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Cancer Research 67, 10519, November 1, 2007. doi: 10.1158/0008-5472.CAN-07-0079
© 2007 American Association for Cancer Research
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Immunology

Activation of Toll-like Receptor 4 on Tumor Cells In vitro Inhibits Subsequent Tumor Growth In vivo

Virginia Andreani, Gerardo Gatti, Lucio Simonella, Virginia Rivero and Mariana Maccioni

Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, Córdoba, Argentina

Requests for reprints: Mariana Maccioni, Haya de la Torre y Medina Allende, Ciudad Universitaria, Córdoba 5016, Argentina. Phone: 54-351-434-4973/76, ext. 4-123; Fax: 54-351-433-3048; E-mail: mmaccioni{at}bioclin.fcq.unc.edu.ar.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although an eruption of information on the role of Toll-like receptor 4 (TLR4), the main receptor for bacterial lipopolysaccharide, in activating macrophages and dendritic cells has emerged, very little is known about the role of TLR4 present on epithelial cells from sterile environments like tumors. The main goal of this work was to investigate the consequences of TLR4 activation present on tumor cells in two different animal models of cancer: the Dunning rat prostate cancer and the B16 murine melanoma models. We show that (a) activating TLR4 signaling in two different tumor cell lines in vitro modifies the tumor outgrowth in vivo; (b) this effect is not due to a direct consequence of TLR4 signaling on the proliferation/apoptosis balance of the tumor cells; (c) the T-cell compartment is somehow involved in the described phenomenon because the inhibitory effect observed is not seen in athymic nude mice; and (d) tumor-infiltrating lymphocytes purified from tumors induced by TLR4-activated cells show strong induction of IFN{gamma} transcript in detriment of interleukin-10 transcript, suggesting a change in their functionality. We hypothesize that TLR4 signaling in tumor cells in vitro induces the expression of proinflammatory mediators, which could dramatically alter the maturation state of dendritic cells present at the site of inoculation, switching the type of immune response elicited against the tumor. These results open up new avenues for understanding the role of TLR4 in tumor cells and for identifying potential new therapy strategies for cancer. [Cancer Res 2007;67(21):10519–27]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Toll-like receptors (TLR) conform a family of receptors that mediate the recognition of microbial/pathogen-associated molecular patterns that have been evolutionarily conserved in specific classes of microbes (1). TLR4, the first one described, recognizes the bacterial lipopolysaccharide (LPS), whereas members of the TLR9 subfamily, such as TLR7, TLR8, and TLR9, sense the microbial RNA and DNA. Binding of these pathogen-associated molecular patterns to TLRs triggers a complicated series of events leading to an increased expression of proinflammatory genes (2). In the recent years, an eruption of information on the role of TLRs in activating macrophages and dendritic cells has emerged (2). Although originally thought to be restricted to innate immune system cells, the presence of different TLRs, particularly TLR4, has been reported in several other cell types. TLR4 has been shown to be present in mucosal epithelia such as intestinal (3–5), tracheobronchial (6), genitourinary tract (7, 8), bladder (9), oral (10), and ocular epithelial cells (11). Even immune-privileged organs like those of the central nervous system are not exempt of TLR4 expression (12). However, the meaning of such expression, particularly in organs physiologically preserved in sterility, remains unclear. A variety of endogenous substances including fibrinogen, fibronectin extra domain, hyaluronic acid, and, perhaps, heat shock proteins can trigger TLR4 (13–15). These interactions have been proposed to initiate inflammation, maintain epithelial cell integrity, and promote recovery from acute injury (16, 17).

The use of TLR ligands as adjuvants to reinforce the immune response against tumors is among the first and oldest strategies used in anticancer immunity. This type of approaches has been empirically used long before TLRs were described, with the idea of activating TLRs-expressing antigen-presenting cells (18). In contrast, the significance of TLR activation on tumor cells has scarcely been explored yet. The main goal of this work was to investigate the consequences of TLR4 activation on tumor cells in two different animal models of cancer: the Dunning rat prostate cancer and the B16 murine melanoma models. We have recently shown that the rat prostate adenocarcinoma MAT-LU cells constitutively express TLR4 and CD14 and are perfectly capable of responding to LPS, activating nuclear factor {kappa}B (NF-{kappa}B) transcription factor, inducing inducible nitric oxide synthase expression, secreting nitric oxide, and up-regulating numerous chemokine genes like MCP1, MIP1{alpha}, IP10, RANTES, and IL-8 (19). Because s.c. inoculation of MAT-LU cells into syngeneic male Copenhagen rats results in the development of prostate tumors, we hypothesize that changes promoted by TLR4 signaling on tumor cells could modify the tumor outgrowth in vivo. We show that stimulating MAT-LU cells with LPS in vitro, before inoculation, produces significant inhibition of tumor growth in Copenhagen rats but not in athymic nude mice. The same effect could be observed when B16 cells, capable of inducing melanomas once inoculated into C57BL/6 mice, were previously treated with LPS. These results open up new avenues for understanding the role of TLRs, particularly TLR4, in tumor cells and for identifying potential new therapy strategies for cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and reagents. LPS from Escherichia coli 055:B5 (Sigma-Aldrich) or Ultra-pure LPS from E. coli K12 (InvivoGen) were used. Monophosphoryl lipid A (used as a TLR4 agonist) and LPS from msbB E. coli mutant, which lack the myristoyl fatty acid moiety of lipid A (used as an antagonist to wild-type E. coli LPS), were also purchased from InvivoGen. The goat polyclonal anti-TLR4 antibody (L-14), the rabbit polyclonal anti-CD14 (M-305), and the mouse monoclonal antibodies (mAb) anti-NF-{kappa}B p65 subunit (F-6), anti–extracellular signal–regulated kinase (ERK)-1/2 (K-23), and anti–p-ERK1/2 (E-4) were from Santa Cruz Biotechnology. Secondary antibodies conjugated with FITC (BD PharMingen), Alexa 546 and Alexa 488 (Molecular Probes), or horseradish peroxidase (Sigma-Aldrich) were used.

Cell lines. MAT-LU (part of the John Hopkins Special Collection; ref. 20) and B16 cell lines were obtained from American Type Culture Collection. MAT-LU cells (a rat adenocarcinoma cell line with characteristics of prostate epithelial cells) and B16 cells (melanoma) were maintained in culture in DMEM (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Natocor), 2 mmol/L L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin (Life Technologies) as previously described (20). Cells were stimulated in vitro with LPS for 48 h, washed exhaustively, and inoculated s.c. into syngeneic hosts.

Animals. Copenhagen rats were purchased from Charles River Laboratories. Athymic nude mice and C57BL/6 mice were purchased from Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Argentina. C57BL/10ScNJ were purchased from The Jackson Laboratory. C57BL/10ScN mice have a deletion of the Tlr4 gene (Tlr4lps-del) that results in the absence of both mRNA and protein and thus in defective response to LPS stimulation. Animals were maintained at the Animal Resource Facility of the CIBICI-CONICET (Centro de Investigaciones en Bioquímica Clínica e Inmunología, CONICET) in accordance with the experimental ethics committee guidelines.

Interleukin-6 ELISA. Interleukin (IL)-6 protein levels in cultured cell supernatants were quantified using IL-6 Immunoassay kit (e-Bioscience) according to the manufacturer's protocol.

In vivo tumor challenge. Prostate tumors and melanomas were established in syngeneic hosts (Copenhagen rats and C57BL/6 mice, respectively) or in nude mice by s.c. injection of 1 x 106 cells in 250 µL of sterile PBS into the right flank. Tumor development was monitored everyday by measuring tumor perpendicular diameters with a metric caliper. Tumor volume was estimated as d2 x D x 0.5, where d and D are the minor and major diameters, respectively. For ethical reasons, animals were sacrificed when tumors reached a volume of >2 cm3.

Reverse transcription-PCR analysis. A standard reverse transcription-PCR (RT-PCR) assay was used in this study. Briefly, total RNA was isolated from MAT-LU cells, B16 cells, or CD3+ tumor-infiltrating lymphocytes (TIL) using Trizol reagent (Life Technologies) according to the manufacturer's protocol. Reverse transcription reactions were done using 2 to 3 µg of total mRNA in a 25-µL mixture. Total RNA was first incubated with 0.5 µg of oligo(dT) primer (Promega) for 10 min at 65°C and allowed to stand at room temperature for 2 min. Samples were then incubated with 1.25 mmol/L deoxynucleotide triphosphates (Promega), 10 units of RNasin inhibitor (Boehringer Mannheim), and 16 units of avian myeloblastosis virus reverse transcriptase (Promega) for 1 h at 42°C in reverse transcriptase buffer. The cDNA obtained was subjected to PCR amplification using the PCR protocols already described (19) for rat ß-actin, rat CXCL8, rat CCL5, rat CXCL10, rat CCL2, rat CCL3, rat CCL4, IL-10, and IFN{gamma} (21) and for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mouse CCL5, mouse CXCL10, mouse CCL2, mouse CCL3, and IL-6 (22). The conditions were chosen so that none of the RNAs analyzed reached a plateau at the end of the amplification protocol (in most reactions, 30 cycles of amplification were used). The PCR products were visualized in 2% agarose gels with ethidium bromide staining.

Confocal microscopy. Cells were plated onto 12-mm circular coverslips in 24-well plates (5 x 105 per well) and cultured overnight. Then the cells were incubated with or without stimulus for different time periods. After the period of stimulation was completed, coverslips were washed twice in PBS, then fixed with 3% formaldehyde in PBS for 10 min at room temperature. Following fixation, coverslips were washed twice in PBS and cells were then permeabilized by treatment with 0.2% Triton X-100 in PBS for 10 min, washed thrice with PBS, and blocked with PBS containing 1% bovine serum albumin (BSA) plus Tween 20 (0.1% v/v; blocking buffer) for 1 h at room temperature in a humid chamber. Cells were then incubated overnight at 4°C with blocking buffer containing the primary antibodies. Cells were then washed with PBS-Tween 20 (0.1% v/v) and incubated for 2 h at room temperature in blocking buffer with the corresponding secondary antibodies conjugated with Alexa 546, Alexa 488, or FITC. Coverslips were washed thrice, mounted with Prolong Antifade (Molecular Probes), and visualized with a confocal laser scanning microscope LSM 510 (Zeiss) using LSM 510 software for image analysis.

Western blot. Cells were pelleted at 500 x g and then lysed at 4°C with agitation for 30 min in 20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L Na2EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß-glycerophosphate, 1 mmol/L Na3VO4, and protease inhibitor mixture (BD PharMingen). For Western blot analysis, cellular extracts were combined with 3x SDS loading buffer [187.5 mmol/L Tris-HCl (pH 6.8), 6% SDS, 30% glycerol, 0.03% bromophenol blue, 125 mmol/L DTT]. Proteins were resolved by SDS-PAGE and then electrotransferred onto Immobilon-P membranes (Millipore). Nonspecific binding sites were blocked by incubating membranes in TBS containing 0.05% Tween 20 and 5% (w/v) dry nonfat milk. Immunoblots were visualized with the enhanced chemiluminescence system (Amersham Bioscience).

Apoptosis assays. MAT-LU and B16 cell apoptosis was evaluated by a double staining procedure with the FITC Annexin V binding assay and propidium iodide (BD Biosciences) and flow cytometry. For the gated cells, the percentages of Annexin V–negative or Annexin V–positive cells and propidium iodide–negative or propidium iodide–positive cells, as well as double-positive cells, were evaluated based on quadrants determined from single-stained and unstained control samples.

Proliferation assay. To evaluate cell proliferation, MAT-LU cells were cultured for 48 h with or without different concentrations of LPS. Then, cultures were pulsed with 1 µCi/mL [methyl-3H]thymidine (specific activity, 50 Ci/mmol; New England Nuclear) for the last 18 h. Results are expressed as cpm ± SD of triplicate determinations.

Obtainment of tumor-infiltrating cells. Tumors were removed from Copenhagen rats and single-cell suspensions were prepared by enzymatic digestion. Resected tumors were weighed, minced into small (1–2 mm3) pieces with a scalpel, and immersed in 10 mL of digestion mixture [5% FBS in RPMI 1640, 0.5 mg/mL collagenase A (Roche Diagnostic), 0.2 mg/mL hyaluronidase type V (Sigma-Aldrich), and 0.02 mg/mL Dnase I (Sigma-Aldrich) per 0.25-mg tumor tissue]. The mixture was incubated at 37°C for 45 min on a rotating platform. The resulting cell suspensions were filtered sequentially through 70- and 40-µm cell strainers (BD Falcon) and washed with 5% FBS in RPMI 1640 and submitted to Ficoll-Hypaque (Sigma) gradient centrifugation.

Flow cytometry. Cells were incubated with phycoerythrin-Cy5–conjugated mouse anti-rat CD4, R-phycoerythrin–conjugated mouse anti-rat CD3, and FITC-conjugated mouse anti-rat CD8a mAbs (BD PharMingen) for 30 min at 4°C in fluorescence-activated cell sorting (FACS) wash buffer (EDTA 5 mmol/L, sodium azide 0.1%, BSA 1% in PBS). Stained cells were analyzed with a Cytoron Absolute (Ortho Diagnostic System).

Purification of CD3+ TILs. Cells were labeled with a FITC-conjugated mouse anti-rat CD3 (clone G4.18; BD Biosciences) and subsequently magnetically labeled with anti-FITC microbeads (Miltenyi Biotec). CD3+ cells were positively selected by magnetic sorting according to the manufacturer's directions. Purified population was >90% CD3+ as measured by flow cytometry.

Statistical analysis. Statistical analysis were done using the LSD Fisher test and the InfoStat software (developed by Statistics Department, National University of Córdoba). P < 0.05 was considered significant. Kaplan-Meier analysis was done using GraphPad Prism4 software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TLR4 signaling in MAT-LU cells. As shown in other tumor cell lines, MAT-LU cells constitutively express significant levels of TLR4, TLR2, and CD14 and are perfectly capable of responding to LPS (19). To further confirm the specificity of our previous findings, we stimulated MAT-LU cells with monophosphoryl lipid A, a derivative of lipid A from Salmonella minnesota R595 LPS that acts as an agonist to LPS. Whereas LPS is a complex heterogeneous molecule, its lipid A portion is relatively similar across a wide variety of pathogenic strains of bacteria (23, 24). Lipid A has been shown to activate TLR4 and has proved to be a potent, yet apparently nontoxic, vaccine adjuvant (25). Thus, we cultured MAT-LU cells on coverslips, subjected them to LPS or lipid A, and carried out indirect immunofluorescent staining for the p65 subunit of NF-{kappa}B. Nuclear localization of this transcription factor subunit was analyzed by confocal microscopy. As shown in Fig. 1A , nuclear localization of NF-{kappa}B was already evident after 30 min of exposure to LPS (Fig. 1A). Moreover, the incubation of MAT-LU cells with lipid A (Agonist) also induces NF-{kappa}B p65 translocation. We also tested the effect of the msbB E. coli mutant LPS, which lacks the myristoyl fatty acid moiety of lipid A and displays a 1,000- to 10,000-fold reduction in the ability to activate NF-{kappa}B. It acts as an antagonist to wild-type E. coli LPS through direct interaction with TLR4 (26, 27). As expected, stimulation of MAT-LU cells with the antagonist molecule did not activate NF-{kappa}B (Fig. 1A, Antagonist). When MAT-LU cells were cultured simultaneously with LPS and LPS from msbB E. coli mutant, the translocation of the p65 subunit to the nucleus was strongly inhibited (LPS + Antagonist, Fig. 1A). Similar inhibition on NF-{kappa}B translocation was observed when lipid A was incubated simultaneously with the antagonist molecule (Agonist + Antagonist, Fig. 1A). These results corroborate that activation of MAT-LU cells and translocation of NF-{kappa}B to the nucleus are a consequence of the direct interaction of LPS with its receptor, TLR4. Furthermore, we confirmed the activation of the TLR4 signaling pathway by analyzing the phosphorylation of downstream signaling molecules like ERK. The kinetics of phosphorylation of ERK in MAT-LU cells after LPS stimulation was very similar to that reported in macrophages, being evident as soon as 10 min after stimulation (Fig. 1B). Activation of TLR4 signaling pattern induces the expression of several chemokine genes like CCL5 (RANTES), CXCL10 (IP10), and CCL3 (MIP1{alpha}), which is readily visible 6 h after LPS stimulation (Fig. 1C). Although some basal expression of CCL2 (MCP1) and CXCL8 could be observed in nonstimulated cells, there was at least a 1-fold increase in their mRNA levels, with peaks at 24 and 48 h of LPS stimulation, respectively (Fig. 1C). Transcription levels of CCL4 (MIP1ß) were kept unchanged. Similar experiments were done with a highly purified preparation of LPS commercially available. As shown in Fig. 1D, this highly purified LPS and lipid A were both capable of up-regulating the expression of chemokine genes like CCL3 and CCL5.


Figure 1
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  		Figure 1. TLR4 signaling in MAT-LU cells. A, MAT-LU cells were cultured with LPS (1 µg/mL), lipid A (Agonist; 1 µg/mL), LPS from msbB E. coli mutant (Antagonist; 10 µg/mL), LPS + Antagonist (0.1 and 10 µg/mL, respectively), and Agonist + Antagonist (1 + 10 µg/mL) for 30 min. Cells were then immunostained with anti-NF-{kappa}B p65 mAb and FITC-labeled secondary antibodies to analyze nuclear translocation of NF-{kappa}B. Nuclei were counterstained with 4',6'-diamidino-2-phenylindole (DAPI). NF-{kappa}B nuclear localization results in colocalization of green and blue signals resulting in cyan fluorescence. Micrographs were obtained at x60 magnification. B, Western blot analysis for ERK and p-ERK protein expression from nonstimulated MAT-LU cells (Basal) and MAT-LU cells stimulated with 1 µg/mL LPS at the indicated times. C, mRNA expression levels of CXCL8, CCL5, CXCL10, CCL2, CCL3, CCL4, and ß-actin evaluated by RT-PCR, following stimulation of MAT-LU cells with LPS (1 µg/mL) for the indicated time periods. D, mRNA expression levels of CCL3, CCL5, and ß-actin evaluated by RT-PCR, following stimulation of MAT-LU cells with LPS (1 µg/mL), Ultrapure LPS (1 µg/mL), and lipid A (Agonist; 1 µg/mL) for 24 h.

 
These findings show that well-known ligands of TLR4, like LPS or lipid A, are capable of triggering the TLR4 signaling pathway in MAT-LU cells, activating proinflammatory genes.

TLR-4 signaling inhibits tumor growth in a syngeneic model of prostate cancer. To evaluate if triggering TLR4 in vitro could modify the tumor outgrowth in vivo, we used a well-characterized syngeneic in vivo model of rat prostate cancer: the Dunning rat prostate cancer model (28). In this model, s.c. inoculation of tumor cells into the right flank of male Copenhagen rats routinely results in the development of primary tumors. Therefore, MAT-LU cells treated or not with LPS during 48 h were exhaustively washed and inoculated into two groups of Copenhagen rats (1 x 106 per rat): Basal group, injected with nonstimulated MAT-LU cells (n = 8), and LPS group, injected with LPS-stimulated MAT-LU cells (n = 11). The growth of tumors was followed everyday. Rats were sacrificed on day 20 and the tumor volume was evaluated. As it can be seen in Fig. 2A , a significant inhibition of tumor growth as well as a marked decrease in the size of the tumors at the time of sacrifice was observed when tumors were induced by LPS-stimulated MAT-LU cells. To verify if the observed inhibition of tumor growth could be due to a direct effect of LPS on the proliferation/apoptosis balance of MAT-LU cells, [3H]thymidine uptake assays and Annexin V/propidium iodide staining were done. The results are shown in Fig. 2C and D. The stimulation of MAT-LU cells with different amounts of LPS for 48 h altered neither the proliferation rate of the cells nor their apoptosis levels, even when doses as high as 100 µg/mL were used. Using the same rationale, we inoculated athymic nude mice with MAT-LU cells treated or not with LPS. Our hypothesis was that if the adaptive immune system was somehow involved in the inhibition of tumor growth observed in Copenhagen rats, no differences should be seen between tumors induced by MAT-LU cells treated or not with LPS in mice lacking T cells. On the contrary, if the LPS treatment was either modifying the proliferation rate of these cells or promoting their apoptosis, the same inhibition of tumor growth should be seen in nude mice. The results are shown in Fig. 2B. Neither tumor growth nor the final tumor volume measured at the time of sacrifice was significantly altered in nude mice inoculated with LPS-stimulated MAT-LU cells (LPS group, n = 10) compared with mice inoculated with nonstimulated MAT-LU cells (Basal group, n = 10). These results suggest that the T-cell compartment is somehow involved in the phenomenon observed.


Figure 2
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  		Figure 2. TLR-4 signaling inhibits tumor growth in a syngeneic model of prostate cancer. Nonstimulated MAT-LU cells (Basal) or MAT-LU cells stimulated with LPS (LPS) for 48 h were s.c. injected in Copenhagen rats (A) and in athymic nude mice (B). The animals were monitored every day for tumor growth. Points, mean tumor size; bars, SD. The animals were killed at day 20 postinjection. Representative of three independent experiments. *, P < 0.05. A, top, representative picture obtained from tumors induced by nonstimulated MAT-LU cells (top) or MAT-LU cells stimulated with LPS (bottom). LPS affects neither the proliferation nor the apoptosis levels of MAT-LU cells in vitro. MAT-LU cells were stimulated with growing concentrations of LPS for 48 h. Proliferation was measured by [3H]thymidine uptake (C) and apoptosis levels were measured by Annexin V (An)/propidium iodide (IP) staining (D). Representative of three independent experiments.

 
LPS-stimulated MAT-LU cells induce a different phenotypic/functional pattern of TILs. We then investigated if the stimulation of MAT-LU cells with lipid A also induced smaller tumors like LPS-stimulated MAT-LU cells did. Therefore, animals were injected with nonstimulated MAT-LU cells (Basal group, n = 5), lipid A–stimulated MAT-LU cells (Agonist group, n = 5), and LPS-stimulated MAT-LU cells (LPS group, n = 5). Tumors were induced and allowed to grow until day 20. A marked inhibition of tumor growth (data not shown) and tumor size at the time of sacrifice was observed in Agonist group compared with Basal group (Fig. 3A ), confirming the results obtained in LPS group.


Figure 3
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  		Figure 3. LPS-stimulated MAT-LU cells induce a different phenotypic/functional pattern of TILs. A, nonstimulated MAT-LU cells (Basal) and MAT-LU cells stimulated with 1 µg/mL LPS (LPS) or 1 µg/mL lipid A (Agonist) were s.c. injected into Copenhagen rats. The animals were monitored every day for tumor growth and sacrificed at day 20 postinjection. Tumor size was calculated as previously described. B, TILs were obtained as previously described in Materials and Methods. Number of cells per gram of tumor was determined by using the following formula: total number of lymphocytes obtained in the lymphocyte gate / tumor weight (g). C, percentages of total CD3+ cells and CD3+CD4+ and CD3+CD8+ cells were evaluated by flow cytometry. Columns, mean (n = 5 per group); bars, SD. *, P > 0.05, compared with Basal group. D, mRNA was obtained from purified CD3+ lymphocytes from individual tumors resected at day 13 (left) and day 20 (right) after inoculation and subjected to RT-PCR to evaluate the expression of IFN{gamma} and IL-10 transcripts.

 
Owing to the apparent involvement of T cells in the inhibition of tumor growth seen in animals inoculated with LPS-stimulated MAT-LU cells, we sought to investigate the characteristics of TILs in the three experimental groups described above. Tumors were resected at the moment of sacrifice and single-cell suspensions prepared from these tumors were analyzed. Interestingly, although not statistically significant, an increase in the number of tumor-infiltrating leukocytes per gram of tumor was observed in LPS and Agonist groups (Fig. 3B). Flow cytometric analysis of the single-cell suspension revealed a substantial population of CD3+ cells in Basal group (38%), which was significantly increased in both LPS and Agonist groups, reaching values of ~60% in both groups (P < 0.05). CD4+ T cells were also enriched in animals from LPS and Agonist groups, whereas the percentages of CD8 cells did not show significant variations (Fig. 3C). It could be argued that the inhibitory effect observed on tumor growth could be due to the kinetics of the immune response (delayed in LPS group) or that the type of immune response being elicited is different. For this reason, we analyzed the lymphocyte infiltrate at earlier points in both groups. The number of TILs per gram of tumor harvested at day 13 was 1,000-fold less compared with the number of TILs per gram of tumor obtained at day 20 in both experimental groups. Again, there was no statistically difference in the number of TILs per gram of tumor between the groups. As previously observed at day 20, higher percentages of CD3+ and CD4+ cells were detected in LPS group (see Supplementary Fig. S1A–C).

We next purified CD3+ cells from tumor-infiltrating leukocytes obtained at days 13 and 20 and analyzed the expression of two cytokines traditionally involved in opposite functions: IL-10, which not only suppresses the antitumor activity of infiltrating leukocytes but can also subvert their function to tumor advantage (29), and IFN{gamma}, which, in contrast, can reverse the balance in favor of antitumor immunity (30). When CD3+ cells were purified at day 13, important levels of IL-10 and IFN{gamma} mRNA could be detected in cells from both experimental groups. However, an already evident increase in the levels of IFN{gamma} transcript was observed in CD3+ cells purified from tumors of LPS group (Fig. 3D, left). This difference is further highlighted at day 20. Certainly, whereas CD3+ cells obtained from tumors of Basal group show important levels of IL-10 transcripts and almost undetectable levels of IFN{gamma} transcripts, CD3+ cells purified from tumors induced by LPS-stimulated MAT-LU cells show increased levels of IFN{gamma} mRNA and diminished transcription of IL-10 (Fig. 3D, right).

These findings suggest that tumors originated from TLR4-stimulated MAT-LU cells have a modified phenotypic/functional pattern of TILs compared with tumors induced by nonstimulated MAT-LU cells.

TLR4 signaling in B16 cells. To ascertain if the observed phenomenon was a particularity of this tumor model, we also carried out similar studies in the B16 mouse model of melanoma. Again, we found that these cells constitutively express CD14 and TLR4 and their levels seem to increase on LPS stimulation (Fig. 4A ). They were also capable of responding to LPS and lipid A by translocation of NF-{kappa}B p65 subunit to the nucleus (data not shown). Stimulation of these cells with LPS for 24 and 48 h also elicited an increase in the mRNA levels of some proinflammatory mediators such as CCL2, CCL3, and CCL5, whereas the levels of some other important chemokine transcripts such as CXCL10 did not show any variation (Fig. 4B). The levels of mRNA and secreted IL-6 were also increased after 48 h of LPS stimulation. The increase in the secretion was abolished when an anti-TLR4 antibody was added to the culture, indicating that this secretion of IL-6 in response to LPS is mediated through TLR4 (Fig. 4C).


Figure 4
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  		Figure 4. TLR4 signaling in B16 cells. A, B16 cells stimulated with 1 µg/mL LPS for 24 h were immunostained with the primary antibodies rabbit antimouse CD14 and goat antimouse TLR4, and then with the corresponding secondary antibodies antirabbit-FITC and antigoat-Alexa 546. To visualize nuclear localization, DAPI staining was used. Micrographs were obtained at x60 magnification. B, mRNA expression levels of CCL2, CCL3, CCL5, CXCL10, IL-6, and GAPDH evaluated by RT-PCR, following stimulation of B16 cells with LPS (1 µg/mL) for the indicated time periods. C, IL-6 levels measured by ELISA in supernatants of B16 cells stimulated or not with LPS. An antibody against mouse TLR4 was added to the culture (15 µg/mL) to show the TLR4 specificity of the IL-6 response to LPS.

 
TLR-4 signaling inhibits tumor growth in the B16 mouse melanoma model. B16 cells were then stimulated with LPS or lipid A for 48 h, washed exhaustively, and injected into syngeneic hosts. As in the MAT-LU prostate cancer model, tumors induced by LPS-stimulated (LPS group, n = 10) or lipid A–stimulated (Agonist group, n = 8) cells had an inhibited growth (Fig. 5A ) compared with those induced by nontreated B16 cells (Basal group, n = 10). When melanomas were induced in nude mice to ascertain that the LPS stimulation on B16 cells was not affecting directly the proliferation or apoptosis levels of the cells, the results obtained were similar to those obtained in the rat prostate tumor model. Again, no significant differences were found in the growth of tumors induced by B16 treated with LPS (LPS group) or nontreated B16 (Basal group) in nude mice (Fig. 5B). Moreover, analysis of the apoptosis levels of LPS-stimulated B16 cells (by Annexin V/propidium iodide staining) showed that, like in the rat tumor cells, LPS stimulation did not induce programmed cell death (see Supplementary Fig. S2).


Figure 5
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  		Figure 5. TLR-4 signaling in B16 cells inhibits tumor growth in a mouse melanoma model. A, nonstimulated B16 cells (Basal) and B16 cells stimulated with 1 µg/mL LPS (LPS) and 1 µg/mL lipid A (Agonist) were s.c. injected into C57BL/6 mice, in nude mice (B), or in C57BL/10ScNJ (Tlr4lps-del) mice (C). The animals were monitored every day for tumor growth and sacrificed at day 20 postinjection. Tumor size was calculated as previously described.

 
To confirm our hypothesis that the diminished tumor growth observed was a consequence of an indirect stimulation of the immune system by TLR4-activated tumor cells rather than a direct effect of possible contaminant LPS on antigen-presenting cells, TLR4-deficient mice were inoculated with B16 cells treated or not with LPS. As observed before, tumors induced by LPS-stimulated B16 cells (LPS group, n = 4) had an inhibited growth (Fig. 5C) compared with those induced by nontreated B16 cells (Basal group, n = 4). These results clearly show that it is the TLR4 triggering in the tumor cells themselves (and not in antigen-presenting cells) that is responsible for the consequent diminution of the tumor growth observed; otherwise, no differences in tumor growth in both experimental groups would have been seen.

Furthermore, activation of the TLR4 pathway in B16 cells by either LPS or lipid A in vitro dramatically alters the survival of tumor-bearing mice from LPS or Agonist group compared with that of Basal group. Indeed, 50% of mice inoculated with LPS- or lipid A–treated cells were still alive by day 60 postinoculation, whereas all the mice inoculated with nontreated B16 cells died at approximately day 45 (Fig. 6A ). Moreover, by approximately day 15, none of the mice from Basal group was free of tumors whereas only 25% and 40% of mice from Agonist and LPS groups, respectively, were still free of tumors by day 60 postinoculation (Fig. 6B).


Figure 6
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  		Figure 6. B16 cells stimulated with LPS increase survival rates in C57BL/6 mice. A, long-term survival of tumor-bearing mice from the Basal, LPS, or Agonist group. Results are compiled from two independent experiments (n = 10 per group). P < 0.002, Kaplan-Meier analysis. B, tumor-free mice recorded as the percentage on a given day.

 
Therefore, TLR4 signaling in two different cell lines and from different species promotes an evident antitumoral effect, at least under the experimental conditions used in this work.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we show that (a) activating TLR4 signaling in two different tumor cell lines in vitro modifies the tumor outgrowth in vivo; (b) this effect is not due to a direct consequence of TLR4 signaling on the proliferation/apoptosis balance of the tumor cells; (c) the T-cell compartment is somehow involved in the described phenomenon because the inhibitory effect observed is not seen in athymic nude mice; and (d) TILs purified from tumors induced by TLR4-activated cells show strong induction of IFN{gamma} transcript in detriment of IL-10 transcript, suggesting a change in the functionality of these tumor-infiltrating cells, at least in one of the tumor models examined.

Because TLRs can modulate both innate and adaptive immunity, stimulation of TLR pathways through their ligands represents a novel opportunity to activate an anticancer immune response. Indeed, classic cancer vaccines have used many TLR ligands as adjuvant immunotherapy for patients with cancer. The most paradigmatic case is Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton, which activates TLR2 and TLR4 and has been injected and emulsified together with tumor debris in experimental models of cancer (31, 32). Bacillus Calmette-Guerin has not only been used as adjuvant but has also been administered alone to patients for postoperative treatments of cancer, producing good prognosis (33). Another example of antitumoral effect mediated by a TLR-agonist is the use of imiquimod, a small-molecule immune response modifier of the imidazoquinoline family that has shown profound antitumoral and antiviral efficacy both in vitro and in clinical applications in vivo (34). Imiquimod combines multiple, presumably synergistic, antitumoral functions. One of them is the induction of apoptosis of tumoral cells through mechanisms independent of TLR signaling (35). However, predominant among its actions is the induction of proinflammatory cytokines through agonistic activity toward TLR7 and TLR8 and, consecutively, activation of the central transcription factor NF-{kappa}B and therefore promotion of tumor-directed cellular immune response (36).

Using the same rationale, other TLR ligands have been explored in experimental models of tumor. Moreover, intratumoral administration of phosphorothioate oligodeoxynucleotides containing CpG motifs (TLR9 ligand) resulted in an 84% reduction of tumor volume in a glioma model in Fischer rats (37). In this model, macrophages played a critical role in the early phase of tumor rejection because the treatment had no effect in rats depleted of macrophages (37). In other tumor models in mice, like the C26 colon carcinoma or the EG-7 thymoma models, CpG showed a significant antitumor effect only when IL-10 or regulatory T cells were blocked simultaneously with intratumoral CpG (38). Similar results were obtained when flagellin, the structural protein subunit of the bacterial flagellum, specifically recognized by TLR5, was peritumorally administered (39). Further, when flagellin was combined with CpG-containing oligodeoxynucleotides, the antitumoral effect was enhanced and tumor growth was completely suppressed (39).

Regarding LPS, the most well-known TLR4 ligand, results are contradictory. On one hand, LPS has previously been reported to have angiogenic activity in a number of angiogenesis models, increasing microvascular permeability and inducing the expression of vascular endothelial growth factor and matrix metalloproteinases (40, 41). In contrast, intratumoral administration of LPS or lipid A analogues seems to have beneficial antitumoral effects, although the mechanisms involved have not been elucidated yet (42). One example of TLR4 agonist successfully used as a therapeutic agent is OK-432. OK-432 and its active component, the lipoteichoic acid–related molecule OK-PSA, are successfully used as immune therapeutic agents in malignancies like head and neck cancer and oral squamous cell carcinoma (43, 44). It has been reported that TLR4 signaling is involved in the regulation of OK-PSA–induced anticancer immunity in tumor-bearing mice and that oral cancer patients who do not express or faintly express TLR4 or MD-2 genes did not obtain satisfactory therapeutic effects in response to OK-432 (44).

Most of the experimental systems detailed above were designed with the idea of activating TLRs present on innate immune system cells and thus activating a more efficient antitumor response. In none of them a clear distinction about the separate contribution of immune cells and epithelial cells has been done yet. Recent data reporting the presence of TLRs, particularly TLR4, in epithelial cells, many tumor cell lines, and clinical samples of tumors (3, 45, 46) raise the question about the meaning of such expression in the tumor environment and about the possibility of manipulating TLR signaling in tumor cells to achieve an antitumoral response.

Concomitant presence of TLR4 and Myd88 has been linked to chemoresistance and poor prognosis in ovarian cancer (46). Huang et al. (45) have shown that blockade of the TLR4 pathway by either TLR4 short interfering RNA or a cell-permeable TLR4 inhibitory peptide delays tumor growth and thus prolongs the survival of tumor-bearing mice (45). They hypothesized that endogenous ligands could activate TLR4-expressing tumor cells and induce the chronic secretion of proinflammatory cytokines and chemokines, well-known predictors of poor prognosis. In contrast, in a two-stage chemical carcinogenesis mouse model in which inflammation mediates the promotion phase of lung cancer, the presence of a functional TLR4 inhibited lung carcinogenesis, suggesting a protective role of TLR4 in this model of cancer (47).

The fact that the inoculation of LPS-stimulated B16 cells in TLR4-deficient mice also elicited smaller tumors further confirms our hypothesis, underlying the importance of TLR4 triggering on tumor cells and ruling out the possibility that trace amounts of contaminant LPS could be directly activating dendritic cells in the host. Our results allow us to hypothesize that TLR4 signaling in tumor cells by an exogenous ligand, like LPS or lipid A, induces the expression of proinflammatory mediators that could, even if transitory, dramatically alter the phenotype of dendritic cells present at the site of inoculation and switch the type of immune response elicited against the tumor. Moreover, the secretion of IL-6– by LPS-stimulated B16 cells could also be contributing to render effector T cells refractory to the suppressive activity of CD4+CD25+ regulatory T cells, as it has been shown in other systems (48).

There is a general consensus that "antigen-primed" dendritic cells migrate from the tumor environment and present tumor-derived antigens to naive T cells in the regional lymph node (49). The local cytokine milieu is crucial for determining the maturation and consequent immunostimulatory or immunosuppressive function of dendritic cells. Thus, this inflammatory milieu generated at very early stages of tumor implantation could have a completely different effect than that chronically induced by endogenous ligands. This could explain why our findings differ so much from those reported by Huang et al. (45), in which TLR4 signaling by endogenous ligands is seen as a mechanism of tumor evasion.

Our results show that tumors begin to be detectable at approximately the same day in both experimental groups of animals and in both tumor models, but then growth diminishes in tumors induced by LPS-treated cells. This fact supports the hypothesis that the generation of an adaptive immunity appearing at days 10 to 12 could be responsible for that. In the same way, the presence of CD3+ infiltrating lymphocytes expressing higher levels of IFN{gamma} gene in detriment of IL-10 implies that a more efficient immune response against the tumor is taking place in tumors induced by LPS-treated cells. It could be argued that downstream events elicited as a consequence of TLR4 signaling, such as tumor necrosis factor-{alpha} production, could be responsible for the effect observed.

Inherited polymorphisms of some TLR4 alleles have been reported to be associated with higher risk to prostate cancer, and a less efficient resolution of infection by innate immune cells has been speculated to be the subjacent cause of this association (50). Our results open up a new perspective to TLR4 signaling that has not been thoroughly explored yet: the role of TLR4 signaling present on the tumor cells themselves. Further studies should be done to understand its meaning and to find potential new therapy strategies for cancer.


   Acknowledgments
 
Grant support: Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) de Investigación Científica y Tecnológica (PICT) 2002 11944, Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET) PEI no. 6557, and Fundación Antorchas 2004 (M. Maccioni). V. Andreani and G. Gatti are Ph.D. fellows of CONICET. M. Maccioni and V. Rivero are members of the Researcher Career of CONICET.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

V. Andreani and G. Gatti contributed equally to this work.

Received 1/ 9/07. Revised 7/24/07. Accepted 8/29/07.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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