Cancer Letters 274 (2009) 160–164

Contents lists available at ScienceDirect

Cancer Letters
journal homepage: www.elsevier.com/locate/canlet

CpG-ODN-based immunotherapy is effective in controlling the growth

of metastasized tumor cells
Han-A Kim a, Hyun-Mi Ko a,b, Hye-Won Ju a, Kyoung-Jin Kim a, Si-Gyun Roh c,
Hern-Ku Lee b, Suhn-Young Im a,*
a
Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea
b
Department of Immunology, Chonbuk National University Institute for Medical Sciences, Chonju 561-756, Republic of Korea
c
Department of Plastic & Reconstructive Surgery, Chonbuk National University Hospital, Chonju 561-756, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG-ODN)
Received 29 April 2008 act as potent immune stimulators by activating innate immunity through toll-like receptor
Received in revised form 27 August 2008 9. These immunomodulatory effects of CpG-ODN have been reported to be associated with
Accepted 9 September 2008
anti-tumor immunity. In this study, we used a murine B16F10 melanoma model and a
CT26 colon cancer model to assess whether CpG-ODN-based immunotherapy was effective
in inhibiting tumor cells that have already metastasized to distant organs. Systemic admin-
istration of CpG-ODN after melanoma cell injection resulted in a significant inhibition of
Keywords:
CpG-ODN
pulmonary colonization. When CpG-ODN was administered after tumor cell injection, it
Tumor metastasis also inhibited pulmonary metastasis of the tumor cells, albeit to a lesser degree in the latter
case. Systemic administration of CpG-ODN after subcutaneous inoculation of CT26 colon
cancer cells diminished pulmonary metastasis from the primary tumor sites. Additionally,
CpG-ODN also inhibited the growth of pulmonary colonization of the colon tumor cells
when CpG-ODN was administered after the primary tumors had been surgically removed.
These data indicate that CpG-ODN was effective in inhibiting pulmonary metastasis of the
B16F10 melanoma and CT26 colon cancer cells, as well as the growth of metastasized
tumor cells. Our results suggest that CpG-ODN-based immunotherapy may be beneficial
in controlling micrometastasis after surgery in clinical settings.
Ó 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction from Mycobacterium bovis activated natural killer (NK) cells

Metastasis, the spread of cells from the primary neo-
plasm to distant sites and their growth there, remains
the principal cause of treatment failure and poor prognosis
in patients with cancer. In a large number of patients with
cancer, metastasis may well have occurred by the time of
diagnosis [1,2]. Most deaths from cancer are due to metas-
tases that are resistant to conventional therapies [1–4].
Synthetic oligodeoxynucleotides (ODN) containing
unmethylated CpG motifs (CpG-ODN) are immune stimula-
tors. Tokunaga et al [5] initially reported that DNA extracted
* Corresponding author. Tel.: +82 62 530 3414; fax: +82 62 530 0848.
E-mail address: syim@chonnam.ac.kr (S.-Y. Im).
to produce IFN and to induce anti-tumor activities. This re-
port also indicated that particular sequences with 50 -
X1X2CGY1Y2-30 (X1; purine, X2; purine or T, Y1 and Y2; pyrim-
idine) motif(s) were essential for these effects. Later,
synthetic CpG-ODN was reported to have similar immuno-
logical effects [6] by activating innate immunity through
toll-like receptor (TLR) 9 [7]. CpG-ODN induces type 1 T
helper (Th1) cells [8,9] and CD8+ cytotoxic T lymphocytes
(CTL) [10] through the activation and maturation of den-
dritic cells [11–13], leading to the secretion of IL-12 and
the expression of co-stimulatory molecules [9]. These
immunomodulatory effects of CpG-ODN have been reported
to be associated with anti-tumor immunity. CpG-ODN has
been shown to have anti-tumor effects when used as a single

0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2008.09.014
 Han-A Kim et al. / Cancer Letters 274 (2009) 160–164
agent in murine models of leukemia, lymphoma, melanoma,
neuroblastoma, and a rodent model of glioma [14–19].
These anti-tumor effects are observed with both local and
systemic injection of CpG-ODN [20].
It has recently been reported that systemic administra-
tion of CpG-ODN prior to tumor cell injection or peritumoral
administration of CpG-ODN in cutaneous melanoma is capa-
ble of inhibiting experimental pulmonary metastasis [21–
23]. However, little is known about how CpG-ODN affects
the growth of tumor cells that have already metastasized
from the primary tumor site to distinct organs.
In this study, we have focused our investigation on the
effect of CpG-ODN on the growth of metastasized tumor
cells. We have found that CpG-ODN was effective in inhib-
iting the growth of metastasized tumor cells.
2. Materials and methods
2.1. Animals
Specific pathogen-free female C57BL/6 and Balb/c mice
were obtained from the Orient Bio. Institute (Seongnam,
Republic of Korea) and were kept in our animal facility
for at least 1 week before use. All mice were used at 8 to
10 weeks of age. All experiments were conducted in accor-
dance with the guidelines of the Chonnam National Uni-
versity Institutional Animal Care and Use Committee.
2.2. Oligodeoxynucleotide (ODN)
Synthetic ODN were prepared with a nuclease-resistant
phosphorothioate backbone by the Bioneer Co. (Daejeon,
Republic of Korea). CpG-ODN 1826, containing two CpG mo-
tifs had the sequences 50 -TCCATGACGTTCCTGACGTT-30
[22]. The control GpC-ODN 1745, in which the CpG motif
was inverted, had the sequences 50 -TCCATGAGCTTCCTGA
GTCT-30 [22]. Twenty micrograms of CpG- or GpC-ODN
was dissolved in 100 ll of PBS, and then injected i.p. into
the mice.
2.3. Cell culture
The B16F10 mouse melanoma, which is metastatic in
the lungs of C57BL/6 mice, was maintained in RPMI 1640
(Invitrogen Co., CA, USA) supplemented with 10% fetal bo-
vine serum (Cambrex Co., Walkersville, MD). The CT26
mouse colon adenocarcinoma, which is metastatic in the
lungs of Balb/c mice, was grown in DMEM (Invitrogen
Co., CA, USA) supplemented with 10% fetal bovine serum.
2.4. Lung colonization assay
Cultured B16F10 tumor cells were washed twice in PBS,
and 1.8  105 tumor cells (>95% viability by trypan blue
exclusion assay), in 100 ll PBS, were injected i.v. into the
C57BL/6 mice (n = 5 for each group). Mice were autopsied
14 days later. Lungs were removed and fixed in Bouin’s
solution (Sigma Chemical Co., St. Louis, MO, USA), and
the number of surface lung colonies was counted under a
dissecting microscope.
161
2.5. Tumor growth
CT26 tumor cells (1  106) were suspended in 100 ll of
PBS and inoculated s.c. into the shaved back of Balb/c mice
(n = 7–10 for each group). Tumor growth was assessed at
twice a week by three-dimensional measurement with a
vernier caliper from the second weeks to the six weeks
after inoculation. Tumor volume was calculated that the
mass of tumor tissue approximates that of a prolate spher-
oid: V = p/6  1  w  h where V = tumor volume (mm3),
l = length (mm), w = width (mm), and h = height (mm)
[24]. Mice were sacrificed on day 42.
2.6. Surgery
CT26 tumor cells (1  106) were inoculated s.c. into the
shaved back of Balb/c mice on day 0. When primary tumors
reached the size of 15  20 mm in diameter, 17 days after
tumor inoculation, the mice were anesthetized with keta-
mine/Rompun (80 and 7 mg/kg body weight, respectively)
and placed on a vertical platform. Tumor masses were sur-
gically removed under anesthetic conditions, and surgical
incisions were closed with sutures. Mice were sacrificed
on day 49.
2.7. Statistical analysis
Data are represented as the mean ± SE. Statistical signif-
icance was determined by one-way ANOVA test (StatView;
Abacus Concepts Inc., Berkeley, CA, USA). All experiments
were conducted at least twice. Reproducible results were
obtained and representative data are, therefore, shown in
the figures.
3. Results
3.1. CpG-ODN inhibits pulmonary metastasis of melanoma and colon cancer
cells
CpG-ODN was administered i.p. before i.v. injection of B16F10 tumor
cells. When CpG-ODN was delivered at -7, -3 or 0 days, it inhibited metas-
tasis up to 62%, 82% and 54%, respectively (Fig. 1). Dual injections of CpG-
ODN (-7 and -3 days) resulted in stronger inhibition than that induced by
a single injection on day -7 or 0, but there was no significant difference com-
pared with group received single CpG-ODN on day -3. Inhibition was not
observed when PBS or control GpC-ODN was used, which demonstrates
the requirement for CpG-ODN for this effect. We determined the optimal
inhibitory concentration of CpG-ODN in a preliminary experiment.
We further investigated the effects of CpG-ODN on the growth and
metastasis of tumor cells using CT26 colon cancer cells. CT26 cells were
inoculated s.c. on day 0. CpG-ODN was i.p. administered either once on
day 18 or twice on days 18 and 25. While CpG-ODN did not inhibit pri-
mary tumor cells growth (Fig. 2A), it significantly inhibited tumor metas-
tasis to the lungs when it was administered twice (Fig. 2B). We could not
perform such an experiment using B16F10 melanoma cells because the
majority of animals that had received the tumor cells s.c. died before
measurable pulmonary metastasis occurred (data not shown).
3.2. CpG-ODN inhibits the growth of tumor cells metastasized to distant
organs
To examine the effect of CpG-ODN on the growth of B16F10 mela-
noma cells that have already metastasized, CpG-ODN was administered
subsequent to i.v. injection of the tumor cells. It was also found to signif-
icantly inhibit the pulmonary colonization (Fig. 3), albeit to a lesser de-
gree compared to that when CpG-ODN was administered prior to the
162 Han-A Kim et al. / Cancer Letters 274 (2009) 160–164

Fig. 1. Inhibition of pulmonary metastasis of B16F10 melanoma tumor

cells by CpG-ODN. C57BL/6 mice (n = 5 for each group) were treated i.p.
with 20 lg of CpG- or GpC-ODN at the indicated time, and B16F10
(1.8  105 cells) were injected i.v. on day 0. Lungs were removed on day
14, and the number of surface colonies was subsequently counted.
*
P < 0.0001; **P < 0.005 compared with GpC-ODN-treated control group.
Values are expressed as means ± SE.
injection of tumor cells. A single injection of CpG-ODN at 3 or 6 days
inhibited the pulmonary colonization up to 45% and 36%, respectively.
The extent of inhibition achieved by twice injections of CpG-ODN (3
and 6 days) was significantly higher than that by a single injection on
day 3 or 6. These data suggest that CpG-ODN may be beneficial to inhibit
the growth of tumor cells that have metastasized to distant organs from
the primary tumor site.
To test this concept, CT26 cells were s.c. injected. After skin tumors
developed to sizes up to 15–20 mm in diameter, the primary tumors were
surgically removed on day 17, and the mice were i.p. injected with CpG-
ODN either once on day 18 or twice on days 18 and 25. Mice were sacri-
ficed on day 49. In the PBS-treated control group, metastasis were ob-
served in 30% of animals (4 of 13) that underwent surgery (Table 1). In
contrast, in the group that received CpG-ODN injection once, metastasis
was observed in only 1 of 11 animals (9.0%), and in the group that re-
ceived CpG-ODN twice, lung nodules were not observed any of the ani-
mals (0 of 11). When metastasis occurred, normal lung architectures
were destroyed by large tumor masses. Collectively, our data indicate that
CpG-ODN is effective in inhibiting the growth of metastasized tumor
cells.
4. Discussion
For immunotherapy of tumors, systemic injections of
CpG-ODN exert an anti-tumor effect [14,15], and local
injections of CpG-ODN into the margin of the tumor also
Fig. 3. Inhibition of the growth of metastasized B16F10 tumor cells by
CpG-ODN. C57BL/6 mice (n = 5 for each group) were treated i.p. with
20 lg of CpG- or GpC-ODN at the indicated time, and B16F10
(1.8  105 cells) were injected i.v. on day 0. Lungs were removed on day
14, and the number of surface colonies was subsequently counted.
*
P < 0.0001; **P < 0.005; ***P < 0.05 compared with GpC-ODN-treated con-
trol group. Values are expressed as means ± SE.
can cure animals bearing small, established tumors [16–
19] when used as a single agent. The toughest challenge
for any type of tumor therapy is to cure established tu-
mors, rather than simply preventing the generation of
new tumors. In this study, to test whether CpG-ODN treat-
ment could accomplish this, we focused on assessing
whether the therapeutic efficacies of this CpG-ODN regi-
men were effective to inhibit the growth of metastasized
tumor cells. When CpG-ODN was administered after
B16F10 tumor cell injection, lung tumor colonization was
significantly reduced, although the inhibition was less
effective compared with that of CpG-ODN administered
before tumor cell injection (Fig. 3). We further demon-
strated this anti-metastatic activity of CpG-ODN by show-
ing that systemic administration of CpG-ODN after
inoculation with CT26 colon cancer cells inhibited pulmon-
ary metastasis of the tumor cells from the primary tumor
site. Our findings were in good agreement with other re-
ports showing that systemic administration of CpG-ODN

Fig. 2. Inhibition of tumor growth and metastasis of CT26 colon cancer cells by CpG-ODN. CT26 (1  106 cells) were injected s.c. into the shaved back of
Balb/c mice (n = 7–10 for each group) on day 0. CpG-ODN (20 lg) was administered i.p. either once on day 18 or twice on days 18 and 25. (A) Tumor volume
was measured 2 times a week. (B) Lungs were removed on day 42, and the number of metastatic nodules was subsequently counted. *P < 0.01 compared
with PBS-treated control group. Values are expressed as median (horizontal bar).
 Han-A Kim et al. / Cancer Letters 274 (2009) 160–164
Table 1
CpG-ODN administered after surgical removal of primary CT26 tumors
inhibits pulmonary metastasis
Expt. No. No. of mice with lung metastasis/total mice
(%)
PBS CpG-ODN
Day 18 Days 18 and
25
1 2/7 (28.6) 1/6 (16.7) 0/6 (0)
2 2/6 (33.3) 0/5 (0) 0/5 (0)
Photographs of lung
tissues
CT26 (1  106 cells) were inoculated s.c. into the shaved back of Balb/c
mice on day 0. Once the tumor reached the size of 15–20 mm in diameter,
17 days after tumor inoculation, it was surgically removed. CpG-ODN
(20 lg) was administered i.p. on day either 18 or days 18 and 25. Mice
were sacrificed on day 49 and compared with PBS-treated control group.
had preventive anti-metastatic effects when CpG-ODN was
administered prior to tumor cells inoculation [20,21] and
that local injections of CpG-ODN into the margin of a tu-
mor also inhibit tumor metastasis to distant organs [22].
However, little is known about the effect of systemic
administration of CpG-ODN on the growth of metastasized
tumor cells. To demonstrate whether CpG-ODN was also
effective in inhibiting the growth of established metasta-
sized tumor cells, CpG-ODN was administered after the
injection of B16F10 tumor cells. CpG-ODN significantly re-
duced lung tumor colonization, although its inhibitory
activity was less prominent compared with that seen when
CpG-ODN was administered prior to tumor cells injection.
We confirmed this effect of CpG-ODN with the observation
that CpG-ODN significantly inhibited the growth of metas-
tasized CT26 colon cells in the lungs when CpG-ODN was
administered systemically following surgical resection of
the primary tumor. Weigel et al. [23] reported improved
survival when CpG-ODN was administered in a state of
minimal disease following surgical resection of a tumor.
These data further support our findings that CpG-ODN is
capable of inhibiting the growth of metastasized tumor
cells.
At present, we do not know the reason why intraperito-
neally administered CpG-ODN has no effect on primary co-
lon cancer cell growth whereas exhibits inhibition on
metastatic tumor cells? In our view, tumor cell number
may affect the differences, i.e., the number of metastasized
tumor cells must be much less compared with subcutane-
ously inoculated cell number. Additionally, differences in
microenvironment between primary tumor site (subcuta-
neous tissue) and secondary organ (lung) which can deter-
mine the degree of distribution of CpG-ODN as well as
CpG-induced anti-tumor immune cells (T, natural killer
and dendritic cells) may also act as factors. Additional
studies are required to address this issue.
In summary, surgical resection is the main therapeutic
strategy for the treatment of malignant solid cancers.
Unfortunately, metastasis may well have occurred by the
163
time of diagnosis, and the majority of these patients tend
to experience recurrence with a poor prognosis, despite
extensive chemotherapy and radiotherapy. Immunother-
apy offers a new treatment option with the potential to im-
prove the overall survival of this patient population. In this
regard, our study demonstrates immunotherapy with CpG-
ODN was effective in controlling established micrometas-
tasis. We hope that the ability of this type of immunopre-
vention to inhibit micrometastasis can eventually be tested
in human clinical trials.
Conflict of interest
None declared.
Acknowledgements
This work was supported by Grant C00348 from the
Korea Research Foundation, and by Grant 0620330-1 from
National R&D Program for Cancer Control, Ministry of
Health and Welfare, Republic of Korea.
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