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- Original Article -
Antitumor immunity by a dendritic cell vaccine encoding
secondary lymphoid chemokine and tumor lysate on murine
prostate cancer
Jun Lu1, Qi Zhang1, Chun-Min
Liang2, Shu-Jie Xia1, Cui-Ping
Zhong2, Da-Wei Wang1
1Department of Urology, Shanghai Jiaotong University Affiliated First People's Hospital, Shanghai 200080, China
2Department of Anatomy, Histology and Embryology, Shanghai Medical College Fudan University, Shanghai 200080, China
Abstract
Aim: To investigate the antitumor immunity by a dendritic cell (DC) vaccine encoding secondary lymphoid chemokine
gene and tumor lysate on murine prostate cancer.
Methods: DC from bone marrow of C57BL/6 were transfected
with a plasmid vector expressing secondary lymphoid chemokine (SLC) cDNA by Lipofectamine2000 liposome and
tumor lysate. Total RNA extracted from SLC+lysate_DC was used to verify the expression of SLC by reverse
transcriptase-polymerase chain reaction (RT-PCR). The immunotherapeutic effect of DC vaccine on murine prostate
cancer was assessed. Results: We found that in the prostate tumor model of C57BL/6 mice, the adminstration of
SLC+lysate_DC inhibited tumor growth most significantly when compared with SLC_DC, lysate_DC, DC or
phosphate buffer solution (PBS) counterparts
(P < 0.01). Immunohistochemical fluorescent staining analysis showed the
infiltration of more CD4+,
CD8+ T cell and CD11c+ DC within established tumor treated by SLC+lysate_DC vaccine
than other DC vaccines (P < 0.01).
Conclusion: DC vaccine encoding secondary lymphoid chemokine and tumor
lysate can elicit significant antitumor immunity by infiltration of
CD4+, CD8+ T cell and DC, which might provide a
potential immunotherapy method for prostate cancer.
(Asian J Androl 2008 Nov; 10: 883_889)
Keywords: dendritic cell; secondary lymphoid chemokine; prostate cancer; tumor lysate
Correspondence to: Dr Jun Lu and Dr Shu-Jie Xia, Department of Urology, Shanghai Jiao Tong University, Affiliated First People's
Hospital, Shanghai 200080, China.
Tel: +86-21-6324-0090 ext. 3161 Fax: +86-21-6324-0825
E-mail: lj0063@msn.com, xsjurologist@163.com
Received 2008-01-28 Accepted 2008-06-01
DOI: 10.1111/j.1745-7262.2008.00431.x
1 Introduction
Prostate cancer is the second leading cause of cancer death in men. Despite the effectiveness of hormone
therapy, most of these patients will eventually develop hormone-refractory disease. Therefore, new investigational
therapies are essential. Immunotheapy could present a novel strategy for hormone refractory prostate cancer (HRPC)
therapy. Dendritic cells (DC) are derived from hematopoietic cells of the bone marrow and are central to the
stimulation of an antitumor T cell response through presentation of tumor antigens. DC present processed peptides in the
context of major histocompability complex (MHC) Class I and Class II molecules to cytotoxic T lymphocyte (CTL)
and express high levels of adhension signals and costimulatory molecules [1]. Studies have been initiated to assess the
therapeutic potential of a DC-based vaccine with prostate tumor antigen-derived peptides or gene products. DC
loaded with the whole antigenic pool of the tumor in the form of protein lysate or total mRNA or even whole tumor
cells themselves in either an allogeneic or a syngeneic
situation is used to strengthen the host antitumor
immune response. The genetic modification of DC,
particularly with chemokine genes, represents a rational
approach to modify the tumor microenvironment that
favors innate or adaptive immunity to prevent or reverse
transcriptase polymerase chain reaction. Chemokine gene
transfer offers the possibility to trigger the recruitment
of initiators and/or effectors of the immune response to
the tumor microenvironment, which is demonstrated in
previous studies on chemokines, such as
macrophage-derived chemokine, macrophage inflammatory protein
and fractalkine [2_5].
Secondary lymphoid chemokine (SLC), a CC chemokine expressed in high endothelial venules and in
T-cell zones of the spleen and lymph nodes, strongly
attracts naive T cells and DC. Co-localization of these
cells within the local tumor environment could enhance
the induction of tumor-specific T cells. High levels of
SLC expression regulates the co-localization of CCR7
expressing antigen presenting DC and naive T cells, thus
facilitating activation and priming of immune responses
[6_8]. In addition to its immunotherapeutic potential,
SLC has potent angiostatic effects [9].
In the present study we evaluated the determinants
and efficacy of the antitumor responses in a murine
prostate cancer model after intratumoral adminstration of DC
encoding secondary lymphoid chemokine and tumor lysate. We utilized tumor lysate's multiple antigen
epitope, which was recognized by DC to induce
extensive T cell reaction, and the capacity of SLC of attracting
T cells, NK cells and DC to the tumor site to generate
systemic antitumor responses.
2 Materials and methods
2.1 Cell line and mice
Murine prostate cancer line
RM-1(H-2Kb) was purchased from the Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences.
Pathogen-free C57BL/6(H-2Kb,I-Ab) male mice
(6_8 weeks of age and 16_18 g in weight) were purchased from the Animal Maintenance Facility of the
Shanghai Medical College at Fudan University
(Shanghai, China).
2.2 Generation of bone marrow dendritic cells and
recombinant pAAV-IRES-hrGFP/SLC plasmid transfection
Bone marrow cells flushed from tibias and femurs
were depleted of erythrocytes by incubating in 0.9%
ammonium chloride for 3 min at 37ºC. The cells were
washed in HBSS and cultured in complete culturing
medium containing 10% FBS (GIBCO-BRL, Gaithersburg,
MD, USA), with 10 ng/mL recombinant mouse GM-CSF and 20 ng/mL recombinant mouse IL-4 (PeproTech,
Rocky Hill, NJ, USA) at 2 × 106 cells/mL. On day 4,
non-adherent cells were harvested by gentle pipetting and
stained with anti-CD11c antibody-conjugated microbeads
(Miltenyi Biotec, Auburn, CA, USA) to magnetically sort
CD11c+ immature bone marrow DC. The purity of the
sorted immature bone marrow DC was consistently greater than 90%, as analyzed by immunofluorescence
staining.
The recombinant pAAV-IRES-hrGFP/SLC plasmid
was obtained from the Department of Anatomy,
Histology and Embryology at Medical College, Fudan University
(Shanghai, China). To optimize the multiplicity of
infection for SLC, every 4 × 105 DC were transduced
with 1 μg recombinant pAAV-IRES-hrGFP/SLC plasmid.
The SLC plasmid was first mixed with Lipofecta-mine2000 (Invitrogen, Carlsbad, CA, USA) for 15 min
and then added to the resuspended DC. After
incubation for 1 h at 37ºC, 7% CO2, the mixture was
washed twice and then cultured in complete RPMI medium
containing recombinant mouse GM-CSF (20 ng/mL), IL-4
(10 ng/mL) until further analyses were performed.
2.3 Dendritic cells loaded with RM-1 tumor cell lysate
After adding the supernatant of tumor cell lysate to
DC tranduced with SLC and incubated for 18 h at
37ºC,7% CO2, the DC were harvested. Thus, the
construction of DC vaccine encoding secondary lymphoid
chemokine and tumor lysate was completed.
2.4 Polymerase chain reaction (PCR) analysis of SLC
Total RNA extracted from SLC+lysate_CDC,
SLC_CDC, Lysate_CDC and DC was used to verify the
expression of SLC by reverse transcriptase polymerase chain
reaction (RT-PCR). According to the sequence of SLCcDNA and multiple clone site of the
pAAV-IRES-hrGFP vector, the primer used for amplification was as
follows: Upper 5'-A TTC TAC AGC TCT GGT CTC ATC CTC A-3', Lower 5'-CG CTC GAG GTC TCT TTT CTA
GCT CCC TCT TTG 3'.
2.5 Establishment and treatment of subcutaneous
tumors
Tumors were established by s.c. injection of 2 ×
106 RM-1 cells into the right suprascapular of C57BL/6 male
mice. On days 5 and 12 after the tumor challenge, DC
vaccine was injected through foot pads and intratumorally,
respectively. Tumors were evaluated by caliber every 2
days by measuring length, width and height (mm), and
the volume of tumors was calculated using the formula:
V(mm3) = 0.52 (length × width × height). Survival was
also monitored. On day 24 after tumor establishment, all
mice were killed and the tumors were extracted and
imbedded in paraffin, then sliced and stained by
hematoxylin and eosin.
2.6 Immunohistochemical fluorescent analysis of tumors
Tumors were imbedded in OTC and processed into 5
μm sections for immunohistochemical staining. The
sections were washed twice for 5 min by PBS. Non-specific proteins were blocked in 0.1 mg/mL BSA.
Sections were then incubated with a monoclonal
Biotin-labeled anti-mouse CD8 Ab, anti-mouse CD4 Ab and a
monoclonal Biotin-labeled anti-mouse CD11 Ab for 24
h at 4ºC. Then the sections were washed three times by
PBS and labeled with phycoerythrin-labeled anti-mouse
IgG (CD8) and StreptAvidin-FITC (CD4, CD11).
2.7 Statistical analysis
Statistical analyses of the data were performed using
the Kruskal-Wallis one-way analysis of variance on ranks,
followed by multiple pairwise comparisons according to
Dunn's method. Survival curves were compared using the
log-rank test. Significance at the P < 0.05 level is
denoted.
3 Results
3.1 Characterization of transfected dendritic cell by
RT-PCR
The SLCcDNA was successfully transfected into DC
by verification of RT-PCR. The SLCmRNA was detected
in group SLC-DC and SLC+lysate_CDC, whereas other groups had no SLCmRNA expression (Figure 1).
3.2 Intratumoral injection of SLC+lysate_CDC inhibits
tumor growth and prolongs the survival of mice
The antitumor efficacy of SLC+Lysate_CDC was evaluated in C57BL/6 male mice with established RM-1
tumors, comparing with SLC_CDC, Lysate_CDC, DC and PBS. On days 5 and 12 after the tumor challenge,
DC vaccine was injected through foot pads and
intratu-morally, respectively. On day 24 after tumor
establishment, all mice were killed and the tumors were extracted. The
growth rate of tumors treated with SLC+lysate_CDC declined significantly (Figure 2A). The average volume
of the tumors in each of the five groups were 266 ± 255
mm3 (SLC+lysate_CDC), 1 204 ± 392
mm3 (lysate_CDC), 1 430 ± 117
mm3 (SLC_CDC), 2 934 ± 286
mm3 (DC), 4 331 ± 2 108
mm3 (phosphate buffer solution [PBS]) and 4 331 ± 2 108
mm3 (PBS) (Figure 2B). Survival was also improved in
SLC+lyate_CDC treated tumors. On day 65 after RM-1 tumor cell inoculation, there were
still three mice treated with SLC+lysate_CDC alive (Figure
2C). From this experiment, we presumed that intratumoral
injection of SLC+lysate_CDC inhibited tumor growth and
prolonged the survival of the mice.
3.3 Intratumoral injection of SLC+lysate_CDC promotes
the infiltration of CD4+,
CD8+ T cells and CD11+DC
The tumors were sliced into sections and stained by
hematoxylin and eosin (HE). The sections were first
observed under microscope (× 100). Both vaccines
induced focal areas of inflammatory cell infiltration.
However SLC+lysate_CDC treated tumors contained a higher infiltration of inflammatory cells than other
tumors (Figure 3).
To better quantitate the degree of T cells and DC
infiltration into the tumors, tumors were imbedded in OTC
and processed into 5 μm sections for
immunohistochemical fluorescent staining. There was a more significant
infiltration ratio of CD4+,
CD8+ T cells and CD11+DC in the SLC+lysate_CDC group than in any other group
(P < 0.01; Figures 4, Table 1).
4 Discussion
Host APC are critical for the cross-presentation of
tumor antigens [10]. However, tumors have the
capacity to limit APC maturation, function, and the infiltration
of the tumor site [11_13]. DC generation and maturation
are inhibited by prostate cancer cells [14] and DC are
eliminated by apoptosis [15] in the prostate tumor mass
[16]. To break this tolerance, the transplantation of
functional DC engineered to overexpress and present
peptides from specific prostate tumor antigen may be
effective. However, as prostate cancer cells are
genetically unstable and represent shifting targets [17],
single-agent vaccines might result in selection of genetic
variants that escape the immune attack. Therefore, in spite
of the fact that clinical benefit has been demonstrated in
patients vaccinated with DC loaded with peptides as well
as a single antigen in the form of a gene or protein, the
general view is that optimal antitumor response requires
polyclonal effector populations directed against a wide
range of tumor epitopes rather than a response restricted
to a single tumor antigen. The use of vaccines
containing mutiple tumor-derived antigens might elicit a broader
antitumor response than single antigen vaccines and
circumvent, to some extent, the problem of immune
escape [18]. This approach, termed polyepitope vaccination, has already been evaluated as a DNA
vaccine [19]. An alternative to this approach is the use of
DC loaded with the whole antigenic pool of the tumor in
the form of protein lysate or total mRNA, or even whole
tumor cells themselves, in either an allogeneic or a
syngeneic situation [20_22]. Another alternative described,
both in mice and humans, is the hybridization of tumor
cells with DC.
However, the application of tumor antigen-pulsed
DC-based vaccines still has limitations and human clinical
trials utilizing this strategy for the treatment of advanced
malignancies have had only modest results. A limitation
is that subcutaneously/intradermally injected DC vaccines
fail to migrate efficiently to secondary lymphoid organs
where they encounter naive T cells, which is a critical
step for the initiation of a primary immune response.
Almost all transferred DC remained at the immunization site
24 h after transfer. Inefficient migration of exogenous
DC to lymphoid organs might lower the frequency of
their encounter with T cells. Therefore, if transferred
DC produce chemokines to intensively attract T cells,
they might prime immune response efficiently, even
though the DC do not migrate to lymphoid organs.
Chemokines are being used in tumor immunotherapy
in animal models with evidence that local chemokine
delivery can increase the number of infiltrating T cells and
mediate delayed tumor growth. Because DC are potent
APC that function as principal activators of T cells, the
capacity of SLC to facilitate the co-localization of both
DC and T cells might reverse tumor-mediated immune
suppression and orchestrate effective cell-mediated
immune responses. Tumor therapies using SLC have proven successful. The ability to induce migration of
naive CD4+, CD8+ T cells, and to expand such cells at
sites of tumor antigen expression provides a powerful
tool for priming T cells responses in potentially
immunosuppressed hosts [23]. Liang et al. [24] found that SLC
induced a significant delay in tumor progression, which
was paralleled by a profound infiltration of DC and
activated CD4+ T cells and
CD8+ T cells (CD3+
CD69+ cells) into the tumor site. Yang
et al. [25] demonstrate that recombinant SLC administered intratumorally leads to
complete tumor eradication, increases in the
CD4+, CD8+ T cells, as well as DC expressing
CD11c+.
Above all, our experiment showed that intratumoral
administration of SLC+lysate_DC vaccine has strong
anti-tumor effects. First, tumor lysate has larger repertoires
of TAAs, which can lessen the possibility of tumor
escape and increase the probability of CTL cross-priming
with antitumor activity. Second, tumor injection of
transferred DC was not only favorable to tumor antigen
presentation by DC, but also resulted in greater
CD4+ and CD8+ T lymphocyte infiltration into tumors through the
chemotactic property of SLC secreted by modified DC.
Finally, the capacity of SLC to facilitate the
co-localization of both DC and T cells might reverse
tumor-mediated immune suppression and orchestrate cell-mediated
immune responses. Our study could provide a new
vaccine strategy for the treatment of prostate cancer and
assist in the ultimate development of a new therapeutic
vaccine for prostate cancer patients.
Acknowledgment
This work was supported by Item of Shanghai Municipal Health Bureau, China (No. 034038).
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