vaccine produced by electrofusion between human osteosarcoma cells and DCs. Methods: In the present study, we eletrofused patient-derived DCs to autologous osteosarcoma cells. The fusion cells possessed the properties of both patient cells. After electrofusion, the cytoplasm of the two cells was integrated, whereas their nuclei remained separate entities. The intracellular structure
was observed on fusion cells under the transmission electron microscope. Results: Coculture of patient-derived peripheral blood mononuclear cells (PBMC) with DC/tumor fusion cells resulted in activation of T cells as assessed by standard cytotoxic T lymphocytes (CTLs) assays.
Key Words: dendritic cell, osteosarcoma, fusion cell, T cell activation, immunotherapy, cytotoxic T lymphocytes, autologous
Dendritic cells (DCs) are the best professional anti-
gen-presenting cells (APCs) and they have been used
extensively in this context because they can increase the
surface expression of major histocompatibility complex
(MHC) antigens of class I and class II, and co-stimulatory
molecules (required for efficient presentation of pep-
tides and stimulation of T cells) [1] and can synthesize a
variety of immunologically important cytokines such as
IL-1, TNF-α, and IL-12. Therefore, DCs have been used
in humans to enhance antitumor immunity by stimulating
the immune system to recognize and destroy malignant
cells. Methods for delivering tumor antigens into DCs are
the focus of intensive investigation in DC-based tumor
vaccines. These include introduction of identified tumor
antigens into DCs by pulsing with peptides or proteins and
transfecting with RNA or DNA [2–6]. In preclinical models,
these DC-based vaccines have induced protective and
therapeutic immune responses against tumors. In clinical
trials, vaccination with lysate- or idiotype-pulsed DCs has
resulted in immunologic and clinical responses [7–11].
Another evolving strategy is the use of fusion
constructs between DCs and tumor cells. With this
technique, an immunogenic hybrid cell can be created
with the properties required for initiation of primary
antitumor immune responses. Theoretically, fusion
of DCs with tumor cells will result in the presentation
of a broad spectrum of tumor antigens, both known
and unidentified, in the context of the potent immune-
stimulatory machinery of the DCs. Indeed, vaccination
of mice with fusion cells has induced protective and
therapeutic antitumor immunity [12–14].However, the
traditional fusion method using polyethylene glycol
(PEG) is often plagued by its too widely ranging ef-
ficiencies, toxicity, poor reproducibility, and varying
susceptibilities among individual tumor cell partners.
Recently an alternative means of generating DC-tu-
mor hybrids by exposing cells to electric fields has been
described. The success of fusion has unequivocally been
verified by a number of analyses including FACS, cytospin,
confocal immunofluorescence, and DNA content. The ef-
ficiency of electrofusion is usually ten to hundreds times
higher than the chemical methods [15–17]. However,
little is known yet about the fusion process, fusion cell
morphology, and the relation between antigen presenta-
tion of fusion cells and induction of antitumor immunity.
The tasks of the present study was to fuse osteosarcoma
cells from patients with bone cancer with autologous DCs,
evaluate an integration of human DCs and tumor cells and
link their properties to T cell activation.
Materials and Methods
Generation of DCs from peripheral blood
mononuclear cells (PBMC). Mononuclear cells
were isolated from the peripheral blood of patients
with osteosarcoma by Ficoll/Hypaque density gradi-
ent centrifugation. The PBMC were cultured in RPMI
1640 medium containing 1% autologous serum for
1 h. The nonadherent cells were removed, and the
T cells were purified by nylon wool separation. The
adherent cells were cultured for 1 week in RPMI 1640
medium containing 1% autologous serum, 1000 U/ml
GM-CSF, 500 U/ml IL-4 and 1000 U/ml recombinant
human tumor necrosis factor-α (TNF-α) (all cytokines
from R&D Systems, USA), to generate DCs. Then the
nonadherent and loosely adherent cell clusters of
proliferating DC were harvested.
Preparation of osteosarcoma cells. Osteosar-
coma cells were obtained from primary tumors. The
tumor tissues were separated in Hank’s balanced
salt solution (Ca++/Mg++ free) containing 1 mg/ml col-
lagenase, 0.1 mg/ml hyaluronidase and then cultured
in RPMI 1640 medium supplemented with 10% heat-
inactivated autologous human serum, L-glutamine
(2 mM), penicillin (100 U/ml) and streptomycin
(100 µg/ml) until they were fused with DCs.
Fusion of DCs with osteosarcoma cells. Au-
tologous DCs were incubated with osteosarcoma
cells at a ratio of 5 : 1 and suspended in 0.3 M glucose
solution containing 0.1 mM Ca(CH3COO)2, 0.5 mM
Mg(CH3COO)2, and 0.3% bovine serum albumin. The
pH of the fusion medium was adjusted to 7.2–7.4 with
L-histidine (all chemicals were from Sigma, USA). After
centrifugation, the cells were resuspended in the same
fusion medium without bovine serum albumin. Routinely,
0.5 ml of cell suspension containing 6 x 106 cells were
processed using a specially designed electroporation
cuvette, precoated on one side with paraffin wax (50 µl
per cuvette). For electrofusion, a pulse generator (model
ECM 2001, BTX Instrument, Genetronics, San Diego,
CA) was used. Electrofusion involves two independent
but consecutive steps. The first reaction is to bring cells
in close contact by dielectrophoresis, which can be
accomplished by exposing cells to an alternating (ac)
electric field of relatively low strength. Then cell fusion
can be triggered by applying a single square wave pulse
to induce reversible cell membrane breakdown in the
zone of membrane contact. For the current study, the
optimal conditions for maximum electrofusion efficiency
without substantial cell death (not lower than 70% vi-
ability by Trypan Blue staining) were found to consist
of two consecutive rounds of an alignment pulse of 50
V for 5 s followed by a fusion pulse of 250 V. The entire
process was repeated a second time to maximize fusion
efficiency. The fusion mixture was allowed to stand for
5 min before suspending in complete medium and then
incubated at 37 °C overnight. The nonadherent cells con-
sisted of mainly DCs, and the adherent cells consisted
of mainly fusion cells and tumor cells. The electrofusion
products were purified by monoclonal antibody CD1α (a
DC marker not expressed on tumor cells) sticking to the
magnetic beads (Miltenyi Biotec, German).
Transmission electron microscopy.For observa-
tion of cell morphology and intracellular structure, cell
preparation was fixed with 1.5% glutaraldehyde in 0.1 M
cacodylate buffer, pH 7.4, for 1 h at 4 °C. The specimens
were washed, treated with 1% osmium tetroxide in 0.1 M
cacodylate buffer, and passed through an alcohol gradi-
ent. They were treated with propylene oxide and embed-
ded. The ultrathin sections were cut with an MT2 Sorvall
ultramicrotome and examined with a JEOL-100-CX
transmission electron microscope (TEM).
Flow cytometry. The patient derived osteosar-
coma cells, DCs and purified fusion cells were washed
and incubated with monoclonal antibodies against
HLA-ABC, HLA-DR, CD14, CD40, CD1α, CD83, CD86,
and MUC1 (all prime antibodies from Serotec Systems,
UK) for 1 h on ice. After washing with PBS, the cells
were incubated with fluorescein isothiocyanate (FITC)/
phycoerythrin(PE)-conjugated goat anti-mouse IgG
(PharMingen, USA) for 30 min. Samples were then
washed, fixed with 2% paraformaldehyde, and ana-
lyzed by FACScan (Becton Dickinson, USA).
Autologous T cell proliferation assay.Autologous
PBMC from the same osteosarcoma patient from whom
fusion cells were derived were purified through nylon wool
to remove APCs and B cells. The T cells were cocultured
with autologous DC/osteosarcoma fusion cells, DCs
mixed osteosarcoma cells and osteosarcoma cells alone
for 5 days in complete RPMI 1640 medium supplemented
with 10% human serum, 20 U/ml human IL-2, 50 µM
2-mercaptoethanol, 2 mM L-glutamine, 10 μM Hepes,
100 U/ml penicillin and 100 μg/ml streptomycin. Then the
cells were pulsed with 1 µCi 3H-Thymidine (New England
Nulear, Boston, MA) per well for 12 h, and T cell prolif-
eration was measures using standard [3H]-thymidine
incorparation. All samples were conducted in triplicate
and expressed as mean ± S.D.
Measurement of CTL activity.
PBMC from osteosarcoma patients were stimulated by co-culturing
with autologous DC/osteosarcoma fusion cells in the
presence of 20 U/ml human IL-2. PBMC cocultured
with DCs mixed with tumor cells, DCs, or tumor cells
alone were used as a control. The stimulated T cells
were harvested at the indicated time, separated by
passing through nylon wool and used as effector
cells in the CTL assay. Autologous osteosarcoma
cells, monocytes, MG63 osteosarcoma cells, LNCap
prostate cancer cells, and K562 cells were labeled
with 51Cr for 60 min at 37 °C. After washing, target cells
(2 x 104) were cocultured with T cells for 5 h at the
indicated cell radio. Supernatants were assayed in a
gamma counter for 51Cr release. Spontaneous release
of 51Cr was assessed by incubation of the targets in the
absence of effectors. Maximum or total release of 51Cr
was determined by incubation of the targets in 0.1%
Triton X-100. The percentage of specific 51Cr release
was determined by the following calculation:
percentage-specific release = [(experimental –
spontaneous)/(maximum - spontaneous)] x 100.
Statistical analysis. Statistical significance was
determined using Student’s t-test.
Results
Morphology and phenotype of DCs, osteosar-
coma and fusion cells.After culturing and induction,
DCs displayed typical morphology with elongated den-
dritic processes (Fig. 1, left panel), whereas osteosar-
coma cells had a thick cell coat and round shape (see
Fig. 1, middle panel). The fusion of osteosarcoma cells
with DCs resulted in a larger hybrid cell with both DCs
and tumor cells (see Fig. 1, right panel) and irregular
surface, suggesting the integration of two or more
cells. Phenotypically, HLA-ABC, HLA-DR, CD14, CD40,
CD1α, CD83, CD86, and MUC1 were detected on the
three populations (Fig. 2). Human DCs expressed
CD1α, but not MUC1 antigens, osteosarcoma cells
expressed tumor-associated MUC1 antigens but not
CD1α, and the purified DC/osteosarcoma fusion cells
highly expressed both CD1α and MUC1 (Fig. 3).
fig. 2. DCs (solid bar), osteosarcoma cells (hatched bar) and
purified DC/osteosarcoma fusion cells (gray bar) from the patient
with osteosarcoma were stained with panels of mAbs and analyzed
by flow cytometry for the expression of the indicated molecules
Stimulation of autologous T cell proliferation by
DC/osteosarcoma fusion cells. To determine the ef-
fects of DCs mixed with osteosarcoma cells or DC/oste-
sarcoma fusion cells in stimulation of T cells, autologous
T cells were cocultured with the mixture or the fusion hy-
brids and their proliferation was measured. As a control,
the T cells were also cocultured with autologous tumor
cells. The results demonstrated little if any evidence for T
cell stimulation by autologous tumor cells, tumor cells, or
the mixture of the two cell types. By contrast, incubation
of T cells with autologous fusion cells was associated with
T cell proliferation (Fig. 4). This finding demonstrates that
fusion of osteosarcoma cells and DCs results in stimula-
tion of a specific T cell response.
fig. 4. Stimulation of T cell by DC/osteosarcoma fusion cells. T
cell were cultured with osteosarcoma cells, osteosarcoma cells
mixed with DCs, or DC/osteosarcoma fusion cells at indicated
ratios of T cells to stimulators
CTL activity against autologous tumors induced
by DC/osteosarcoma fusion cells. To assess the in-
fig. 1. Surface and intracellular structure of cells examined by transmission electron microscopy (× 4000). DCs displayed typical
morphology (left panel); osteosarcoma cells had a thick cell coating and round shape (middle panel); the fusion construct of
osteosarcoma cells with DCs resulted in a larger hybrid cell with both DCs and tumor cells (right panel)
fig. 3. FACS analysis of DCs, osteosarcoma cells and DC/osteosarcoma fusion cells. DCs (left panel), osteosarcoma cells (middle
panel), and DC/osteosarcoma fusion cells (right panel) were stained with anti-MUC1, and anti-CD1α mAbs and analyzed by two-
color flow cytometry
duction of tumor-specific CTLs, T cells were stimulated
for 10 days and then isolated for assaying lysis of au-
tologous tumor cells. T cells incubated with autologous
DCs, osteosarcoma cells, or an unfused mixture of both
exhibited a low level of autologous osteosarcoma cell
lysis (Fig. 5). Also, T cells stimulated with the fusion cells
were effective in inducing cytotoxicity of autologous tu-
mor. These results are consistent with our previous find-
ing that fusion between DCs and tumor cells is critical
for the hybrid cells to acquire the stimulating ability.
fig. 5. Activation of anti-tumor CTLs by autologous fusion cells.
T cell were stimulated with autologous DCs, autologous osteosar-
coma cells, osteosarcoma cells mixed with DCs, or DC/ osteosa-
rcoma fusion cells at indicated ratios of T cells to stimulators
Osteosarcoma-specific CTLs induced by DC/os-
teosarcoma fusion cells. To determine the specificity
of the CTLs induced by fusion cells, multiple targets were
used in a parallel assay. T cells stimulated by DC/osteosa-
rcoma fusion cells lysed aotologous osteosarcoma cells,
but not autologous monocytes, MG63 osteosarcoma
cells, LNCap prostate cancer cells and natural killer-sensi-
tive K562 cells. In addition, the CTL activity was inhibited by
anti-HLA class I antibody, indicating HLA class I-restricted
mechanism. Collectively, these results indicate that the
CTLs induced by DC/osteosarcoma fusion cells are os-
teosarcoma-specific and MHC class I-restricted
fig. 6.Specificity of CTLs generated by autologous fusion cells.
T cell were stimulated with DC/osteosarcoma fusion cells were
incubated with 51Cr-labeled autologous osteosarcoma cells (OS),
autologous monocytes (MC), MG63 cells, LNCaP prostate can-
cer cells, or K562 cells at a ratio 40 : 1 (solid bars). The targets
were also preincubated with an anti-HLA class I antibody (W6/32;
dilution 1 : 100) and then assayed for lysis (hatched bars). CTL
activity was determined by 51Cr release. The results are expressed
as mean ± SD of three replicates
discussion
Osteosarcomas are the prominent primary bone
cancers in humans, excluding hemopoietic malignan-
cies. They mainly affect children and adolescents and
are usually highly aggressive and eventually lethal. In an
attempt to individualize the therapeutic interventions of-
fered to osteosarcoma patients, immunotherapy might
make a contribution to the prevention and cure [18].
In immunotherapy, DC-based vaccine affords a
promising new approach for the clinical response of
cancers and has become an issue of the highest inter-
est. Fused DC-tumor cells present to CD4+ T-helper
cells a high level T cell costimulatory and MHC mol-
ecules, both of which are absent in most tumor cells.
This engagement results in the up-regulation of cell
surface markers on T-helper cells and the secre-
tion of various cytokines. The CD4+ T cell therefore
provides “help” by generating potent CTLs that are
the principal effectors of specific antitumor immune
responses [19–20]. Our current work aimed to explore
an alternative approach to a DC-based vaccine for
osteosarcoma and demonstrate that the electrofusion
cells are functional in inducing osteosarcoma-specific
and MHC class I-restricted CTL activity.
In this study, an electrofusion protocol was em-
ployed and a standard CTLs assay was adopted. Sig-
nificantly, one important advantage of immunization
with electrofusion products is the potential to induce an
immune response against all possible tumor antigens,
known or unknown. Several in vitroand in vivoapplica-
tions have been explored for the use of electrofused
DC-tumor hybrids as APCs [21–23]. From the results
obtained in the present studies, we could conclude that
the fusion cells were effective in inducing anti-tumor
CTLs, which lyse autologous osteosarcoma cells by an
MHC class I-restricted mechanism. Characterization of
the peptides recognized by these CTLs can be used to
identify tumor-associated antigens that are the targets
of the immune response.
Recently, there have been many relevant outcomes
about using allogenic DCs as fusion partners [24–25], for
T cells are potentially activated through both MHC class
I molecules derived from tumor cell and co-stimulatory
and adhesion molecules from allogenic DCs. Allogenic
DCs express many co-stimulatory and adhesion mol-
ecules that provide secondary signals for stimulation of
active T cell populations in the same way and secrete
a variety of cytokines additionally [26–28]. This option
seemed to project a practical advantage, for in a clinical
setting, allogenic DCs can be generated conveniently
from stored peripheral mononuclear cells from normal
healthy volunteers from the general population. How-
ever, there have been little proofs so far that autologous
DC/osteosarcoma fusion cells as tumor vaccine could
be effective in stimulating T cells, so we are determined
to explore the biology and efficacy of electrofusion cell
immunization against osteosarcoma gradually and more
studies on allogenic fusion cells will be investigated.
Unfortunately, the characterization and selection of
DC/osteosarcoma fusion cells remain a challenge due
to the lack of an unique marker for the osteosarcoma
cells. In the present study, we selected a representa-
tive marker based on the phenotype of tumor cells
in the patient. MUC1 was used as a tumor marker in
osteosarcoma patients since peripheral blood derived
DCs expressed minimal MUC1.
In summary, this study has demonstrated that it’s
feasible to generate a large number of DC/osteosa-
rcoma hybrid cells by the electrofusion technique.
Compared with other methods, electrofusion could
be reproducible and the fusion rate tended to be high.
Autologous DCs fused with osteosarcoma cells were
capable of inducing a potent antitumor response and
could be employed to treat the malignant bone tumor
effectively. This approach could conceivably be ap-
plied to a wide range of cancer indications for which
tumor-associated antigens have not been identified.
Acknowledgments
This work is sponsored by the National Natural
Science Foundation (30330610, CHN). We would like
to thank Professor Zhang Dianzhong for his technical
help and Zhang Yunfei for his efforts in interpreting
and analyzing the data. We also thank Dr. Long Hua
for his valuable advice.
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