CpG oligodeoxynucleotides are potent enhancers of radio- and chemoresponses of murine tumors

Radiotherapy and Oncology 80 (2006) 192–198 www.thegreenjournal.com

Experimental radiobiology

CpG oligodeoxynucleotides are potent enhancers of radio- and chemoresponses of murine tumors Kathryn A. Masona,*, Robert Neala, Nancy Huntera, Hisanori Arigaa, Kian Angb, Luka Milasa a

Department of Experimental Radiation Oncology, and bDepartment of Radiation Oncology, The University of Texas, Houston, TX, USA

Abstract Background and purpose: Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanine (CpG) motifs bind to Toll-like receptor 9 (TLR9) and stimulate both innate and adaptive immune reactions and possess antitumor activity. We recently reported that CpG ODN 1826 strongly enhances radioresponse of both immunogenic [Milas L, Mason K, Ariga H, et al. CpG oligodeoxynucleotide enhances tumor response to radiation. Cancer Res 2004;64:5074–7] and non-immunogenic [Mason KA, Ariga H, Neal R, et al. Targeting toll-like receptor-9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy. Clin Cancer Res 2005;11:361–9] murine tumors. Using two immunogenic murine tumors, a fibrosarcoma (FSa) and a mammary carcinoma (MCa-K), the present study explored whether CpG ODN 1826 also improves the response of murine tumors to the chemotherapeutic agent docetaxel (DOC). Materials and methods: CpG ODN 1826 (100 lg) was given sc three times: when leg tumors were 6 mm, when they grew to 8 mm and again 1 week later. DOC (33 mg/kg iv) and local tumor radiation (10 Gy) were given when tumors were 8 mm. Effects of the treatments were assayed by tumor growth delay, defined as days for tumors to grow from 8 to 12 mm in diameter. Results: Treatment with CpG ODN 1826 resulted in strongly enhanced response of FSa tumors to radiation and MCa-K tumors to the chemotherapeutic agent DOC. Enhancement of tumor treatment response was demonstrated by a strong prolongation in the primary tumor treatment endpoint, tumor growth delay. Coincidentally, this treatment also resulted in a higher rate of tumor cure than that observed after tumor radiotherapy or chemotherapy alone. When all three agents were combined the effect was comparable to that of the combination of CpG ODN 1826 with radiation in the case of FSa or of the combination of CpG ODN 1826 with DOC in the case of MCa-K. Conclusion: Overall results show that CpG ODN 1826 can markedly improve tumor response to radiation and chemotherapy (DOC), suggesting that CpG ODNs have potential to be beneficial when used singly or in combination with other standard treatment modalities such as taxane chemotherapy, radiotherapy or both. c 2006 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 80 (2006) 192–198.



Keywords: CpG oligodeoxynucleotides; Radioresponse; Chemoresponse

Tumor growth and its response to radiation or cytotoxic drugs are influenced by many factors related to tumor cells, tumor pathophysiology, and tumor host. The immune system is one of these factors. It has long been recognized that immune deficiency lowers whereas stimulation of the immune system enhances tumor response to conventional therapeutic treatments [5,23,33]. This led to immunotherapy approaches in cancer treatment, administered either as a single treatment modality or in combination with chemotherapy or radiotherapy. In earlier developmental phases of cancer immunotherapy, a frequent approach consisted of non-specific stimulation of anti-tumor immunological reactions of tumor-bearing hosts by bacteria or bacterial extracts such as Bacillus Calmette-Guerin (BCG) and Coryne-



bacterium parvum [23,33]. This type of immunotherapy elicits or augments many facets of immunological reactions including macrophage and natural killer (NK) cell activation, induction of antibody-dependent cytotoxicity, and production of cytokines exerting anti-tumor activity such as interferons (IFN) and tumor necrosis factor-a (TNF-a). These bacteria or their extracts exhibited strong anti-tumor activity on their own in a variety of rodent tumors, and in addition acted either additively or synergistically when combined with chemotherapeutic drugs or radiation [23]. They were tested in clinical settings as well [19] but their therapeutic benefit was only modest when compared to their robust activity in preclinical models. Nevertheless, partial or complete tumor regressions were often seen when

0167-8140/$ - see front matter c 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2006.07.024

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these bacteria were inoculated intratumorally. However, the full exploration of therapeutic potential of whole bacteria or their crude extracts in the clinic was hampered by severe toxicity associated with repeated systemic administration. Recent advances in understanding the cellular localization of immunostimulatory components within bacteria, and discovery of their receptors on immune cells offered prospects for significant improvements in anti-tumor immunotherapy [29]. It was discovered that the immunostimulatory activity of bacteria resides in their DNA [29], notably in unmethylated DNA CpG motifs [12] prevalent in bacterial but not in vertebrate genomic DNA. This discovery enabled synthesis of oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs. Another important recent advancement in immunology was discovery of Toll-like receptors (TLR) on immune cells that recognize and bind different bacterial components or bacterial products such as lipopolysaccharides, proteoglycans, flagellin, and endotoxin, as well as CpG motifs. More than 10 different TLR have been identified of which only TLR9 on plasmacytoid dendritic cells (DC) and B cells recognize CpG ODN. This receptor-mediated signaling pathway activates both innate and adaptive immunological reactions. An important feature of this pathway is that it is associated with much lower toxicity compared to the other TLR signaling pathways [8]. CpG ODNs activate PDC and B cells and thus initiate development of strong innate and adaptive immunological reactions. Activated DC and B cells acquire increased ability to present antigens to T-cells and to secrete different cytokines and chemokines that, within several hours after CpG ODN administration, trigger a wide range of secondary effects, such as NK cell and monocyte activation with anti-tumor activity [30]. Within several days of the initial innate immune response the adaptive immune response ensues, which is distinguished by production of cytotoxic T lymphocytes [27,32] and antibody-producing B cells [4,13,24]. CpG ODNs have been shown to exert anti-tumor activity against different types of rodent tumors, both in preventive and therapeutic settings. Tumor growth delay and prolongation in tumor-host survival were most commonly observed and were more pronounced when treatment was initiated when tumors were small [1–3,7,9,11,15,31]. The antitumor efficacy of CpG ODNs was particularly strong in combination with standard cancer treatment modalities, significantly improving the outcome of surgery [11,31], chemotherapy [11,31], and radiotherapy [22]. Our own recent research explored the efficacy of CpG ODN 1826 in combination with radiation. Using both immunogenic and non-immunogenic syngeneic murine tumors we observed strongly enhanced tumor growth delay and rate of tumor cure in response to both single-dose and fractionated radiation [16,22]. These effects were diminished if mice were immunosuppressed by prior whole body irradiation suggesting that an intact immune system at the time of CpG ODN administration is needed to achieve better anti-tumor efficacy [22]. Tumors treated with both CpG ODN and radiation were heavily infiltrated by host inflammatory cells (lymphocytes and granulocytes) and showed histological changes

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characteristic of massive tumor cell destruction including increased necrosis and reduced tumor cell density. Mice cured of their tumors by combined CpG ODN 1826 plus radiotherapy were found to be highly resistant to subcutaneous (sc) tumor take or development of tumor nodules in the lung from intravenous (iv) injected tumor cells when re-challenged, suggesting the development of an immune memory response. The present study assessed whether CpG ODN 1826 enhances tumor response to single agent docetaxel, one of the most commonly used clinical chemotherapeutics, and the combination of docetaxel and radiation.

Materials and methods Mice and tumors C3Hf/KamLaw male mice bred and maintained in our specific pathogen-free mouse colony were 3–4 months old at the beginning of experiments and housed four or five per cage. Animals used in this study were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the United States Department of Agriculture and Department of Health and Human Services. Two murine tumors were used for these studies: an immunogenic sarcoma, designated FSa, originally induced by methylcholanthrene in this strain of mice [28], and an immunogenic mammary carcinoma, designated MCa-K, that arose spontaneously in a C3H female [20]. The tumors were in their sixth and eighth isotransplant generations, respectively. Solitary tumors were produced in the muscles of the right hind leg by the inoculation of 4.5 · 105 cells. Tumor cell suspensions were prepared by mechanical disruption and enzymatic digestion of non-necrotic tumor tissue [21].

CpG oligodeoxynucleotide (ODN) The active CpG ODN 1826 (sequence TCCATGACGTTCCT GACGTT) was provided by Coley Pharmaceutical Group, Inc. (Wellesley, MA). The compound was diluted with phosphate buffered saline to a concentration of 1 mg/ml and stored at 4 C for up to a week. The drug was injected sc peritumorally in a volume of 0.1 ml to achieve a dose of 100 lg per mouse. CpG ODN 1826 was given to mice three times: when tumors measured 6 mm, when they grew to 8 mm and again 7 days later.

Docetaxel Docetaxel (DOC) was obtained from Aventis Pharmaceuticals (Parsippany, NJ) as a pure crystalline powder. A stock solution of 50 mg/ml was prepared in absolute ethanol and stored at 20 C for the duration of experiments. Treatment solutions were prepared by mixing 1 volume of the ethanolic stock solution, 1 volume of polysorbate 80 (Sigma Chemical Co., St. Louis, MO), and 18 volumes of 5% glucose in water. DOC was given as a single iv dose of 33 mg/kg. Treatment solutions were kept on ice and injected within 10 min of formulation.

CpG ODN enhances tumor radio- and chemoresponse

Tumors were locally irradiated when they grew to 8 mm in diameter. A single dose of 10 Gy was delivered to the tumor-bearing legs using a dual-source 137Cs c-ray unit at a dose rate of 5.43 Gy/min. Unanesthetized mice were immobilized in a jig with tumors centered in the 3 cm diameter field of exposure. When radiation was combined with DOC radiation was given 24 h after DOC.

Tumor growth delay The effects of CPG ODN 1826 on tumor chemo- and radioresponses were determined by tumor growth delay. When tumors grew to 6 mm in diameter, half the mice were treated with CpG ODN 1826 and the remaining mice left untreated. When tumors subsequently grew to 8 mm in diameter CpG ODN 1826 was given 3 h following treatment with either DOC or radiation. To obtain tumor growth curves, three orthogonal tumor diameters were measured at 1–3 day intervals with a vernier caliper, and the mean values were calculated. Regression and regrowth of tumors were followed until tumor diameter reached approximately 14 mm. Tumor growth delay was expressed either as the absolute or normalized growth delay (AGD or NGD). AGD was defined as the time in days for tumors in the treatment arms to grow from 8 to 12 mm in diameter minus the time in days for the tumors in the untreated control to reach the same size. NGD was defined as the time for tumors in groups treated with a combined regimen to grow from 8 to 12 mm minus the time for tumors to reach the same size in mice treated with CpG ODN 1826 alone. Groups consisted of 7–10 mice each. Comparison of tumor growth delay means was carried out by t-test.

Histological analysis Mice were treated with CPG ODN 1826 when MCa-K tumors were 6 mm and 8 mm or with docetaxel when tumors were 8 mm in diameter and then sacrificed 4, 8, 24, 72 or 144 h later. When CPG ODN 1826 was combined with DOC, the second dose of CPG ODN 1826 was injected 3 h after DOC and mice sacrificed 1, 5, 21, 69 or 141 h later. Tumors were removed, fixed in neutral buffered formalin and processed for routine histological examination. Hematoxylin and eosin (H&E)-stained 4-lm sections were assessed microscopically at 400· for the presence of mitosis, apoptosis and infiltration of lymphoid cells as described previously [17]. Groups consisted of three mice each.

Results Anti-tumor efficacy of CpG ODN 1826 The anti-tumor activity of CpG ODN 1826 was tested using two immunogenic murine tumors, FSa and MCa-K, grown as solitary tumors in the hind leg of mice. As shown in Fig. 1 CpG ODN 1826 was effective against both tumors as evidenced by significant delay in tumor growth. To double their size, from 6 to 12 mm in diameter, untreated FSa tumors needed 8.9 ± 0.3 days whereas FSa tumors treated with CpG ODN 1826 needed 17.5 ± 3.4 days (p = 0.013). Sim-

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Days After First Treatment Fig. 1. Effect CpG ODN 1826 on the growth of FSa (A) and MCa-K (B) tumors. Mice bearing tumors in the leg were untreated (s) or treated with CpG ODN 1826 (d). Treatment with CpG ODN 1826 at a dose of 100 lg per mouse was given sc peritumorally once when tumors were 6 mm and 8 mm and 1 week later. Each data point represents the mean size of 7–9 tumors; bars, SE. One mouse with FSa tumor was cured by CpG ODN 1826 treatment and was excluded from the growth delay analysis.

ilarly, while untreated MCa-K tumors doubled their size in 9.6 ± 0.6 days, treated tumors needed 18.8 ± 0.9 days (p < 0.0001). One of 8 FSa tumors treated with CpG ODN 1826 permanently regressed. In all analyses of tumor growth delay, mice cured of their tumor were excluded.

Effect of CpG ODN 1826 on tumor radioresponse To determine whether CpG ODN 1826 potentiates tumor response to radiation, CpG ODN 1826 was given as above and 10 Gy single dose local tumor irradiation was performed when tumors were 8 mm in diameter. Fig. 2 plots the growth curves starting from the time of tumor irradiation (Day 0). CpG ODN 1826 delayed growth of FSa by 7.8 ± 3.2 days (AGD) (p = 0.017), 10 Gy only delayed it by 2.3 ± 0.5 days (p = 0.001), and the combined CpG ODN 1826 plus radiation treatment delayed growth by 15.6 ± 5.5 days (p = 0.004) (Fig. 2A). The combined treatment effect was more than the additive effects of single treatments, suggesting synergy of the two agents. The radiation enhancement factor (EF) was 3.39. In addition to enhancing radiation-induced tumor

K.A. Mason et al. / Radiotherapy and Oncology 80 (2006) 192–198

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(p = 0.013) in mice treated with CpG ODN 1826, and 10.0 ± 3.4 days (p = 0.009) for those treated with the combination. In this last group 1 of 10 mice were cured. In contrast to FSa, MCa-K tumor was much more sensitive to DOC, and responded dramatically to the combination of CpG ODN 1826 plus DOC in which case 50% of mice (5 of 10) were cured (Fig. 3B). CpG ODN 1826 delayed MCa-K growth by 5.5 ± 0.4 days (AGD), DOC delayed it by 24.6 ± 0.9 days (p = 0.001), and the combined CpG ODN 1826 plus DOC treatment delayed it by 38.4 ± 3.4 days (p = 0.0001). The EF caused by CpG ODN 1826 was 1.34.

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To assess the effect of CpG ODN 1826 on tumor response to the combined DOC plus radiation treatment, mice were treated with three injections of CpG ODN 1826 starting

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growth delay, CpG ODN 1826 combined with 10 Gy caused permanent tumor cure in 4 of 10 mice (40%). In contrast to exerting a dramatic effect on FSa radioresponse, CpG ODN 1826 had no appreciable effect on MCa-K tumor radioresponse (Fig. 2B). CpG ODN 1826 delayed MCa-K growth by 5.5 ± 0.4 days (AGD) (p = 0.0001), 10 Gy delayed it by 7.6 ± 0.6 days (p = 0.0001), and the combined CpG ODN 1826 plus radiation treatment delayed it by 9.5 ± 0.8 days (p = 0.001). Thus, even less than the additive effect of the two cytotoxic agents was achieved.

Effect of CpG ODN 1826 on tumor response to DOC The effect of the combined treatment of DOC and CpG ODN 1826 on both tumors is shown in Fig. 3. DOC exerted a weak effect against FSa, regardless of whether mice received CpG ODN 1826 or not (Fig. 3A). AGD was 0.6 ± 0.3 days (p = 0.258) in mice treated with DOC, 7.8 ± 3.2 days

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Fig. 2. Effect CpG ODN 1826 on tumor radioresponse. Mice bearing FSa (A) or MCa-K (B) tumors in the leg were untreated (s) or treated with CpG ODN 1826 (d), 10 Gy single dose local tumor irradiation (h) or a combination of CpG ODN 1826 plus 10 Gy (j). Treatment with CpG ODN 1826, at a dose of 100 lg per mouse, was given sc peritumorally once when tumors were 6 mm and 8 mm and 1 week later. Irradiation was performed when tumors were 8 mm in diameter. Each data point represents the mean size of 6–9 tumors; bars, SE. Four of 10 FSa tumors were cured by treatment with CpG ODN 1826 plus radiation and were excluded from the growth delay analysis.

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Days After Docetaxel Fig. 3. Effect of CpG ODN 1826 on tumor response to DOC. Mice bearing FSa (A) or MCa-K (B) tumors in the leg were untreated (s), or treated with CpG ODN 1826 (d), DOC (n) or a combination of CpG ODN 1826 plus DOC (m). Treatment with CpG ODN 1826, at a dose of 100 lg per mouse, was given sc peritumorally once when tumors were 6 mm and 8 mm and 1 week later. DOC was given iv at a dose of 33 mg/kg when tumors were 8 mm in diameter. Each data point represents the mean size of 5–9 tumors; bars, SE. Treatment with CpG ODN 1826 plus DOC cured 1 of 10 FSa tumors and 5 of 10 MCa-K tumors. Mice cured of their tumors were not included in the growth delay analysis.

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CpG ODN enhances tumor radio- and chemoresponse

when tumors were 6 mm in diameter, 33 mg/kg DOC or 10 Gy when tumors were 8 mm in diameter, or with all three agents in which case local tumor irradiation was delivered 1 day after docetaxel. This 1-day interval between DOC and irradiation was selected on the basis of earlier studies showing that this treatment schedule resulted in significant enhancement of tumor radioresponse [18]. The effect of this combination on both tumors is shown in Fig. 4. This dose and schedule of DOC in combination with radiation was highly effective in enhancing radioresponse of MCa-K (Fig. 4B) but not of FSa tumors (Fig. 4A). In MCa-K tumors DOC treatment resulted in an AGD of 24.6 ± 0.9 days, radiation treatment in an AGD of 7.6 ± 0.6 days and the combined DOC plus radiation treatment in an AGD of 43.5 ± 8.2 days (p = 0.0001). The EF of this synergistic effect was 2.49. This

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Lymphocytic infiltration of MCa-K tumors treated with CpG ODN 1826 plus DOC

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MCa-K tumors treated with CpG ODN 1826 and DOC were histologically analyzed at different times after the end of the treatment (see Materials and methods). DOC induced mitotic arrest of tumor cells, most prominent at 4 and 8 h after DOC treatment (Fig. 5B). At later times, mitotic arrest declined and only a few mitoses were present 2 days after DOC. Considerable apoptosis of tumor cells was also observed. In tumors treated with both CpG ODN 1826 and DOC the most prominent feature was infiltration with host mononuclear cells, most of which had the morphological appearance of lymphocytes. The infiltration started about 2 days after DOC and was particularly prominent 6 days after DOC (Fig. 5C). Lymphocytic infiltration was accompanied by marked tumor cell depopulation. Remaining tumor cells exhibited extensive cellular polymorphism including multinucleated cells, polyploid cells, cells with large swollen nuclei, and apoptoses.

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combined effect was not further increased by CpG ODN 1826 administration as this triple agent treatment resulted in an AGD of 37.6 ± 3.2 days (p = 0.584). It should be noted however that this triple agent therapy resulted in a somewhat higher rate of tumor cure (7 of 10 mice cured; 70%) than the DOC plus radiation treatment (5 of 9 mice cured; 56%). DOC alone was weakly effective against the FSa tumor (AGD of 0.6 ± 0.3 days) and anti-tumor efficacy of radiation was not improved: AGD of 2.6 ± 0.3 days compared to the AGD of 2.3 ± 0.5 days (p = 0.423) after 10 Gy alone (Fig. 4A). The AGD in mice treated with all three agents was 10.5 ± 2.8 days (p = 0.001), attributable to the combined effect of CpG ODN 1826 plus radiation. In this triple-agent combination group 5 of 10 (50%) mice were cured of their tumors.

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Days After Docetaxel Fig. 4. Effect of CpG ODN 1826 on tumor response to DOC plus radiation. Mice bearing FSa (A) or MCa-K (B) tumors in the leg were untreated (s), or treated with DOC (n), local tumor irradiation (h), DOC plus irradiation (m) or a combination of all three agents (j). CpG ODN 1826 (100 lg) was given when tumors were 6 mm and 8 mm and 1 week later. DOC (33 mg/kg) and local tumor irradiation (10 Gy) were given when tumors were 8 mm in diameter. In the combination treatment, irradiation was given 1 day after DOC. Each data point represents the mean size of 3–10 tumors; bars, SE. Tumors cured by the treatments were excluded from the growth delay analysis. Five of 9 MCa-K tumors treated with DOC plus radiation were cured. When CpG ODN 1826 was combined with DOC and radiation, 5 of 10 FSa tumors and 7 of 10 MCa-K tumors were cured.

The results showed that treatment of mice bearing large established tumors with CpG ODN 1826 resulted in remarkably enhanced response of FSa tumors to radiation and MCa-K tumors to the chemotherapeutic agent DOC. Enhancement of tumor treatment response was demonstrated by a strong prolongation in tumor growth delay, which was the primary treatment endpoint in this study. In addition, a higher rate of tumor cure than that from tumor radiotherapy or chemotherapy alone was also observed. When all three agents were combined the effect was comparable to that of the combination of CpG ODN 1826 with radiation in the case of FSa or of the combination of CpG ODN 1826 with DOC in the case of MCa-K. Our recent investigations [16,22] showed that the enhanced tumor response to radiation by CpG ODN 1826 is predominantly mediated by the host immunological response. CpG ODN 1826 was much less effective in mice immunocompromised by whole body irradiation [22]. CpG ODN 1826 induced heavy infiltration of irradiated tumors with host inflammatory cells associated with massive tumor cell lysis [22]. The present study showed

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Fig. 5. Histological appearance of MCa-K tumors untreated (A) or treated with DOC (B) or with a combination of CpG ODN 1826 plus DOC (C). CpG ODN 1826 (100 lg) was given when tumors were 6 mm and 8 mm in diameter and DOC (33 mg/kg) was injected when tumors were 8 mm in diameter. Histological sections were obtained, stained with H&E and observed microscopically at 400·. B, mitotic arrest of tumor cells at 4–8 h after DOC treatment; C, lymphocytic infiltration accompanied by heavy tumor cell depopulation at 6 days after DOC in tumor treated with both CpG ODN 1826 and DOC.

qualitatively that tumors treated with both CpG ODN 1826 and DOC also displayed heavy infiltration by lymphoid mononuclear cells of lymphocyte morphology. This

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infiltration was associated with profound tumor cell depletion and visible morphological characteristics of tumor cell lysis. The specific nature of the anti-tumor immune rejection response induced by CpG ODN 1826 was not addressed in our experiments. Other studies reported that CpG ODNs induce an antigen-specific anti-tumor T-cell response [10,14,25]. Injection of CpG ODN creates a Th1-like cytokine/chemokine milieu and lymphadenopathy in the draining lymph nodes [10,14]. Among cells within the enlarging lymph nodes are dendritic cells that express increased levels of co-stimulatory molecules and major histocompatibility complex (MHC) [14]. CpG ODN activation of DC promotes strong memory T-cell responses [25]. CpG ODN-primed mice respond to subsequent antigen injection in the same anatomic region with a strong Th1-based response and high levels of cytotoxic T lymphocytes (CTL) even several weeks after the CpG ODN injection [10,14]. Based on these findings we hypothesize that exposure of tumors to radiation or DOC releases antigens from dying tumor cells in mice treated with CpG ODN that are taken up by activated DC leading to the induction of a tumorspecific T-cell response. This notion is supported by our earlier finding that mice cured of their tumors by combined CpG ODN 1826 plus radiotherapy were more resistant to tumor cell re-challenge than mice cured by radiotherapy only [16]. An interesting observation was reported that CpG ODNs protect mouse spleen cells and macrophages from radiation damage, and that the increase in radioresistance of these lymphoid cells was accompanied by upregulation of Bcl-xS/L and Bcl-2 [26]. Thus, CpG ODN-activated host inflammatory cells may acquire resistance to treatment with radiation or DOC that enables their persistence in killing tumor cells. A highly intriguing, but at present unexplainable, observation was that the host immune system activated by CpG ODN 1826 affected differently the response to both radiation and DOC between FSa and MCa-K tumors. Enhanced radioresponse, but not chemoresponse, was exhibited by the FSa tumor, whereas enhanced tumor chemoresponse, but not radioresponse, was exhibited by the MCa-K tumor. Differences in tumor response could be related to the type of treatment-induced host immune reaction or tumor specific sensitivity to the immune response. The interactions between the immune system and radiation that may affect tumor response are multiple and complex [6]. Radiation and chemotherapeutic agents may alter tumor immunogenicity, up-regulate expression of inflammatory mediators, induce immunomodulatory cytokines and initiate both T cell dependent and T cell independent cascades of anti-tumor immune responses, all of which are likely to be augmented when radiation is combined with immunomodulating agents. In conclusion, the results of our study together with those we previously reported [16,22] have established that CpG ODN 1826 can markedly improve tumor response to radiation and chemotherapy (DOC), suggesting that CpG ODNs have potential to be beneficial when used singly or in combination with other standard treatment modalities such as taxane chemotherapy, radiotherapy or both.

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Acknowledgement This work was supported by a Laboratory Study Agreement with Coley Pharmaceutical Group, Inc., and National Cancer Institute Grants CA06294 and CA16672. * Corresponding author. Kathryn A. Mason, Department of Experimental Radiation Oncology, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009, United States. E-mail address: kmason@mdanderson. org

Received 8 May 2006; received in revised form 18 July 2006; accepted 19 July 2006; Available online 14 August 2006

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