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Experimental Therapeutics, Preclinical Pharmacology |
University of Minnesota Cancer Center, Department of Pediatrics, Division of Pediatric Hematology/Oncology and Bone Marrow Transplantation, Minneapolis, Minnesota 55455 [B. J. W., B. R. B.]; Department of Surgery, Mayo Clinic and Foundation, Rochester, Minnesota 55905 [D. A. R.]; Department of Veterans Affairs Medical Center and the Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242 [A. M. K.]; and Coley Pharmaceutical Group, Wellesley, Massachusetts 02481 [A. M. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSSurgical Resection of TumorStatisticsRESULTSDISCUSSIONREFERENCES |
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Experimental Design: The systemic administration of CpG 2006 was tested beginning on day 9 or 19 when tumors were not yet palpable or palpable, respectively, and after surgical resection of the tumor. CpG was also administered in combination with the chemotherapeutic agents, cyclophosphamide (CY) and topotecan, or surgical resection.
Results: Systemic CpG prolonged survival when begun at day 9 but had no effect with a large tumor burden. CpG administered after surgical resection of tumor significantly improved survival of mice (P < 0.03). On day 9, CY plus CpG 2006 resulted in improved survival compared with CY alone (70% versus 41%, respectively). Survival was significantly improved when CpG 2006 was administered systemically with CY beginning on day 19 (15% versus 0% survival). The administration of CpG 2006 with topotecan significantly improved survival in mice with large tumors. Cell-depletion studies demonstrated that the antitumor effects of systemically administered CpG 2006 combined with CY were predominantly T cell dependent.
Conclusions: These data are the first to show that immune stimulatory agents such as CpGs may enhance the antitumor effects of chemotherapeutic agents and improve survival after surgical resection of a solid tumor.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSSurgical Resection of TumorStatisticsRESULTSDISCUSSIONREFERENCES |
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The essential components of an immune response to tumor are tumor antigen presentation by an APC3 (B cell, macrophage, or DC) and the interaction of these APCs with T cells to produce a specific CTL response. DCs are the most potent APCs and are critical for inducing antigen-specific antitumor immune responses. Enhancing T-cell/APC interaction by augmenting APC number and/or function or by enhancing T-cell stimulation may be effective in increasing antitumor immune responses.
To enhance an antitumor immune response to solid tumors, strategies to augment APC or T-cell number and/or function have used cytokines such as Flt3L, granulocyte/macrophage-colony stimulating factor, and IL-2 and IL-12 (4, 5, 6, 7)
. Krieg (8)
have described the use of a synthetic ODN containing CpG motifs that may enhance APC function when administered systemically or at the site of tumor. These CpG motifs, characteristic of prokaryotic DNA, are unmethylated DNA sequences containing characteristic CpG dinucleotides flanked by two 5' purines and two 3' pyrimidines (5'-purine-purine-CG-pyrimidine-pyrimidine-3'). These CpG motifs are thought to be responsible for initiating a potent Th1-like innate immune response to intracellular pathogens in mice, primates, and humans (8, 9, 10, 11)
. CpG ODNs have been shown to activate APCs (particularly tissue DCs), leading to up-regulation of costimulatory molecules and the secretion of cytokines (IFN-
, IL-12, IL-6, and tumor necrosis factor-
) necessary for productive Th1/Tc1-cell activation (8
, 12
, 13)
. CpG ODNs have been shown to have antitumor effects when used as a single agent in murine models of leukemia, lymphoma, melanoma, neuroblastoma, and a rodent model of glioma (14, 15, 16, 17, 18, 19)
. These antitumor effects are observed both with local and systemic injection of CpGs. The predominant effector cells responsible for the antitumor effect have varied and include NK cells, T cells, and/or macrophages, depending on the model system studied.
To determine whether CpG ODN administration could lead to an immune response to murine rhabdomyosarcoma implanted in an orthotopic location, CpG ODN was administered systemically in a setting of either minimal or gross tumor. Because agents such as CpG ODN will rarely be used as single agents in cancer treatment, we sought to determine whether coadministration with the chemotherapeutic agents CY or topotecan would improve antitumor responses in murine rhabdomyosarcoma. CY has been a major component of rhabdomyosarcoma treatment for >30 years (1) . CY has been shown to augment immunotherapy strategies in model systems including in murine rhabdomyosarcoma (20, 21, 22) . Topotecan is a new agent in the treatment of rhabdomyosarcoma (23) . The mechanism of action of these two drugs is different, and they are both in active clinical trials for the treatment of rhabdomyosarcoma. As in all solid tumors, surgical resection has a significant role in the treatment of rhabdomyosarcoma. Surgery greatly minimizes the burden of disease, and in this state of minimal disease, immunotherapy strategies such as CpG ODNs may be most effective. We report that CpG ODNs combined with chemotherapy or with surgical resection of tumor result in potent antitumor effects, even when therapy is initiated in a setting of bulk disease.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSSurgical Resection of TumorStatisticsRESULTSDISCUSSIONREFERENCES |
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Cells and Cell Culture Reagents
The murine embryonal rhabdomyosarcoma cell line, 76-9 (MHC I weakly positive, MHC II negative; a kind gift from R. Evans, The Jackson Laboratory) was maintained in RPMI 1640 supplemented with 1 mM penicillin/streptomycin, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, and 10% fetal bovine serum (HyClone, Ogden, UT). The cells were maintained at 37°C and 5% CO2.
Immunohistochemistry of in Vivo Tumors
Mice were injected with 76-9 tumor cells (103 cells/mouse) into the left hind limb muscle. One week after injection, the tumor was removed, placed in OCT, and rapidly frozen in liquid nitrogen. The tissue samples were cut into 6-µm sections, and the histological features evaluated using H&E staining. Subsequent sections were stained for the tumor markers actin, desmin, and myogenin. Slides were evaluated using a Leica light microscope with a x40 objective.
CpG ODNs
Phosphorothioate-modified ODN 2006 was provided by the Coley Pharmaceutical Group. ODNs had undetectable endotoxin levels using the limulus amebocyte lysate assay. ODN 2006 was diluted in Tris-EDTA buffer and further diluted in PBS for in vivo use. The ODN 2006 sequence was: TCGTCGTTTTGTCGTTTTGTCGTT. ODNs were injected at 100 µg/dose. This dose was determined to be optimal for the induction of antitumor responses in several systems without the toxicity seen at higher doses. Approximately 4% of the fully phosphorothioate-modified ODN dose is present in the spleen during the first 24 h after injection (24)
, resulting in a splenic concentration of 4 µg/ml.
In Vitro Cytotoxicity Studies.
The 76-9 tumor cells were grown in log phase suspended at 0.1 x 106 cells/ml and plated at 2 ml/well in 6-well plates. CpG 2006 was added at concentrations of 0, 1, 3, 10, 30, or 100 µg/ml. The viability and the cell growth were assessed by trypan blue exclusion 24, 38, and 72 h after the addition of CpG ODN.
In Vivo Tumor Response.
Cohorts of mice (8–10/group) were injected i.m. in the left hind limb muscle with 76-9 cells at a dose of 103/mouse. Cells were injected in 50-µl volumes in sterile PBS. CpG 2006 (100 µg/mouse) was administered systemically (i.p.; 200 µl) on days 9, 12, 16, and weekly for 4 weeks or days 19, 22, 26, and weekly for 4 weeks. In cohorts of mice receiving combined chemotherapy and immunotherapy, CY (200 mg/kg) was administered via i.p. injection 9 or 19 days after tumor injection, or topotecan (10 mg/kg) was administer i.p. 19 days after tumor injection. Nine days was chosen to represent minimal or subclinical disease because tumor is histologically present but not palpable. At 19 days, tumors are large and easily measurable, representing clinically significant tumor burden. Tumor volumes were measured twice weekly using digital Varier calipers. Tumor volumes were calculated as [length x (width)2]/2. The date of death is recorded as the date when mice spontaneously succumbed to tumor or were sacrificed because of a moribund state, ulcerated tumor, or when the tumor measured 1500 mm3 (a volume at which the tumor is not going to regress) in accordance with institutional animal care guidelines. All mice were necropsied at the time of death to confirm the presence of tumor.
In Vivo Depletion of Effector Cells.
To determine the effector cell populations responsible for a CpG + CY-induced immune response, mice were treated with irrelevant rat IgG, anti-NK 1.1 (clone PK136), or anti-CD4 (clone GK1.5) and anti-CD8 (clone 2.43) depleting mAbs given i.p. at a dose of 400 µg each starting on day 8 or 18 (1 day before CpG and/or CY administration) and continued weekly. This mAb administration schedule routinely results in >95% depletion of the targeted cell population for >1 week beyond discontinuation of mAb injections.
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Surgical Resection of Tumor |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSSurgical Resection of TumorStatisticsRESULTSDISCUSSIONREFERENCES |
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Statistics |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSSurgical Resection of TumorStatisticsRESULTSDISCUSSIONREFERENCES |
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSSurgical Resection of TumorStatisticsRESULTSDISCUSSIONREFERENCES |
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). The tumor also expressed the rhabdomyosarcoma tumor markers of actin, desmin, and myogenin (Fig. 1)
.
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. There is a dose-response curve in mortality rates when the tumor dose is increased from 101 to 103 and 105 cells/mouse. The tumor readily metastasizes to the lungs when injected into the hind limb muscle with >80% of mice having pulmonary metastatic disease at the time of death or maximal tumor size (data not shown).
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CpG 2006 Administered Systemically in Combination with CY Enhances the Antitumor Response in the Setting of Minimal Tumor Burden.
CY is an effective agent in the treatment of rhabdomyosarcoma but will not cure the majority of patients when used as a single agent (1)
. Evans (22)
has shown previously that CY is effective in the murine model of rhabdomyosarcoma in reducing tumor burden. To simulate CY responses in patients, a relatively high CY dose (200 mg/kg) was used for tumor reduction. CY led to significantly improved survival when administered 9 days after tumor injection (49% versus 4% in untreated controls at day 107, P 
0.00004); however, 50% of the mice ultimately succumbed to tumor progression (Fig. 3A)
.
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). When combined with CY administered on day 9, CpG 2006 improved the survival of mice (Fig. 3C)
. The systemic administration of CpG 2006 improved the survival of mice when given in combination with CY compared with mice receiving CY alone (70% versus 41%, respectively, P = 0.07). Mice surviving on day 108 were rechallenged with 103 tumor cells in the contralateral hind limb. There was no difference in the mortality rates between mice that received CY alone versus those receiving a combination of CY/CpG, with 90% of the mice succumbing to the tumor rechallenge 40–50 days after injection (data not shown).
CpG 2006 Administered Systemically in Combination with CY Enhances the Antitumor Response in the Setting of Large Tumor Burden.
The use of immunotherapy strategies to treat tumors has been most successful when the tumor burden has been minimal. However, it is not always possible to have a state of minimal residual disease. Therefore, we evaluated the effect that combining CpG ODN with CY would have in the setting of large tumors. The lysis of the tumor cells by the CY should provide more available tumor antigen to be taken up and processed by APCs that have been activated by CpG ODN administration. CY significantly delayed the time to mortality when administered to mice with measurable tumor but did not lead to overall improvement in survival compared with controls (8% versus 3%, respectively; Fig. 4A
). To determine whether systemic CpG administration had any antitumor effects as a single agent in these mice with large tumors, CpG 2006 therapy was initiated on day 19 after tumor injection, continued on days 19, 22, and 26, and then weekly four times. CpG 2006 as a single agent did not have any antitumor effect when systemically administered at this later time point (Fig. 4B)
. Combined CpG and CY administered on day 19 rescued 15% of mice versus uniform lethality in either CpG-treated (Fig. 4B)
or CY-treated (Fig. 4C)
recipients. The fact that combined therapy with systemic CpG and CY lead to improved survival in mice with measurable tumors is further evidenced by the regression of tumor in some mice and the overall slowing of tumor growth in mice treated with combination therapy. The combined administration of CpG 2006 and CY led to tumor regression and slowing of tumor growth (Fig. 4D)
. These data suggest that there may be an additive or synergistic effect of the systemically administered CpG, even in the setting of large tumors.
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or total (Fig. 5B)
loss of the antitumor effect. Depletion of NK cells did not alter survival in either setting. Therefore, under these conditions T cells were found to be the predominant cell involved in mediating an antitumor response in mice that received systemic CpG + CY. There was no effect of depleting T cells or NK cells on the antitumor effects of CY as a single agent, suggesting that at the dose used in these studies, the predominant effect of CY is direct tumor lysis (data not shown).
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. This antitumor effect was further improved in mice that received CpG 2006 and topotecan, with 41% of the combination treated mice surviving compared with mice that received topotecan alone (P = 0.09). These data suggest that the combined antitumor effects of CpG and chemotherapy are not unique to one class of chemotherapeutic agents but may be applicable to several classes of drugs.
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. The survival of mice that underwent surgical resection of tumor is significantly improved with the administration of CpG 2006 beginning on the day of surgery. Mice that were given CpG 2006 after surgery had a 68% survival compared with the 39% for surgery-only controls (P < 0.03). Surviving mice were rechallenged 160 days after tumor injection in the contralateral hind limb (103 cells/mouse), with 14% of CpG-treated mice surviving compared with 0% of controls (P = 0.5; data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSSurgical Resection of TumorStatisticsRESULTSDISCUSSIONREFERENCES |
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Given that CpG ODNs in general stimulate the innate immune system, antitumor studies have focused on developing CpG ODNs as adjuvants for tumor-associated antigens or cellular vaccinations (25, 26, 27, 28) . The published literature using CpG ODN as a single agent in models of cancer indicates that systemic or local CpG ODN administration alone can induce tumor regression in established tumors (14, 15, 16, 17, 18, 19) . Different CpG ODNs each have distinct functions on the immune system, depending on the exact ODN sequence and modifications of the ODN backbone, which makes extrapolation to literature studies using CpG ODNs other than the one used here not possible. We have shown recently that CpG 2006, when administered systemically before tumor challenge, has a significant antitumor protective effect against murine acute myelogenous leukemia (14) . In our model of rhabdomyosarcoma, CpG 2006 had a very modest antitumor effect as a single agent when administered systemically after rhabdomyosarcoma was established (day 9). At earlier time points (days 0, 2, and 5), CpG 2006 has also resulted in antitumor responses.4 Given that the antitumor effect of CpG 2006 and other CpG ODNs has not lead to complete tumor regression in several model systems but has led to enhanced effects when used as an adjuvant with antibodies, tumor vaccines, and peptides, it is possible that CpG ODNs will function best as adjuvants to other therapies for the treatment of malignancies.
In our studies, the antitumor effect of CpG 2006 as a single agent was only evident when administered systemically with minimal tumor burden early after tumor injection (day 9) or after surgical resection of the tumor. When administered with the cytotoxic agent CY, the antitumor response with systemic CpG ODN was greatly enhanced. The same synergistic effect was also seen with intratumoral rather than systemic administration of CpG in the setting of minimal disease burden but not with large tumors.4 The exact mechanism for this enhanced antitumor response with combination CpG/CY therapy has not been fully elucidated, although the combined effect clearly is T-cell dependent. CpG ODNs are known to activate APCs and enhance the capacity of APCs to stimulate both CD4+ and CD8+ T-cell responses (29, 30, 31) . Immature DCs treated with CpG ODN and cocultured with irradiated tumor cells have been shown to provide protection against tumor challenge in vaccinated mice (32) . Therefore, it is likely that in vivo APC stimulation induced by CpG 2006 contributed to the antitumor effects in our model of rhabdomyosarcoma. The administration of CY causes lysis and apoptosis of the tumor cells. The administration of CpG leads to activation of DCs that take up these apoptotic and possibly live tumor cells and present the tumor antigens within the context of MHC molecules to CTLs, which then can eliminate remaining tumor cells. This T-cell-dependent antitumor effect of combined immunotherapy with a cytotoxic agent is somewhat counter to the well-known effects of T-cell immunodeficiency resulting from a cytotoxic chemotherapeutic agent (33) .
NK cells can also be activated by CpG administration. Activated NK cells are directly cytotoxic to the tumor cells. These activated DCs and NK cells produce IL-12 and IFN-, which help to further activate T cells and NK cells for tumor lysis (12
, 13
, 34, 35, 36)
. IFN- may also cause up-regulation of MHC class I on the surface of the tumor cells, making them a better target for T-cell killing. Incubation of 76-9 tumor cells with 1000 units/ml of IFN- for 24 h increases the expression of MHC class I from approximately 5 to 60% (data not shown). The same phenomenon may be occurring in vivo after CpG administration. In general, CpG ODNs with phosphorothioate-modified backbones are not as efficient at directly inducing NK-cell function but are more efficient in inducing IL-12 than those with nonmodified backbones (35
, 37)
. CpG 2006 is known to be a potent inducer of IL-12 in B6 mice at the biological concentrations likely achieved in this study.4
76-9 tumor cells transduced to produce IL-12p70 are markedly less tumorigenic than control transduced cells.4
We hypothesize that the antitumor effects of CpG 2006 in our tumor model may relate to its ability to induce relatively strong IL-12 production. Studies are currently under way to determine whether the antitumor effects of CpG 2006 observed in our model are dependent on IFN- and/or IL-12.
By using a variety of CpG ODNs in different tumor model systems, antitumor effects have been shown to be mediated predominantly by NK cells, T cells, or macrophages (14
, 18
, 37
, 38)
. Our data indicate that in the rhabdomyosarcoma model using CpG 2006 and CY, the primary effector cell is the T cell. This may be attributable to the large tumor volume that is lysed by the CY, providing more tumor antigen, which in turn is more likely to generate a T-cell response. Despite the requirement for T cells in the antitumor response, there is no significant memory cell response attained with the combined CpG/CY administration. There may be several reasons for the failure to attain a memory cell response. It has been reported recently that NKT cells, a subset of CD8+ T cells, express NK-inhibitory receptors that recognize multiple MHC class I molecules (39
, 40)
. The exact role of NK-inhibitory receptors on NKT cells is unclear; however, it has been suggested that the presence of the NK-inhibitory receptors leads to an inability of naive T cells to effectively generate a cytolytic response or develop memory phenotype (41)
. CTLs isolated from tumor-infiltrating lymphocytes of patients with melanoma contained a subpopulation of CTLs with high expression of NK-inhibitory receptors. The in vitro cytolytic function of these NK-inhibitory receptor-positive CTLs was low; however, function was restored when the receptors where blocked using receptor-specific mAbs (42)
. These data suggest that the presence of the NKT cells may modulate the T-cell response to tumor. Another possible mechanism inhibiting T-cell memory response may be related to the presence of inhibitory or regulatory T-cell populations, which are thought to be capable of inhibiting the generation of potent antitumor memory cell development (43
, 44) . A third possibility is that some memory response was generated, but that this response was of insufficient magnitude to slow the rapidly growing tumor cells; the 76-9 cell line has a doubling time of 
16 h in vitro. Studies are under way to investigate the possible inhibitory mechanisms responsible for the lack of a strong memory response and to determine whether these inhibitory mechanisms could be overcome to generate a lasting memory against rhabdomyosarcoma.
CY is a chemotherapeutic agent used routinely in the treatment of rhabdomyosarcoma. It has also been shown to have potent immune-modulating properties when used at low dose alone or in combination with immunotherapy strategies (20) . The dose of CY used in these studies is higher than that classically used to generate an immune-potentiating response. Lower doses of CY were evaluated, and at 75 mg/kg minimal antitumor responses were observed when combined with systemic administration of CpG 2006 in the subclinical model, and at doses <75 mg/kg, no effect of CY alone or in combination with CpG 2006 was observed.4 Depletion of T cells or NK cells in mice that received CY alone did not show any change in the antitumor effects of CY at a dose of 200 mg/kg. This suggests that the effect of the CY is related to cytotoxic properties at the higher dose of 200 mg/kg rather than a direct immune-modulating effect of the CY. Recently published data suggest that the local administration of CpG with CY in a rat model of glioma decreases tumor size (45) . This expands the potential role for combination therapy to other tumors. The fact that CpG 2006 administered with the chemotherapeutic agent topotecan also improves survival in mice with large tumors suggests that the combined antitumor effects are not limited to one class of agents. Our data suggest that combining CpG ODN with different chemotherapeutic agents may lead to enhanced antitumor effects that are generalizable to a broad range of chemotherapy agents.
An important aspect of our studies is the finding that CpG in combination with chemotherapy or surgical resection has antitumor effects even in the setting of large tumor burden. In clinical practice, it is unlikely that CpGs will be administered as a single agent to patients with rhabdomyosarcoma in the absence of any chemotherapeutic agent. Our studies clearly suggest that the combined use of CpGs with chemotherapy or surgical resection may in fact increase the antitumor response observed with either modality used independently. The timing of the administration of the CpGs in conjunction with chemotherapy or surgical resection is probably critical with regard to sustaining a response in the setting of responsive disease. It may also be necessary to have repeated dosing during hematopoietic recovery to maximally stimulate the responding cells. Because CpGs are potent activators of innate immunity and these cells are one of the first cells to recover function after intensive chemotherapy or bone marrow transplant, the combination of CpGs with chemotherapy for the treatment of malignancy is an attractive one.
In conclusion, we have shown, for the first time, that CpGs combined with CY have enhanced antitumor effects in rhabdomyosarcoma that is greater than either agent used independently. We have also shown that CpGs, as a single agent, improve survival in the setting of minimal disease after surgery. The implications of these data could be substantial for the ongoing clinical application of CpGs in cancer immunotherapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by grants from the Children’s Cancer Research Fund and Children’s Oncology Group; NIH Grants R01 CA-72669 and P01 CA66579; a Career Development Award from the Department of Veterans Affairs; and a grant by the Coley Pharmaceutical Group. 
2 To whom requests for reprints should be addressed, at University of Minnesota, MMC 366, 420 Delaware Street SE, Minneapolis, MN 55455. Phone: (612) 626-1926; Fax: (612) 624-3913; E-mail: weige007{at}tc.umn.edu
3 The abbreviations used are: APC, antigen-presenting cell; DC, dendritic cell; IL, interleukin; ODN, oligodeoxynucleotide; NK, natural killer; CY, cyclophosphamide; mAB, monoclonal antibody.
4 B. J. Weigel and B. R. Blazar, unpublished data.
Received 2/ 3/03; revised 3/19/03; accepted 3/19/03.
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