SUCCESSFUL TREATMENT OF INTRACRANIAL GLIOMAS IN RAT BY OLIGODEOXYNUCLEOTIDES CONTAINING CPG MOTIFS1
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Vol. 6, 2469 –2473, June 2000 Clinical Cancer Research 2469
Successful Treatment of Intracranial Gliomas in Rat by
Oligodeoxynucleotides Containing CpG Motifs1
Antoine F. Carpentier,2,3 Jun Xie,3 Karima properties have been linked to a much higher frequency of
Mokhtari, and Jean-Yves Delattre unmethylated CpG dinucleotides (CpG motifs) in bacterial
Biologie des Interactions Neurones/Glie, Institut National de la Santé DNA than in vertebrate DNA (5). CpG motifs flanked by two 5⬘
et de la Recherche Médicale U-495, Université Pierre Marie Curie, purines and two 3⬘ pyrimidines appear as the most potent
and Fédération de neurologie Mazarin [A. F. C., J. X., J.-Y. D.], and immunostimulatory sequences (5). Synthetic ODNs containing
Laboratoire de Neuropathologie Raymond Escourolles [K. M.], such hexamers have immunological effects similar to those seen
Hôpital de la Pitié-Salpêtrière, 75651 Paris Cedex 13, France
with bacterial DNA and are promising vaccine adjuvants (5–7).
Malignant glioma patients exhibit depressed in vitro and in
ABSTRACT vivo reactivity of peripheral blood and tumor-infiltrating lym-
Phosphorothioate oligodeoxynucleotides with CpG mo- phocytes (8, 9), which has been attributed to the decreased IL-2
tifs (CpG-ODNs) activate various immune cell subsets and sensitivity of CD4⫹ lymphocytes (10) and local secretion, by
induce production of numerous cytokines. To evaluate glioma cells, of the immunosuppressive factors, transforming
whether CpG-ODNs can induce rejection of established tu- growth factor-2, prostaglandin E2, and IL-10 (11–13). Treat-
mors, Lewis rats were inoculated intracerebrally with syn- ment with an immunostimulatory agent that would reverse this
geneic CNS-1 glioma cells and subsequently injected with immunosuppressive tumor environment might allow the rejec-
CpG-ODNs into the tumor bed. Although all of the control tion of glioma cells by the immune system. Thus, we evaluated
rats (n ⴝ 14) died within 23 days, 88% of the animals (n ⴝ whether an ODN with a CpG-ODN could lead to glioma rejec-
8) treated with a single CpG-ODN injection 5 days after tions. Immunocompetent rats were inoculated with syngeneic
tumor inoculation showed long-term survival (>90 days; CNS1 glioma cells and subsequently treated with CpG-ODNs
P < 0.002). CpG-ODNs increased tumoral infiltration with directly into the tumor bed to bypass the blood-brain barrier
macrophage/microglial cells, CD8, and natural killer lym- (14).
phocytes. CpG-ODN-cured animals were further protected
against a second tumor challenge. CpG-ODNs had no effect
on a s.c. CNS1 tumor in nude mice, which suggested that MATERIALS AND METHODS
CpG-ODN is not directly cytotoxic and that immunostimu- Oligonucleotides. Purified single-stranded ODNs were
lation is required for the antitumoral effect. These findings purchased from Genset (Paris, France). The sequences used in
suggest that intratumoral injections of CpG-ODNs repre- this study were CpG-ODN, 5⬘-TGACTGTGAACGTTC-
sent a new immunotherapeutic approach in human gliomas, GAGATGA, which contains two CpG dinucleotides, one being
which overcome the need for the selection and purification part of an immunostimulatory sequence (5⬘-AACGTT), and
of a tumoral antigen. IMM-ODN, 5⬘-TGACTGTGAAGGTTAGAGATGA, in which
both CpG motifs had been mutated, as described by Roman et
INTRODUCTION al. (6). Lipopolysaccharide levels in the ODNs, assessed by the
Bacterial DNA can activate in vivo various immune cell Limulus assay, were ⬍1 ng/mg (BioWhittaker, Emerainville,
subsets, including B cells, macrophages, or NK4 cells, and France).
induce production of a wide variety of cytokines, such as tumor Glioma Cell Line and in Vitro Toxicity Assays. The
necrosis factor-␣, IFN-␥, or IL-12 (1– 4). These immunogenic murine glioma cell line CNS-1 (15), kindly provided by Dr.
W. F. Hickey (Hanover, NH), was cultivated in RPMI 1640
supplemented with 10% FCS (Boehringer, Meylan, France). For
toxicity assays, 10,000 CNS-1 cells were plated in 25-cm2
culture flasks, and ODNs were added (at 5 ⫻ 10⫺6 or 5 ⫻ 10⫺7
Received 11/30/99; revised 3/6/00; accepted 3/7/00. M) on day 1 of culture. The cells were harvested on day 3 by
The costs of publication of this article were defrayed in part by the
trypsinization and counted on a Malassez hematocytometer. Cell
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to viability was then checked by trypan blue exclusion. The ex-
indicate this fact. periments were performed in triplicate, and results were ex-
1
Supported by Assistance Publique-Hopitaux de Paris, Université Paris pressed as the means ⫾ SD.
VI, and Institut National de la Santé et de la Recherche Médicale.
2 Tumor Implantation and in Vivo Treatment. For in-
To whom requests for reprints should be addressed, at Fédération de
neurologie Mazarin, 47 Boulevard de l’hôpital, Hôpital de la Salpêtrière, tracerebral tumor implantation, anesthetized 6- to 7-week-old
75013 Paris, France. Fax: 011-33-1-44-23-81-85; E-mail: antoine. male Lewis rats (CERJ, Lyon, France) were placed in a ster-
carpentier@psl.ap-hop-paris.fr. eotactic frame, and a burr hole was drilled 2 mm posterior and
3
A. F. C. and J. X. contributed equally to this work.
4 4 mm lateral to the bregma. The brain was punctured by a
The abbreviations used are: NK, natural killer; IL, interleukin; ODN,
phosphorothioate oligodeoxynucleotide; CpG-ODN, ODN with an im- Hamilton syringe, and the needle was inserted 5 mm deep.
munostimulatory CpG motif. Viable glioma cells (1 ⫻ 105), suspended in 10 l of RPMI
Downloaded from clincancerres.aacrjournals.org on February 19, 2021. © 2000 American Association for Cancer
Research.2470 Treatment of Gliomas by CpG Oligodeoxynucleotides
Fig. 1 Survival curves of Lewis rats inoculated intracerebrally with Fig. 2 Survival curves of Lewis rats inoculated intracerebrally with
CNS1 glioma cells and injected into the tumor bed with 100 g of CNS1 glioma cells and injected into the tumor bed with various doses
CpG-ODN on day 1 (– 䡠 – 䡠 –), day 5 (䡠䡠䡠䡠), day 9 (– – –), or with sodium of CpG-ODN or sodium chloride (----) on day 1. Long-term survival
chloride (----). Survival was increased in CpG-ODN-treated animals (⬎90 days) was increased to 60% (n ⫽ 5; P ⬍ 0.03), 40% (n ⫽ 5; P ⫽
with a long-term survival (⬎90 days) of 67% (n ⫽ 6; P ⬍ 0.01), 88% 0.22), and 0% (n ⫽ 5; P, not significant), respectively, for rats treated
(n ⫽ 8; P ⬍ 0.002), and 29% (n ⫽ 7; P ⬍ 0.07), respectively, for rats with 50 (– – –), 10 (– 䡠 – 䡠 –), and 1 g (䡠䡠䡠䡠), whereas all of the control
treated on days 1, 5, and 9, whereas all of the control animals (n ⫽ 14) animals (n ⫽ 5) died within 3 weeks.
died within 23 days.
1640, were injected. The ODNs, dissolved to the appropriate
concentration in 7 l of saline, were injected similarly.
Six-week-old male nude mice (CERJ, Lyon, France) were
injected s.c. with 105 CNS1 cells into the right flank. Five days
later, mice were injected into the tumor bed either with 50 l
sodium chloride or 100 g of ODNs dissolved in 50 l of saline.
Tumor volumes were assessed with a caliper every 4 days using
the formula: /6 ⫻ length ⫻ width2 (16).
Histology/Immunohistochemistry. For histology and
Fig. 3 Survival curves of rats (n ⫽ 5) previously cured by CpG-ODN
histochemistry analysis, the rats were killed just before the injections (– – –) and subjected to a second intracranial tumor challenge
expected date of death. After induction of anesthesia, the ani- with 106 CNS1 cells, 12 weeks after completion of the initial treatment.
mals underwent intracardiac perfusion with 2% paraformalde- All of them survived without any treatment, whereas all of the control
hyde in PBS. The brains were then fixed for 2 h in the same rats (----, n ⫽ 4) died (P ⬍ 0.001).
fixative and either embedded in paraffin for staining with H&E
or snap-frozen and stored at ⫺80°C. For immunohistochemis-
try, frozen sections (10 m) were thawed, incubated at room vehicle or with 7 l of vehicle alone on day 1 as control (Fig.
temperature for 1 h with 10% normal goat serum (Jackson 1). The control animals injected with vehicle (n ⫽ 14) showed
ImmunoResearch, West Grove, PA), and subsequently incu- a median survival time of 15 days, all dying within 23 days.
bated for 2 h with the primary antibody. The following primary Survival was increased in CpG-ODN-treated animals with long-
antibodies (all from Serotec, Oxford, UK) were used: OX8 term survival (⬎90 days) in 67% (n ⫽ 6; P ⬍ 0.01), 88% (n ⫽
(1:100) directed against CD8-positive T cells; 3.2.3 (1:100) 8; P ⬍ 0.002), and 29% (n ⫽ 7; P ⬍ 0.07) for those treated on
against NK cells, and ED1 (1:500) against microglial cells and days 1, 5, and 9, respectively, after tumor implantation. The
macrophages. The sections were incubated additionally for 1 h difference in terms of survival between animals treated on day
with FITC-conjugated goat antimouse IgG1 (1:50; Clini- 5 or 9 with CpG-ODNs was statistically significant (P ⫽ 0.03).
sciences, Montrouge, France) and were examined under a Leica None of the surviving animals exhibited neurological disabili-
fluorescent microscope. Quantitative analysis of labeled cells ties, and histopathological studies of six animals killed on day
was performed using two different sections for each sample. In 90 showed no tumor cells but showed enlarged ventricles in the
each section, the number of positive cells in three random fields vicinity of the former tumor site. Tumor growth was found to be
measuring 0.135 mm2 were counted by an investigator who was the cause of death for all of the nonsurviving animals.
blinded to the animal history. To assess the minimal dose of required CpG-ODNs, rats
Statistics. Statistical significance for survival was as- were injected intracerebrally 1 day after CNS1 tumor graft with
sessed using the Kaplan-Meier analysis. Comparisons of cellular 50, 10, or 1 g of CpG-ODN in 7 l of vehicle (Fig. 2). Long-term
infiltrates were done by the Mann-Whitney U test. survival (⬎90 days) was observed in 60% (n ⫽ 5; P ⬍ 0.03),
40% (n ⫽ 5; P ⫽ 0.22), and 0% (n ⫽ 5; P, not significant) for
RESULTS rats treated with 50, 10, and 1 g, respectively, whereas all of
Injections of CpG-ODNs into Intracerebral Glioma. the control animals injected with the vehicle died (n ⫽ 5).
In the first set of experiments, each rat was inoculated intrac- To assess whether CpG-ODNs could also act on a distant
erebrally with 105 CNS1 cells and then injected 1, 5, or 9 days intracerebral tumor, rats were grafted with 105 CNS1 cells into
later in the same location with 100 g of CpG-ODN in 7 l of the right cerebral hemisphere in two different locations (4-mm
Downloaded from clincancerres.aacrjournals.org on February 19, 2021. © 2000 American Association for Cancer
Research.Clinical Cancer Research 2471
Fig. 4 Histological analysis of tumors in rats implanted on day 0 with CNS1 cells, injected with saline (a and c) or CpG-ODN (b and d) on day 5,
and killed on day 6. CNS1 tumor cells invade the normal parenchyma in a control rat (a), whereas in a CpG-ODN-treated animal (b), viable tumor
cells are surrounded by an area of pyknotic cells and a cicatricial tissue, where macrophages and lymphocytes are seen but where tumor cells are
sparse. Normal parenchyma is seen on the right. Inset, enlargement of the pyknotic area showing apoptotic cells (H&E staining, ⫻200; inset, ⫻400).
Immunohistochemical analysis (⫻400) with OX8 antibodies showing higher tumoral infiltration with CD8 T lymphocytes in CpG-ODN-injected
animals (d) than in control rats (c).
distance) and also were injected 5 days later in only one of the In Vitro Toxicity of CpG-ODNs. To investigate whether
two locations with 50 g CpG-ODN in 7 l of vehicle or in 7 the effect of CpG-ODNs could be mediated by a direct toxicity
l of vehicle alone. The control animals (n ⫽ 6) all died within on glioma cells, 10,000 CNS1 cells were plated in 25-cm2
25 days, whereas long-term survival (⬎90 days) was seen in culture flasks. CpG-ODN or IMM-ODN was added at two
44% of those treated with CpG-ODNs (n ⫽ 7; P ⬍ 0.05). different concentrations (5 ⫻ 10⫺6 or 5 ⫻ 10⫺7 M) on day 1 of
Second Challenge with CNS1 Cells in Rats Cured by culture, and the cells were counted on day 3. No inhibition of
CpG-ODNs. Twelve weeks after completion of the initial proliferation was seen in either of the concentrations tested
treatment, some of the surviving animals (n ⫽ 5) that had not when compared with the controls (mean ⫾ SD: 97,000 ⫾ 1,500
been killed for histological evaluation were subjected to a sec- for control; 73,000 ⫾ 1,000 and 88,000 ⫾ 1,500 for 5 ⫻ 10⫺6
⫺7
ond intracranial tumor challenge with 106 CNS1 cells. All of M and 5 ⫻ 10 M CpG-ODN, respectively; 77,000 ⫾ 2,000 and
them survived without any treatment, whereas all of the control 99,000 ⫾ 1,000 for 5 ⫻ 10⫺6 M and 5 ⫻ 10 M CpG-ODN,
rats (n ⫽ 4) died (P ⬍ 0.001; Fig. 3). Histopathological studies respectively).
did not reveal any evidence of residual tumor cells in the Treatment with CpG-ODNs in Nude Mice. To further
surviving rats. assess that the effects of CpG-ODN did not result from a direct
Intratumoral Treatment with an ODN That Lacked the cytotoxicity on CNS1 cells but rather involved the immune
CpG Motifs. To assess whether the tumor rejection observed system, nude mice that had been implanted s.c. with 105 CNS1
with CpG-ODNs was dependent on the CpG motifs, rats were cells were divided into two groups (n ⫽ 4 in each) and were
grafted intracerebrally with 105 CNS1 cells and then injected injected on day 5 at the same location with a single injection of
24 h later in the same location with 7 l of vehicle (n ⫽ 5) or either 50 l of sodium chloride or 100 g of CpG-ODN. No
with 100 g of IMM-ODN that lacked the CpG motifs (n ⫽ 5). significant differences in tumor volumes measured on day 12
IMM-ODN injections did not affect median survival time when were seen between the two groups (mean tumor volume ⫾ SE:
compared with controls (14 versus 15 days), and all of the rats 628 ⫾ 158 mm3 for saline; 613 ⫾ 74 mm3 for CpG-ODN; P ⫽
in both groups died. 0.56).
Downloaded from clincancerres.aacrjournals.org on February 19, 2021. © 2000 American Association for Cancer
Research.2472 Treatment of Gliomas by CpG Oligodeoxynucleotides
though a diffusion of CpG-ODN through the cerebral spinal
fluid cannot be ruled out. We have recently reported that CpG-
ODN injections could also induce rejection of syngenic neuro-
blastoma in mice through an immune mechanism (16). The
efficacy of CpG-ODNs in both gliomas and neuroblastomas
suggests that the antitumoral properties of CpG-ODNs may be
applied to several types of cancers, the spectrum of which
remains to be defined. Intratumoral injections of the MY-1
extract of Mycobacterium bovis have been shown previously to
inhibit tumor growth in murine models of carcinoma (17).
Because the activity of MY-1 was dependent on the presence of
bacterial DNA, at least part of the effects of MY-1 might be
attributable to the immunostimulatory CpG motifs included in
bacterial DNA. However, MY-1 failed to improve survival in a
Fig. 5 Numbers of OX8- (CD8 T cells), 3.2.3- (NK cells), and ED1- mouse glioma model (18). The superior efficacy of CpG-ODNs
(macrophages and microglial cells) positive tumor-infiltrating cells in described in the present study may be related to the amplifica-
CpG-ODN-treated animals (f) and in control rats (䡺; mean per 0.135- tion of immunostimulatory sequences and the enhanced stability
mm2 field ⫾ SE). The rats were implanted on day 0 with CNS1 cells,
injected with CpG-ODN or saline on day 5, and killed on day 6 (ⴱ, P ⬍ of ODNs (19) when compared with crude bacterial DNA.
0.05). The mechanisms responsible for the antitumoral effects
could result from a direct cytotoxicity of CpG-ODNs. However,
no cytotoxicity on CNS1 cells was detected in vitro, and their
antitumoral effects were abrogated in nude mice, which strongly
Recruitment of Macrophage/Microglial, NK, and CD8 suggests that eradication of glioma cells were immune-medi-
Tumor-infiltrating Cells by CpG-ODN Injections. To study ated. The increased tumoral infiltration with macrophage/micro-
the effects of CpG-ODN on tumors, rats implanted on day 0 glial cells, CD8, and NK lymphocytes seen after CpG-ODN
with CNS1 were injected with CpG-ODN or with saline on day
injections when compared with the control group support this
5. The rats were killed on day 6, and the brains were removed
hypothesis. Interestingly, rats that were cured by CpG-ODN
for histological evaluation. On H&E-stained brain tissue sec-
injections were protected additionally against a new tumor chal-
tions, CpG-ODN-treated animals showed intratumoral infiltra-
lenge, which showed that a long-term immunity was primed.
tion and large areas of apoptotic cells at the periphery of the
tumors, whereas the controls showed less infiltration and no CpG-ODN has been reported to induce endogenous release of
apoptosis (Fig. 4). The number of OX8- (CD8 T cells), 3.2.3- IL-12 by macrophages (4). Because IL-12 displays antitumor
(NK cells), and ED1- (macrophages and microglial cells) posi- effects in murine glioma models (20, 21), the CpG-ODNs’
tive tumor-infiltrating cells were, respectively, 4.5-, 4.4-, and antitumoral effects could be mediated, at least in part, by IL-12
3-fold higher in CpG-ODN-treated animals than those cells in secretion. CpG-ODNs carry out the advantage over IL-12 alone
the controls (mean of positive cells per 0.135-mm2 field ⫾ SE: to trigger a sustained expression of IL-12 for at least 8 days (22),
132 ⫾ 9 versus 29 ⫾ 7 for OX8⫹ cells, P ⬍ 0.05; 71 ⫾ 3 versus whereas the half-life of exogenous IL-12 is ⬍10 h (23).
16 ⫾ 5 for 3.2.3⫹ cells, P ⬍ 0.05; and 207 ⫾ 23 versus 68 ⫾ Despite the susceptibility of the Lewis strain to develop
19 for ED1⫹ cells, P ⬍ 0.05; Figs. 4 and 5). No significant experimental acute encephalomyelitis, rats injected with CpG-
differences were seen between control animals injected with ODN showed no short- or long-term neurological impairment.
sodium chloride and noninstrumented animals killed on day 6 Histological studies of the brains of cured animals showed no
after tumor inoculation (mean of positive cells per 0.135-mm2 abnormalities other than enlarged ventricles in the vicinity of the
field ⫾ SE: 35 ⫾ 9 for OX8⫹ cells; 20 ⫾ 5 for 3.2.3⫹ cells; original tumor sites. Direct injections of CpG-ODN in normal
and 82 ⫾ 19 for ED1⫹ cells). Histological evaluation of the rat brains yielded only minimal necrosis along the needle tracks
surrounding parenchyma and contralateral hemisphere was nor- (data not shown). Although additional toxicological studies are
mal, except for scattered reactive microglial cells in the ipsilat- mandatory, CpG-ODN injections seemed thus far to have no
eral hemisphere of the CpG-ODN-injected animals. deleterious effect on the normal brain parenchyma.
CpG-ODNs are promising adjuvants for immunization
DISCUSSION against selected antigens such as hepatitis B (6, 24) or for
These data show that rejection of a syngeneic intracerebral immunization against a tumor antigen in a murine lymphoma
glioma can be achieved by intratumoral injections of CpG- model (25). Our data show that direct injections of CpG-ODN
ODNs. Although none of the control animals survived the tumor alone into tumors represent a simple means of achieving ther-
challenge, ⬎85% of the rats treated 5 days after the tumor apeutic effects without the need for selection and purification of
inoculation showed long-term survival and tumor eradication. tumor antigens. Human gliomas display a locally invasive pat-
Moreover, increased survival was seen in rats bearing two tern of growth and rarely metastasize, which makes local treat-
separate tumors but treated only at one tumor site, which sug- ment clinically relevant. Intratumoral injections of CpG-ODN,
gests that the treatment may also be active on tumor cells therefore, may represent a new immunotherapeutic approach in
located at some distance from the CpG-ODN injections, al- gliomas and warrant additional studies in other malignancies.
Downloaded from clincancerres.aacrjournals.org on February 19, 2021. © 2000 American Association for Cancer
Research.Clinical Cancer Research 2473
ACKNOWLEDGMENTS tor- in glioblastoma. Preferential production of transforming growth
We thank B. Zalc, J. J. Hauw, M. Mallat, C. Jacques, P. F. Pradat, factor-2. J. Immunol., 143: 3222–3229, 1989.
and E. Altman for their helpful comments during the redaction of the 13. Huettner, C., Paulus, W., and Roggendorf, W. Messenger RNA
manuscript. expression of the immunosuppressive cytokine IL-10 in human gliomas.
Am. J. Pathol., 146: 3173–3122, 1995.
14. Saijo, Y., Perlaky, L., Wang, H., and Busch, H. Pharmacokinetics,
REFERENCES tissue distribution, and stability of antisense oligodeoxynucleotide phos-
1. Cowdery, J. S., Chace, J. H., Yi, A. K., and Krieg, A. M. Bacterial phorothioate ISIS 3466 in mice. Oncol. Res., 6: 243–249, 1994.
DNA induces NK cells to produce IFN-␥ in vivo and increases the 15. Kruse, C. A., Molleston, M. C., Parks, E. P., Schiltz, P. M.,
toxicity of lipopolysaccharides. J. Immunol., 156: 4570 – 4575, 1996. Kleinschmidt-DeMasters, B. K., and Hickey, W. F. A rat glioma model,
2. Ballas, Z. K., Rasmussen, W. L., and Krieg, A. M. Induction of NK CNS-1, with invasive characteristics similar to those of human gliomas:
activity in murine and human cells by CpG motifs in oligodeoxynucle- a comparison to 9L gliosarcoma. J. Neuro-oncol., 22: 191–200, 1994.
otides and bacterial DNA. J. Immunol., 157: 1840 –1845, 1996. 16. Carpentier, A. F., Chen, L., Maltonti, F., and Delattre, J. Y. Oli-
3. Halpern, M. D., Kurlander, R. J., and Pisetsky, D. S. Bacterial DNA godeoxynucleotides containing CpG motifs can cure neuroblastoma in
induces murine interferon-␥ production by stimulation of interleukin-12 mice. Cancer Res., 59: 5429 –5432, 1999.
and tumor necrosis factor-␣. Cell. Immunol., 167: 72–78, 1996.
17. Tokunaga, T., Yamamoto, H., Shimada, S., Abe, H., Fukuda, T.,
4. Chace, J. H., Hooker, N. A., Mildenstein, K. L., Krieg, A. M., and Fujisawa, Y., Furutani, Y., Yano, O., Kataoka, T., and Sudo, T. Anti-
Cowdery, J. S. Bacterial DNA-induced NK cell IFN-␥ production is tumor activity of deoxyribonucleic acid fraction from Mycobacterium
dependent on macrophage secretion of IL-12. Clin. Immunol. Immuno- bovis BCG. I. Isolation, physicochemical characterization, and antitu-
pathol., 84: 185–193, 1997. mor activity. J. Natl. Cancer Inst., 72: 955–962, 1984.
5. Krieg, A. M., Yi, A. K., Matson, S., Waldschmidt, T. J., Bishop, 18. Nakaichi, M., Takeuchi, A., Shitara, N., and Takakura, K. The
G. A., Teasdale, R., Koretzky, G. A., and Klinman, D. M. CpG motifs
antitumor activity of the DNA fraction from Mycobacterium bovis BCG
in bacterial DNA trigger direct B-cell activation. Nature (Lond.), 374:
(MY-1) for glioblastoma. J. Vet. Med. Sci., 57: 583–585, 1995.
546 –549, 1995.
19. Crooke, R. M. In vitro toxicology and pharmacokinetics of anti-
6. Roman, M., Martin-Orozco, E., Goodman, J. S., Nguyen, M. D.,
sense oligonucleotides. Anticancer Drug Des., 6: 609 – 646, 1991.
Sato, Y., Ronaghy, A., Kornbluth, R. S., Richman, D. D., Carson, D. A.,
and Raz, E. Immunostimulatory DNA sequences function as T helper- 20. Kishima, H., Shimizu, K., Miyao, Y., Mabuchi, E., Tamura, K.,
1-promoting adjuvants. Nat. Med., 3: 849 – 854, 1997. Tamura, M., Sasaki, M., and Hakakawa, T. Systemic interleukin 12
7. Lipford, G. B., Sparwasser, T., Bauer, M., Zimmermann, S., Koch, displays anti-tumour activity in the mouse central nervous system. Br. J.
E. S., Heeg, K., and Wagner, H. Immunostimulatory DNA. sequence- Cancer, 78: 446 – 453, 1998.
dependent production of potentially harmful or useful cytokines. Eur. 21. Jean, W. C., Spellman, S. R., Wallenfriedman, M. A., Hall, W. A.,
J. Immunol., 27: 3420 –3426, 1997. and Low, W. C. Interleukin-12-based immunotherapy against rat 9L
8. Brooks, W. H., Netsky, M. G., Normansell, D. E., and Horwitz, D. A. glioma. Neurosurgery, 42: 850 – 856, 1998.
Depressed cell-mediated immunity in patients with primary intracranial 22. Krieg, A. M., Love-Homan, L., Yi, A. K., and Harty, J. T. CpG
tumors. Characterization of a humoral immunosuppressive factor. J. DNA induces sustained IL-12 expression in vivo and resistance to
Exp. Med., 136: 1631–1647, 1972. Listeria monocytogenes challenge. J. Immunol., 161: 2428 –2434, 1998.
9. Miescher, S., Whiteside, T. L., Carrel, S., and Von Fliedner, V. 23. Atkins, M. B., Robertson, M. J., Gordon, M., Lotze, M. T.,
Functional properties of tumor-infiltrating and blood lymphocytes in DeCoste, M., DuBois, J. S., Ritz, J., Sandler, A. B., Edington, H. D.,
patients with solid tumors: effects of tumor cells and their supernatants Garzone, P. D., Mier, J. W., Canning, C. M., Battiato, L., Tahara, H.,
on proliferative responses of lymphocytes. J. Immunol., 136: 1899 – and Sherman, M. L. Phase I evaluation of intravenous recombinant
1907, 1986. human interleukin 12 in patients with advanced malignancies. Clin.
10. Elliott, L. H., Brooks, W. H., and Roszman, T. L. Inability of Cancer Res., 3: 409 – 417, 1997.
mitogen-activated lymphocytes obtained from patients with malignant 24. Davis, H. L., Weeranta, R., Waldschmidt, T. J., Tygrett, L., Schorr,
primary intracranial tumors to express high affinity interleukin 2 recep- J., and Krieg, A. M. CpG DNA is a potent enhancer of specific
tors. J. Clin. Invest., 86: 80 – 86, 1990. immunity in mice immunized with recombinant hepatitis B surface
11. Lauro, G. M., Di Lorenzo, N., Grossi, M., Maleci, A., and Guidetti, antigen. J. Immunol., 160: 870 – 876, 1998.
B. Prostaglandin E2 as an immunomodulating factor released in vitro by 25. Weiner, G. J., Liu, H. M., Wooldridge, J. E., Dahle, C. E., and
human glioma cells. Acta Neuropathol. (Berl.), 69: 278 –282, 1986. Krieg, A. M. Immunostimulatory oligodeoxynucleotides containing the
12. Bodmer, S., Strommer, K., Frei, K., Siepl, C., de Tribolet, N., Heid, CpG motif are effective as immune adjuvants in tumor antigen immu-
I., and Fontana, A. Immunosuppression and transforming growth fac- nization. Proc. Natl. Acad. Sci. USA, 94: 10833–10837, 1997.
Downloaded from clincancerres.aacrjournals.org on February 19, 2021. © 2000 American Association for Cancer
Research.Successful Treatment of Intracranial Gliomas in Rat by
Oligodeoxynucleotides Containing CpG Motifs
Antoine F. Carpentier, Jun Xie, Karima Mokhtari, et al.
Clin Cancer Res 2000;6:2469-2473.
Updated version Access the most recent version of this article at:
http://clincancerres.aacrjournals.org/content/6/6/2469
Cited articles This article cites 23 articles, 10 of which you can access for free at:
http://clincancerres.aacrjournals.org/content/6/6/2469.full#ref-list-1
Citing articles This article has been cited by 17 HighWire-hosted articles. Access the articles at:
http://clincancerres.aacrjournals.org/content/6/6/2469.full#related-urls
E-mail alerts Sign up to receive free email-alerts related to this article or journal.
Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Subscriptions Department at pubs@aacr.org.
Permissions To request permission to re-use all or part of this article, use this link
http://clincancerres.aacrjournals.org/content/6/6/2469.
Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
Rightslink site.
Downloaded from clincancerres.aacrjournals.org on February 19, 2021. © 2000 American Association for Cancer
Research.You can also read
