoietic cell populations that are found on day 14 of ex vivo expansion were separately analyzed for their potential to
influence growth of PKH-labeled tumor cells. In the presence of serum
together with high numbers (5 × 105/mL) of 14-day ex
vivo expanded CD34+ cells, numbers of PKH-labeled tumor
cells increased up to 2.5-fold compared with control cultures (Fig 4B,
left panel). In contrast, during this 72-hour period, no increases in
tumor cell numbers were detected in the presence of CD34+
BPC (Fig 4B, left panel). Therefore, mature hematopoietic cells at
higher cell densities may harbor a risk to stimulate tumor cell growth.
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| Fig 4.
Influence of hematopoietic cells on primary breast cancer
cells during ex vivo expansion. (A) Culture-enriched mammary carcinoma
cells (solid symbols) and CD34+ enriched BPCs (open
symbols) were cocultured at a ratio of 1:10 in 3-mL flasks in the
presence of SCF, IL-1, IL-3, IL-6, and EPO with (squares) or without
(circles) 30 ng/mL of TGF-1. Tumor cells were enumerated by
immunocytochemistry using anti-CK and Ber-Ep4 antibodies. Mean values
using cells from three different patients are shown. (B) Ten
PKH-26-labeled tumor cells were suspended in 100 µL of
FCS-containing IMDM or serum-free medium together with the indicated
numbers of enriched CD34+ BPCs that had either not been
precultured (CD34+) or that had been ex vivo expanded
for 14 days in the presence of SCF, IL-1, IL-3, IL-6, and EPO
(expanded). After 3 days, the entire cultures were examined under a
fluorescence microscope and all cells displaying a red fluorescence
were counted. Results are the mean values ± SD of triplicate cultures
and show a representative experiment.
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Influence of serum on ex vivo proliferation and survival of breast
cancer cells.
Ex vivo expansion of CD34+ blood progenitor cells has been
efficiently performed in serum-free, chemically defined
media.15,25,38 To investigate the influence of serum-free
culture conditions on the behavior of epithelial-derived tumor cells
during ex vivo expansion, primary breast cancer cells were inoculated
into serum-free medium. To exclude that during the precultivation
period tumor cells had adapted to serum and that they were influenced
by serum withdrawal, primary breast cancer cells used in these
experiments were not culture-enriched, but were directly inoculated
immediately after isolation from patients. Whereas tumor cells survived
during a culture period of 7 to 14 days or started to grow in
serum-containing cultures, culture under serum-free conditions resulted
in a 10- to 100-fold depletion of breast cancer cells (Fig
5). Whereas prolonged culture until day 30 resulted in an increase in the absolute numbers of tumor cells in
serum-supplemented cultures, a sharp decrease in tumor cell numbers was
recorded in the serum-free cultures. Similar survival curves of tumor
cells as in the serum-free medium preparation were observed in cultures
containing -MEM without FCS (data not shown). No difference was
evident between cultures (serum-free or serum-supplemented) when
performed in the presence or absence of SCF + IL-1 + IL-3 + IL-6 + EPO,
indicating that the hematopoietic cytokines do not influence the
survival (in serum-free cultures) or the proliferation (in
serum-supplemented cultures) of mammary carcinoma cells (Fig 5). When
low numbers of PKH-labeled breast cancer cells were cocultured with
hematopoietic cells for 3 days in serum-free medium, very little or no
influence of hematopoietic cells on the survival of tumor cells was
seen, irrespective of the cell density used or if fresh
CD34+ cells or more mature ex vivo expanded cell
populations were analyzed (Fig 4C). These results demonstrate that
breast cancer cells may be efficiently eliminated in serum-free culture
medium ex vivo, irrespective of the presence of hematopoietic growth
factors.
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| Fig 5.
Influence of culture in SFM on survival of primary
mammary carcinoma cells. Freshly isolated cells from malignant
effusions were directly inoculated into 24-well plates at a cell
density of 0.1 to 1 × 106/mL into the indicated culture
media with or without cytokines (SCF, IL-1, IL-3, IL-6, and EPO;
S136E) as indicated. Independent cultures were set up in duplicates for
the individual time points. After trypsinization both adherent and
nonadherent cells were subjected to immunocytochemical analysis of
tumor cells as described in Materials and Methods. Results represent
mean values. FCS: -MEM + 10% fetal calf serum.
|
|
| |
DISCUSSION |
Tumor cells have been found in bone marrow or mobilized peripheral
blood from patients with solid tumors, eg, breast cancer, in both
unprocessed and in CD34+ selected
harvests.4,12,13 Contaminating breast cancer cells have
been found viable and capable of ex vivo growth4,22,23 and
may therefore be of clinical concern.9-11 For ex vivo
expansion of autologous hematopoietic progenitor cells, culture
conditions have been optimized to generate large numbers of progenitor
cells.14,39 Because results of a systematic analysis of the
behavior of primary solid tumor cells during ex vivo expansion cultures
have not been available so far, we investigated the influence of
variables in culture conditions on mammary cancer cell growth,
survival, and clonogenicity.
Because the incidence of contaminating tumor cells in autologous
transplants is very low, we used mammary carcinoma cells from malignant
effusions. We were able to grow breast cancer cells from 6 of 11 aspirates. Other or similar approaches performed from other
investigators to isolate proliferating tumor cells from effusion fluids
have also only been successful in a proportion of the samples
used.40,41 Sharp et al23,42 and Ross et
al4 were able to culture proliferating breast cancer cells
isolated from bone marrow or mobilized blood specimens. Recently,
Emerman et al43 could successfully grow primary breast
cancer cells in liquid culture from 4 of 7 bone marrow specimens and 3 of 4 pleural effusions. Ethier et al44 also found growth of
pleura-derived tumor cells in 3 of 7 samples from patients with
metastatic breast cancer. The breast cancer origin of the cells from
our 6 specimens that showed in vitro growth was documented and
monitored by their positive immunostaining with anticytokeratin and
Ber-Ep4 antibodies and negative immunostaining with anticalretinin
antibodies. These antibodies in combination have been shown to
distinguish epithelial tumor cells from mesothelial or mesenchymal
cells in serous fluids.28-30 In addition, erbB2-protein
overexpression, which has been described as a characteristic feature of
a proportion of mammary carcinomas,31 was recorded in part
of our cultured samples.
Initially, tumor cells grew very slowly, but in later passages their
growth rate increased. Thus, our primary tumor cells displayed a
similar growth behavior as tumor cells isolated from the site of the
primary or metastatic tumor40,44,45 or to the
micrometastatic mammary carcinoma cells isolated from bone marrow.22,46 Our primary cultures terminated spontaneously after 8 to 18 weeks of culture, and we did not obtain continuous cell
lines. The incidence of clonogenic cells in semisolid medium (range,
0.025% to 0.75%) detected in our tumor cell population also
represents previously described incidences in tumor
biopsies47 or bone marrow samples.4
Our results indicate that under serum-containing conditions, primary
tumor cells may remain viable and proliferate ex vivo. We did not find
significant stimulatory or inhibitory effects of the combination of
cytokines, SCF + IL-1 + IL-3 + IL-6 + EPO, on the growth rate of
primary breast cancer cells. Previous studies using pre-established
carcinoma cell lines found that IL-1 and IL-6 had an inhibitory
influence,48-50 whereas IL-3, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been found to stimulate the
growth of tumor cells.37,51-54 Using primary breast cancer cells, Emerman et al55 found no effect of hematopoietic
stimulatory cytokines, which is in line with our results. We cannot
exclude the possibility that each factor alone may positively or
negatively influence the ex vivo growth of the primary breast cancer
cells; however, the ultimate response to the cytokine combination that has been shown to mediate ex vivo hematopoietic expansion16 was neither growth-stimulatory nor growth-inhibitory.
Tumor cell growth could be stimulated by mature hematopoietic cells at
high cell densities in the presence of serum. These results are in
analogy to the findings of Vogel et al,20 who suggested
that direct cellular interactions between ex vivo expanded hematopoietic cells and solid tumor cells result in a better survival of tumor cells in the later phases of the ex vivo culture compared with
earlier phases. This underscores a supportive effect of more mature
hematopoietic cell types on in vitro tumor cell survival. Also, Emerman
et al43 have shown that excess numbers of hematopoietic cells as present in LTBMC are able to stimulate the growth of mammary
carcinoma cells. It is also known that, within LTBMC, high levels of
TGF-1 are prevalent.56 These findings can explain our
observation that the suppressive effect of TGF-1 on tumor cells that
is recorded in our experiments in the absence of CD34+
cells and that was also seen by other investigators with various epithelial tumor cell types57-59 is counteracted in the
presence of excess numbers of hematopoietic cells.
We have observed that primary mammary carcinoma cells undergo rapid
elimination during culture in serum-free medium. This effect persisted
upon culture for extended time periods, indicating that the tumor cells
could not adapt their in vitro growth to the serum-free conditions. A
similar decrease of tumor cells was observed using both a basal culture
medium (-MEM) containing essentially only salts, glucose, amino
acids, and vitamins or a serum-free medium preparation that, in
addition, also contains supplemental bovine serum albumin, cholesterol,
and transferrin. These results indicate that the medium supplements,
which are of importance for the survival of hematopoietic
cells,25 do not contribute to survival of breast cancer
cells. We also found that the presence of hematopoietic cytokines in
the serum-free cultures could not protect the primary mammary tumor
cells from elimination. Thus, in addition to the results shown in Fig
2A, where influences on the proliferation of tumor cells by cytokines were determined, the survival of primary breast cancer cells, assayed
in the cultures in serum-free medium, is not affected by the
hematopoietic stimulatory cytokines.
Taken together, we have shown that culture conditions used for ex vivo
expansion influence tumor cell survival and growth. A strong
suppression of tumor cells was achieved in serum-free medium
preparations. In contrast, ex vivo expansion for prolonged time periods
reaching relatively high cell densities of mature hematopoietic cells
may harbor a risk of stimulating tumor cell growth. These results will
be of importance for ex vivo expansion of hematopoietic transplants for
maximal efficiency of tumor cell purging. Our findings should be
essential when designing clinical trials that investigate the value of
tumor cell purging in patients with breast cancer.
| |
ACKNOWLEDGMENT |
The authors thank Prof Dr R. Mertelsmann for his continuous
encouragement and conceptional advice. We also thank Dr M. Schmidt for
providing Western Blot analyses and E. de Lima-Hahn for excellent technical support.
| |
FOOTNOTES |
Submitted February 2, 1998;
accepted September 24, 1998.
A.S. and W.B. contributed equally to this work.
Supported by the Deutsche Forschungsgemeinschaft through
Sonderforschungsbereich 364 (Project A1 to R.H.).
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 R. Henschler, MD, Abt.
Hämatologie, Medizinische Universitätsklinik, Hugstetter
Strasse 55, D-79106 Freiburg, Germany.
| |
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Abstract
Introduction