Simultaneous TLR2 inhibition and TLR9 activation synergistically suppress tumor metastasis in mice.

AIM
To develop a rational immunotherapy against tumor metastasis by combining a Toll-like-receptor 2 (TLR2)-neutralizing antibody with a TLR9 agonist CpG ODN, and then investigate the mechanism of action for this combinational regimen.


METHODS
After mouse melanoma B16-F10 cell inoculation, female C57BL/6 mice were treated with either CpG ODN (0.5 mg/kg) or the anti-TLR2 antibody (200 μg/kg), or with a combination of the two agents. Pulmonary metastases were evaluated by counting metastatic nodes on the lung surface using anatomical microscopy. Flow cytometry was used to evaluate the cytotoxicity of the immune cells in tumor-draining lymph nodes, the cell population in the spleen, and the infiltration of immune cells within the lungs. Cytokine and enzyme expression in the lung tissue was evaluated using ELISA or immunostaining.


RESULTS
Anti-metastatic effects were detected in mice treated with either CpG ODN or the anti-TLR2 antibody alone. However, treatment with CpG ODN plus the anti-TLR2 antibody synergistically suppressed the metastasis as compared with treatment with either single agent. The combinational treatment resulted in enhanced infiltration of natural killer cells and cytotoxic T cells, reduced recruitment of type 2 macrophages and Tregs, and decreased expression of immunosuppressive factors including TGF-β1, cyclooxygenase-2 and indoleamine 2,3-dioxygenase, thus stimulated tumor cytotoxicity and suppressed metastasis. The anti-metastatic effect of the combinational regimen was further confirmed in spontaneous metastatic mouse model of Lewis lung carcinoma.


CONCLUSION
Our studies suggest that combining a TLR9 agonist with an anti-TLR2 antibody, which eliminates immunosuppressive factors from the tumor environment, is critical for an effective anti-metastatic immunotherapy.


Introduction
During the last two decades, significant advances have been made in the field of cancer immunotherapy, and numerous strategies have been developed to provide a tumor-specific immune response [1] . However, the success of these strategies in clinical trials remains limited. Increasing evidence has shown that not only are there insufficient numbers of antitumor effector cells but that there are also too many immunosuppressive factors in tumor environment, which culminates in the failure to eradicate tumor cells [2,3] . Thus, a rational anti-cancer immunotherapeutic strategy should include both anti-tumor immune elements and elements able to eliminate tumor-induced immunosuppressive factors [4] . Toll-like receptors (TLRs) recognize pathogen-or damageassociated molecular patterns (PAMPs or DAMPs) to initiate innate and adaptive immune responses against pathogens and cancer cells [5] . In addition to their importance in pathogen and transformed cell elimination, TLRs are of great importance in mediating chronic inflammation and immune tolerance [6] . TLR targeting has been promising as an effective regimen for disease treatment. The TLR9 agonist CpG ODN was shown to have encouraging anti-tumor activity mediated by simulation of anti-tumor immunity in a number of experimental models. TLR9 agonists induce the activation of plasmacytoid DCs and B cells and stimulate potent Th1-type immune responses. Therefore, TLR9 agonists have been used or are currently under development as monotherapies or in combination with other anti-cancer therapies [7] . However, there have been disappointing results from phase III trials combining TLR9 agonists with chemotherapeutic agents for treatment of nonsmall-cell lung cancer, with little evidence of tumor regression www.nature.com/aps Yan J et al Acta Pharmacologica Sinica npg or remission compared with the control [7,8] . A recent study has suggested that immune suppression induced by tumor cells inhibits the activation of the immune system by TLR9 agonists [9] .
Recent data indicate that many endogenous molecules released by transformed or damaged cells act as alarm signals to stimulate TLRs and induce chronic inflammation to facilitate tumor growth and metastasis [10] . TLR2 has been identified as one of the important mediators inducing the immunosuppressive response [11] . Studies by Kim et al and our own group indicate that blocking TLR2 activity is a novel therapeutic strategy for anti-metastasis that combats the immunosuppressive microenvironment [12,13] . These findings collectively suggest that a combination of a TLR2-neutralizing antibody with a TLR9 agonist CpG ODN may produce greater antimetastatic activity than either treatment alone. In this study, we demonstrate that a TLR9 agonist CpG ODN, which can initiate anti-tumor immunity, combined with a TLR2-neutralizing antibody, which can eliminate inhibitory immune factors from tumor tissue, synergistically act to induce an intense anti-metastatic effect compared with either agent alone. Our studies suggest that combining an immune stimulatory agent with an agent that eliminates immunosuppressive factors from the tumor environment is a rational strategy for designing an effective immunotherapeutic regimen against tumor metastasis.

Cell culture
The mouse melanoma cell line B16F10 and the Lewis lung carcinoma cells were cultured in RPMI-1640 (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 2 g/L Na 2 CO 3 , 100 units/mL penicillin, 50 µg/mL gentamicin, and 10% FBS at 37 °C in 5% CO 2 . These two cells were kindly donated by Prof Rui HAN  To generate a mouse model of pulmonary metastasis, B16F10 cells were trypsinized and resuspended in a PBS solution at a density of 6.25×10 5 cells/mL. Then, 200 μL of the suspension was injected into the lateral tail vein of each mouse. The mice were euthanized with an overdose of anesthesia on the 21st day after inoculation, and a whole lung was extracted for calibrating the lung index by lung weight (mg) per body weight (g). An anatomical microscopic metastasis quantization was performed by counting the metastatic nodes on the surface of the whole lung. The lungs were then fixed with 4% paraformaldehyde to prepare for histological analysis.
B16F10 melanoma cells were inoculated on day 0. The TLR2-neutralizing (200 µg/kg), anti-IgG antibody (200 µg/kg), CpG (0.5 mg/kg) and CpG control (0.5 mg/kg) were injected on day 3. The treatment with CpG or CpG control was repeated every three days and the treatment with TLR2neutralizing or anti-IgG antibody was repeated every seven days [12,14] . The sham and control mice received a PBS vehicle treatment alone.
To determine the impact of the TLR2-neutralizing antibody plus CpG treatment on tumor cell growth and spontaneous metastasis, 2×10 6 Lewis lung carcinoma cells were resuspended in 100 µL PBS and subcutaneously injected into the right flank of mice to establish tumors. Tumor volume was calculated as (length×width) 2 /2 every four days. When small lumps ranging in diameter from 1 to 2 mm were palpated on the 7th day, the mice were randomly divided into four equally sized groups. The dosage and frequency of the TLR2-neutralizing antibody, IgG, CpG, and CpG control were the same as the B16 experiment. Mice treated with cyclophosphamide (40 mg/kg, ip) every two days were used as a positive control. At 24 days post-tumor inoculation, the mice were sacrificed by excessive anesthesia. The primary tumors and the lungs were surgically removed and weighed. The lung metastatic nodes were counted using anatomical microscopy.

Immunohistochemistry
The left lower lobe of the lung was isolated, fixed, paraffin embedded and coronally sliced into 4-µm sections. The protocols for immunohistochemical staining for IDO and COX2 have been described previously [12] (the dilution was 1:250 for primary and secondary antibodies). The protein signal intensity was determined by Image-Pro Plus image analysis software. The reported final integrated optical densities (IOD) represent averages from 10 non-overlapping images of each lung specimen.

Cytotoxicity assay
Tumor draining lymph nodes (TDLNs) from B16 mice treated with the indicated agents were isolated and tested for cytotoxicity against B16F10 tumor cells using a standard flow cytometry assay [15] . To determine the cytotoxicity of immune cells from TDLNs, B16F10 melanoma cells were labeled with 5 µmol/L of CFSE [5-or 6-(N-Succinimidyloxycarbonyl)-3',6'-O,O'-diacetylfluorescein, Dojindo Mol Technol, Inc] and cultured with the TDLNs at 3 different effector to target (E:T) ratios (50:1, 25:1, and 12.5:1) in triplicate wells for 4 h. The cells were harvested and stained with propidium iodide (PI). Dead target cells were detected as PI + CFSE + cells using flow cytometry. Cytotoxicity, defined as the percent cytotoxicity in each sample, was calculated based on each E:T ratio. The dose-response curves of percent-specific lysis versus E:T ratios for each TDLN sample were evaluated.

ELISA for cytokines in lung tissue
The right lung lobes were lysed in PBS supplemented with a complete protease inhibitor cocktail. The lung tissue homogenate was diluted with lysis buffer to a final protein concentration of 500 μg of protein per mL. The concentrations of IFN-γ and TGF-β1 in the lung were detected using ELISA kits in accordance with the manufacturer's instructions.

Statistical analysis
Differences between groups were assessed by ANOVA. Survival curves were compared by the log-rank test. Our results are presented as the means±standard error (SEM). P values < 0.05 were considered statistically significant.

Results
TLR2-neutralizing antibody and CpG ODN act in synergy to suppress metastasis and improve the survival rates of mice Our group and others have previously reported that blocking TLR2 activity attenuates pulmonary metastasis by inhibiting the immunosuppressive tissue environment [12,13] . We wondered if a combination of a TLR2-neutralizing antibody with a TLR9 agonist CpG ODN would exert increased inhibition of tumor metastasis. Lung metastasis was evaluated in mice 3 weeks after tail vein injections with B16 melanoma cells. The number of metastatic nodules in the lungs of mice in the combination treatment group was notably reduced in comparison with the mice treated with the CpG ODN or the TLR2neutralizing antibody alone upon both visual ( Figure 1A) and numerical analysis ( Figure 1B). As shown in Figure 1B, the number of metastatic nodes in the lungs of mice treated with the CpG ODN plus the TLR2-neutralizing antibody was markedly attenuated compared with that of the IgG plus CpG ODN control group, the CpG ODN group and the TLR2neutralizing antibody group. The number of metastatic nodes in the lungs of mice treated with either the CpG ODN or the TLR2-neutralizing antibody was also found to be significantly lower than that of the PBS group and the IgG plus CpG ODN control group. Consistent with these data, the average weight indices of the lungs of PBS-treated mice were markedly increased (15.3‰±1.8‰ vs 7.4‰±0.2‰, P<0.01), whereas the application of CpG ODN resulted in markedly decreased lung weight indices (11.6‰±0.7‰ vs 15.3‰±1.8‰, P<0.05). Further, the application of the TLR2-neutralizing antibody alone (10.5‰±0.6‰) or in combination with CpG ODN (7.8‰±0.4‰) efficiently attenuated the lung weight indicesas compared with the PBS group, the IgG plus CpG control group and the CpG ODN group. The average lung index of the mice that received combination therapy was found to be significantly lower than that of the anti-TLR2 group.
To compare the anti-metastatic efficacy of the CpG ODN, the TLR2-neutralizing antibody, and the combination of the CpG ODN and the TLR2-neutralizing antibody, animals in the individually treated groups were monitored for survival time after inoculation with B16 melanoma cells. As shown in Figure 1D, 50% of B16-bearing mice treated with PBS or IgG plus the CpG ODN died by the 29th day after tumor cell inoculation, and all died by the 35th or 36th day. The treatment of B16-bearing mice treated with the TLR2-neutralizing antibody increased the animals' survival rates (50% survival on the 35th day, 0% survival on the 42th day). For the mice treated with the CpG ODN alone, the survival rates decreased to 50% on the 32th day and to 0% on the 40th day. However, the treatment of B16-bearing mice with the CpG ODN plus the  Therefore, the combination regimen synergistically increased the ratio of CD8/CD4 T cells in the spleen ( Figure 2C). Why did the CpG ODN treatment alone fail to induce an identical degree of tumor cell cytotoxicity as the combination treatment? We suspected that some fraction of the CD4 + T cells induced by the CpG ODN suppressed the cytotoxicity of the NK cells and the CD8 + T cells. Indeed, the CpG ODN treatment and the combination treatment decreased the frequency of Th2 cells induced by splenic tumor cells ( Figure 2D). Treg cells represent a fraction of CD4 + T cells that play a significant role in suppressing anti-tumor immunity. Inoculation of tumor cells resulted in a significant increase in the numbers of Tregs in the spleen. The anti-TLR2 antibody treatment alone and the combination therapy both suppressed the numbers of Tregs. However, the number of splenic Tregs in CpG ODN-treated mice was much higher than that of the anti-TLR2 antibody-treated or the combination regimen-treated mice ( Figure 2E). These data indicate that the combination regimen produced greater tumor cell cytotoxicity compared with that of either single agent alone by inducing more NK and CD8 + T cells while simultaneously reducing the numbers of Th2 cells and Tregs in the spleen.
Treatment with TLR2-neutralizing antibody plus CpG ODN enhances the antitumor immunity and reduces immune suppression in the tumor microenvironment To find whether the differential immune responses induced by A number of studies indicate that IFN-γ can induce antitumor activity through STAT1 activation and autophagy induction [14,18,19] . By contrast, TGF-β1, IDO and COX2 are immunosuppressive factors that can promote tumor progression through the activation of Treg cells and suppression of T cell proliferation and responses [20] . As shown in Figure 4, there was a significant decrease in the expression of IFN-γ and a significant increase in the expression of TGF-β1, IDO and COX2 in the lung tissues of PBS-treated B16-bearing mice compared to normal mice. Treatment of mice with the CpG ODN plus the TLR2-neutralizing antibody (98.5±22.6 pg/mL), the CpG ODN alone (159.6±33.1 pg/mL) or the TLR2-neutralizing antibody alone (80.2±9.5 pg/mL) resulted in a significant increase in the expression of IFN-γ compared to PBS treatment (41.6±5.1 pg/mL) ( Figure 4A). The treatment of mice with CpG ODN or the TLR2-neutralizing antibody alone did not result in any change in the expression of TGF-β1. However, the CpG ODN plus anti-TLR2 combination treatment (262.4±63.6 pg/mL) significantly reduced the expression of TGF-β1 in the lung tissues compared with the PBS treatment (668.2±112 pg/mL) and CpG ODN treatment (596.3±72.3 pg/mL) ( Figure 4B). Furthermore, the expression of IDO and COX2 in the lung tissues from mice treated with the CpG ODN plus the anti-TLR2 antibody was markedly decreased compared with the PBStreated, CpG ODN-treated and anti-TLR2-treated mice ( Figure  4C and 4D). These data suggest that the combination regimen produces a synergistic anti-metastatic effect compared to treatment with the CpG ODN or the anti-TLR2 antibody alone by increasing anti-tumor factors and reducing pro-tumor factors in lung tissues.
Treatment with the TLR2-neutralizing antibody plus CpG ODN suppresses the spontaneous metastasis of Lewis lung carcinoma To verify the antitumor effect of the TLR2-neutralizing antibody plus the CpG ODN, we examined whether this regimen could suppress the growth and spontaneous metastasis of Lewis lung carcinoma cells. Lewis lung carcinoma cells were transplanted into the right flanks of the mice. The chemotherapeutic agent CTX, used as positive control, inhibited the growth (1.5±0.2 vs 5.3±0.6 g, P<0.001) and spontaneous metastasis (5.6±1.7 vs 24.6±3.5, P<0.001) of the Lewis lung carcinoma cells ( Figure 5A). As shown in Figure 5B, the average weight of the primary tumor 24 days post-inoculation was 5.3±0.6 g in the PBS-treated mice, 5.5±0.5 g in the isotype IgG plus CpG ODN control-treated mice, and 4.7±0.5 g in the mice treated with the TLR2-neutralizing antibody plus the CpG ODN (P>0.05) ( Figure 5B). However, the number of metastatic  nodes in the lungs of the mice treated with the TLR2-neutralizing antibody plus CpG ODN was markedly decreased by 53.3%±11.3% as compared with the PBS-treated and isotype IgG plus CpG ODN control-treated mice ( Figure 5C). The lung index was also reduced in the combination-treated mice ( Figure 5D). These results suggest that combining the TLR2neutralizing antibody with the CpG ODN is not sufficient to attenuate the growth of established tumor but is efficient in the attenuation of tumor metastasis.

Discussion
The major finding of this study is that a rational combination of a TLR2-neutralizating antibody, which eliminates the inhibitory immune factors from tumor tissue, and the TLR9 agonist CpG ODN, which can increase the anti-tumor stimulatory factors, is more effective in inhibiting experimental and spontaneous metastasis than either agent alone. Indeed, the synergistic anti-metastatic effects of the two agents are the result of enhanced immune cytotoxicity against the tumor cells. This . Treatment with either agent alone also induced a similar pattern of changes in the immune response, but the magnitudes observed were significantly less than those of the combination regimen. However, the combination regimen does not suppress tumor growth. This result may be due to the following: 1) the importance of TLR2 and TLR9 on host immune cells and tumor cells for positively modulating metastatic behavior compared to their minimal influence on primary subcutaneously implanted tumor growth [12,13,21,22] ; 2) the failure of the combination regimen to promote sufficient immune cell infiltration into the primary tumor to produce a potent cytotoxic suppression of tumor growth due to the anatomical site of administration [23] ; 3) the inability of the therapeutic administration of the combination regimen to overcome the immunosuppressive barrier after the suitable microenvironment for tumor growth has been established [14] .
The TLR9 agonist CpG ODN is a promising anti-cancer immunotherapy based on its ability to safely stimulate Th1dominant innate and adaptive immunity in humans [7] . Nevertheless, tumors employ multiple means of suppressing or evading antitumor immunity [24] and cannot be overcome by the CpG ODN treatment alone. For example, the CpG ODN is a potent inducer of IL-10 from DCs and has been found to protect Streptozocin-induced autoimmune diabetes by the induction of IDO and Tregs [25] . Indeed, administration of the CpG ODN in the current study increased the frequency of NK and CTL cell infiltration, IFNγ secretion, and M1 cell differentiation but did not suppress the number of Tregs in the spleen. Further, the TLR9 agonist CpG ODN increased the expression of TGF-β1, IDO, and COX-2 and promoted the infiltration of Treg cells into the tumor microenvironment after tumor inoculation. The Tregs, other immune-suppressive cells, cytokines and enzymes subsequently counteract the initiation and perpetuation of antitumor cytotoxicity either by direct cell contact or by secreting suppressive cytokines. Thus, the cytotoxicity of the immune cells from CpG ODN-treated B16-bearing mice is diminished even though the numbers of NK and CTL cells are increased in the spleen. Xiong Z et al recently demonstrated that CpG ODN immunotherapy in a breast cancer model prevents but fails to entirely eradicate established brain metastases despite a significant increase in brain-infiltrating T and natural killer cells in treated mice relative to saline controls [26] . These findings indicate that CpG ODN treatment alone can stimulate anti-tumor effectors but fails to reverse the immunosuppressive responses in the tumor microenvironment. Therefore, the anti-metastatic activity of CpG ODN monotherapy is limited.
TLR2 is an unique member of the TLR family because it triggers an immunosuppressive response in vivo [27] . Activation of TLR2 promotes IL-10 production by innate immune cells in vitro and results in the proliferation of Tregs in vitro and in vivo [28] . Treg numbers in the circulation of TLR2-deficient mice are reduced compared with their WT littermate controls [10] . Furthermore, the activation of the TLR2 signal may be crucial for tumor cells to escape killing by the host [29] . Huang et al recently found that activation of TLR2 on cancer cells by Listeria monocytogenes promotes tumor growth [30] , while Kim et al reported that activation of TLR2 by an extracellular matrix proteoglycan versican of Lewis lung carcinoma stimulates pro-tumor inflammation and metastasis [13] . We recently demonstrated that the level of TLR2 expressed on B16F10 melanoma cells determines their invasive activity in response to the endogenous factors released from tumor cells [12] . However, contradictory observations by others indicate that TLR2 activation is able to induce inflammatory cytokines and activate NK cells in vitro and in vivo and protect mice from tumor development and progression [31,32] . In the current study, we find that blocking TLR2 effectively reverses the immunosuppressive characteristics of the microenvironment by suppressing the expression of TGF-β1, IDO, and COX2 and attenuating the infiltration of M2 and Tregs cells. This scenario is an example of how immunotherapy is able to break self-tolerance by depleting immunosuppressive factors in the tumor microenvironment to generate effective anti-tumor immunity.
In summary, our studies indicate that developing an optimal immunotherapeutic strategy in which combining an immune stimulator with an agent eliminating inhibitory immune factors is critical for obtaining a desired therapeutic effect against tumor metastasis. Our studies provide novel insights into the development of the rational immunotherapeutic strategies against cancer.