Activated macrophages promote Wnt signalling through tumour necrosis factor-α in gastric tumour cells

The activation of Wnt/β-catenin signalling has an important function in gastrointestinal tumorigenesis. It has been suggested that the promotion of Wnt/β-catenin activity beyond the threshold is important for carcinogenesis. We herein investigated the role of macrophages in the promotion of Wnt/β-catenin activity in gastric tumorigenesis. We found β-catenin nuclear accumulation in macrophage-infiltrated dysplastic mucosa of the K19-Wnt1 mouse stomach. Moreover, macrophage depletion in ApcΔ716 mice resulted in the suppression of intestinal tumorigenesis. These results suggested the role of macrophages in the activation of Wnt/β-catenin signalling, which thus leads to tumour development. Importantly, the conditioned medium of activated macrophages promoted Wnt/β-catenin signalling in gastric cancer cells, which was suppressed by the inhibition of tumour necrosis factor (TNF)-α. Furthermore, treatment with TNF-α induced glycogen synthase kinase 3β (GSK3β) phosphorylation, which resulted in the stabilization of β-catenin. We also found that Helicobacter infection in the K19-Wnt1 mouse stomach caused mucosal macrophage infiltration and nuclear β-catenin accumulation. These results suggest that macrophage-derived TNF-α promotes Wnt/β-catenin signalling through inhibition of GSK3β, which may contribute to tumour development in the gastric mucosa.


Introduction
The canonical Wnt signalling pathway (Wnt/b-catenin pathway) operates by stabilizing b-catenin (Taketo, 2006). The phosphorylation of b-catenin by glycogen synthase kinase 3b (GSK3b) results in degradation through the ubiquitin pathway. The binding of Wnt ligands to a Frizzled receptor leads to inhibition of the b-catenin degradation complex consisting of APC, AXIN and GSK3b thereby allowing the nuclear translocation of stabilized b-catenin followed by transcriptional activation of the Wnt target genes. Wnt/b-catenin signalling has an important function in the maintenance of intestinal stem cells and progenitor cells (Korinek et al, 1998;van de Wetering et al, 2002), and its activation causes gastrointestinal tumour development (Oshima et al, 1995(Oshima et al, , 2006Fodde et al, 2001).
The activation level of Wnt/b-catenin signalling has been reported to be a determining factor for the multiplicity of intestinal polyposis in several types of Apc gene mutant mice (Gaspar and Fodde, 2004;Li et al, 2005). Moreover, nuclear bcatenin accumulation is predominantly observed in the invasion front of colon cancer in comparison to the non-invasive tumour area (Brabletz et al, 1998). Furthermore, sensitivity of embryonic stem (ES) cells for differentiation is inhibited by increase of Wnt/b-catenin signalling activity (Kielman et al, 2002). These results, taken together, suggest that the promotion of the Wnt/b-catenin activity beyond the threshold level is required for tumorigenesis, invasion and the maintenance of cell stemness.
On the other hand, it has been established that inflammation has an important function in cancer development (Coussens and Werb, 2002). The activation of tumour necrosis factor (TNF)-a or NF-kB pathway is required for the development of hepatocellular carcinoma (Pikarsky et al, 2004) and intestinal tumours (Greten et al, 2004;Popivanova et al, 2008) through the induction of growth factors and suppression of apoptosis. Although several signalling pathways have been reported for the promotion of Wnt/b-catenin activity (Fodde and Brabletz, 2007), it has not been elucidated yet whether the inflammatory response contributes to the promotion of Wnt/b-catenin signalling during tumorigenesis.
Gastric cancer is the second most common cancer in the world and it is closely associated with Helicobacter pylori infection, which leads to chronic inflammation (Correa, 2003). Moreover, the activation of the Wnt/b-catenin signalling is found in about 30% of gastric cancer (Clements et al, 2002), suggesting that Wnt activation is one of the major causes for gastric cancer development. We recently showed that cooperation of Wnt signalling and prostaglandin E 2 (PGE 2 ) pathway causes gastric cancer development in a transgenic mouse model (Oshima et al, 2006). Because the activation of PGE 2 signalling induces infiltration and activation of macrophages in the gastric mucosa (Oshima et al, 2004, these results collectively suggest that activated macrophages promote the Wnt/b-catenin signalling activity, which thus contributes to gastric cancer.
We herein show that TNF-a derived from activated macrophages promotes the Wnt/b-catenin activity in gastric cancer cells through the suppression of GSK3b. Moreover, Helicobacter infection in the Wnt1 transgenic mice resulted in macrophage infiltration and the activation of Wnt/b-catenin signalling in the gastric mucosa, which thus led to gastric tumorigenesis. These results suggest that activated macrophages in inflammatory microenvironment have an important function in gastric tumorigenesis through the promotion of the Wnt/b-catenin activity.

Results
Macrophage infiltration and b-catenin nuclear accumulation in gastric dysplasia of K19-Wnt1 transgenic mice K19-Wnt1 transgenic mice expressing Wnt1 in the gastric epithelial cells develop sporadic dysplastic lesions in the glandular stomach (Oshima et al, 2006). In these dysplastic lesions, inflammatory cells infiltrated the submucosa, whereas cell infiltration was rarely detected in the adjacent normal mucosa ( Figure 1A). By immunostaining, we found macrophage infiltration in the dysplastic mucosa, whereas tissue macrophages were only sparsely scattered in the normal mucosa ( Figure 1B and H). We also found strong nuclear staining of b-catenin in the epithelial cells of the dysplastic mucosa ( Figure 1C). Notably, b-catenin-accumulated epithelial cells were physically associated with stromal macrophages in the dysplastic lesions ( Figure 1E). In contrast, mild b-catenin accumulation was found only in the cell proliferation zone of the normal mucosa ( Figure 1I). We confirmed the increase in the b-catenin-positive index in the dysplastic lesion to be associated with the increased number of infiltrated macrophages ( Figure 1F). Moreover, the number of Ki-67-positive proliferating cells increased in the dysplastic mucosa when compared with that in the adjacent normal region ( Figure 1D and J). The mean Ki-67 labelling indices in the dysplastic lesions and normal mucosa were 43 and 15%, respectively (P ¼ 0.007). We also confirmed by BrdU incorporation analysis that cell proliferation was increased in dysplastic lesions ( Figure 1G). On the basis of these results, we hypothesized that macrophage infiltration by inflammatory responses causes promotion of Wnt/ b-catenin signalling activity in gastric epithelial cells, which probably leads to dysplastic changes in the gastric mucosa.

Requirement of macrophages for intestinal tumour development in Apc D716 mice
To examine whether infiltrated macrophages are required for tumorigenesis, we performed crossing experiments using op/op mice, in which the number of macrophages significantly decreased in the majority of tissues because of Csf1 mutation (Cecchini et al, 1994). We first examined whether op/op mutation suppressed macrophage infiltration in the inflamed stomach using K19-C2mE mice, which develop gastritis with heavy macrophage accumulation (Oshima et al, 2004). However, mucosal macrophages were still found in the stomach of the K19-C2mE op/op compound mice (Supplementary Figure 1). We therefore crossed op/op mice with Apc D716 mice, a model for intestinal polyposis caused by Wnt activation (Oshima et al, 1995). Apc D716 mice develop numerous polyps in entire intestinal tract (Figure 2A), and macrophages were infiltrated in the polyp stroma ( Figure 2B). Interestingly, Apc D716 op/op compound mice showed dramatic suppression of intestinal polyposis ( Figure 2C and E), and macrophages were not found in the polyp tissues ( Figure 2D). Moreover, the number of polyps 41 mm significantly decreased in Apc D716 op/op compound mice by 80% compared with Apc D716 mice. Although it remains to be elucidated as to whether the loss of the CSF-1 function affects tumorigenesis, these results suggest that macrophages were required for the growth of Wnt/b-catenin-activated tumour cells. Consistently, the BrdU labelling index also decreased in Apc D716 op/op mouse tumours ( Figure 2F).

Fluctuation of Wnt/b-catenin signalling activity in gastric cancer cells
We next examined the regulation of the Wnt/b-catenin signalling activity by in vitro experiments using human gastric cancer cells, AGS and Kato-III. AGS cells harbour a heterozygous mutation in the b-catenin gene (CTNNB1) at codon 34 (Caca et al, 1999;Supplementary Figure 2), whereas CTNNB1 is amplified in Kato-III cells (Suriano et al, 2005). Consistent with the previous results (Nojima et al, 2007), both cell lines showed an elevated b-catenin/TCF transcriptional activity as detected by a TOPFLASH assay (Supplementary Figure 3). By immunocytochemistry, the cytoplasmic and nuclear accumulation of b-catenin was found in both AGS and Kato-III cells ( Figure 3A, left). We also found the nuclear localization of active form of b-catenin, unphosphorylated on Ser37 and Thr41 (van Noort et al, 2002; Figure 3A, right), thus indicating the activation of Wnt/b-catenin signalling. However, several cells showed a weak b-catenin staining intensity ( Figure 3A, arrowheads), suggesting that the level of Wnt/b-catenin signalling activity varies in populations.
To examine the Wnt/b-catenin activity in the live cells, we transfected a TOPEGFP vector that expressed a green fluorescent protein (GFP) in response to b-catenin/TCF transcriptional activity ( Figure 3B), and established AGS-GFP and Kato-III-GFP cell lines. We confirmed that GFP expression was induced in b-catenin accumulated cells after the transient transfection of mutant CTNNB1 (S33A) expression vector ( Figure 3C). Importantly, we directly found by time-lapse video analysis that the Wnt/b-catenin activity was not constant but fluctuated in individual AGS-GFP cells (Supplementary Video 1).
To confirm the fluctuation of Wnt/b-catenin activity, we isolated by cell sorting the high-GFP (top 5%) and low-GFP (bottom 5%) populations corresponding to high and low Wnt/b-catenin signalling activity, respectively, from AGS-GFP and Kato-III-GFP cells ( Figure 3D, left). Immediately after isolation, both populations showed distinct GFP intensities ( Figure 3D, centre). Importantly, however, the GFP intensities of the isolated populations returned to a similar distribution to the unsorted cells after culturing for 5 days ( Figure 3D, right). These results confirmed that the activation level of Wnt/b-catenin signalling fluctuates in each cell.
We next examined the level of Wnt/b-catenin activity in cell cycle-synchronized Kato-III-GFP cells. However, the ratio of a high-GFP population did not change during cell cycling, thus indicating Wnt/b-catenin fluctuation to be independent of the cell cycle status (Supplementary Figure 4).

Promotion of Wnt/b-catenin activity in gastric cancer cells by activated macrophages and TNF-a
We next examined whether the activity of Wnt/b-catenin signalling is promoted by activated macrophages using the GFP reporter-transfected cells. We prepared a conditioned medium of macrophage cell line, RAW264, activated with lipopolysaccharide (LPS) for 24 h; (hereafter, CM-LPS ( þ )). A conditioned medium from unstimulated RAW264 cells was used as the control (CM-LPS (À)). When AGS-GFP and Kato-III-GFP cells were stimulated with CM-LPS ( þ ), the high-GFP population (corresponding to top 2% in the untreated control cells) significantly increased in a CM concentrationdependent manner ( Figure 4A and B, left and top, red area). In contrast, stimulation of the cells with CM-LPS (À) or LPS alone did not affect the Wnt/b-catenin activity in either cell line ( Figure 4A and B, middle and bottom). These results indicate that the soluble factor(s) derived from activated macrophages promote the Wnt/b-catenin activity in gastric cancer cells.
TNF-a is a key mediator for inflammation and is produced by activated macrophages. Notably, treatment of the cells with a neutralizing antibody against TNF-a significantly suppressed the effect by CM-LPS ( þ ) to enhance the Wnt/b-catenin activity in both cell lines ( Figure 4C). Furthermore, direct treatment with TNF-a increased the level of Wnt/b-catenin activity ( Figure 4D). We detected the expression of both TNF R1 and TNF R2 on AGS and Kato-III cells as well as in the primary cultured gastric epithelial cells (Supplementary Figure 5A and C and data not shown). Importantly, the blockade of either receptor by neutralizing antibody significantly suppressed the TNF-a-induced Wnt/ b-catenin activation (Supplementary Figure 5B). These results suggest that TNF-a signalling through both TNF R1 and TNF R2 is thus involved in the promotion of the Wnt/ b-catenin activity. By immunohistochemistry, we found most TNF-a-expressing cells in gastric tumours to be macrophages, whereas CD11c-positive dendritic cells were rarely detected (Supplementary Figure 6A). Importantly, treatment of the K19-Wnt1 mice with anti-TNF-a-neutralizing antibody resulted in a decrease of the b-catenin immunostaining intensity in the dysplastic lesions (Supplementary Figure 6B). Moreover, we examined the Wnt-promoting effect of other inflammatory cytokines, IL-1b, IL-6 and IL-11, which have an important function in gastric tumorigenesis (Howlett et al, 2005;Fox and Wang, 2007). However, these cytokines did not promote Wnt/b-catenin activity in gastric cancer cells, although IL-1b slightly increased the high-GFP population in AGS-GFP cells (Supplementary Figure 7). These results, taken together, suggest that TNF-a is one of the important macrophage-derived factors that promote the Wnt/b-catenin activity in gastric epithelial cells and cancer cells.

Suppression of b-catenin phosphorylation by TNF-a through the inhibition of GSK3b in gastric cancer cells
By a flow cytometry analysis, we found that the level of unphosphorylated b-catenin increased significantly in Kato-III cells when cells were treated with CM-LPS ( þ ) or TNF-a ( Figure 5A). By western blotting, we confirmed the increased level of unphosphorylated b-catenin in Kato-III cells stimulated with CM-LPS ( þ ) or TNF-a ( Figure 5B). These results suggested that CM-LPS ( þ ) or TNF-a suppressed the phosphorylation of b-catenin, resulting in Wnt/b-catenin promotion. Significantly, treatment with TNF-a resulted in an increase of GSK3b phosphorylation on Ser9 both in AGS and Kato-III cells ( Figure 5C), which caused the suppression of the GSK3b activity (Cross et al, 1995). The mean relative band intensities of the phosphorylated GSK3b to the control level were 2.03±0.14 (P ¼ 0.009) and 1.68±0.31 (P ¼ 0.03) in the AGS and Kato-III cells, respectively. Interestingly, a strong immunostaining signal for phosphorylated GSK3 was found in a sub-population of Kato-III cells that also showed nuclear accumulation of b-catenin at the same time ( Figure 5D). These results suggest that the promotion of Wnt/b-catenin signalling is regulated by the GSK3b phosphorylation status in gastric cancer cells. Akt is an important kinase for phosphorylation of GSK3b at Ser9 (Sharma et al, 2002). Importantly, the active form of Akt, phosphorylated on Ser473, increased significantly after TNF-a stimulation in both AGS and Kato-III cells ( Figure 5C). The mean relative band intensities of the phosphorylated Akt to the control level were 2.08 ± 0.16 (P ¼ 0.005) and 1.81 ± 0.19 (P ¼ 0.002) in the AGS and Kato-III cells, respectively. These results suggest that TNF-a accelerates the GSK3b phosphorylation through the activation of the Akt pathway.

NF-jB-independent promotion of Wnt/b-catenin activity in gastric cancer cells
To examine whether NF-kB is involved in Wnt/b-catenin promotion by activated macrophages, we transfected IkBsuperrepressor (IkBSR) expression vector into AGS-GFP cells and established two sublines, in which TNF-a-induced NF-kB activation was suppressed ( Figure 6A). Notably, the TNF-a-or CM-LPS ( þ )-induced promotion of the Wnt/bcatenin activity did not change in either of IkBSR-transfected AGS-GFP cells ( Figure 6B), thus suggesting the existence of an NF-kB-independent mechanism for the TNF-a-induced Wnt/b-catenin promotion.
When Kato-III cells were treated with the protein synthesis inhibitor cycloheximide, the unphosphorylated b-cateninpositive population decreased gradually in an incubation time-dependent manner, probably due to the phosphorylation of b-catenin by GSK3b ( Figure 6C, black lines). However, the simultaneous treatment with CM-LPS ( þ ) significantly suppressed the cycloheximide-induced decrease of this population ( Figure 6C, red lines). Therefore, it is also possible that activated macrophages suppress b-catenin phosphorylation independent of protein synthesis, which is consistent with the results that the transcription factor NF-kB pathway is not involved in the suppression of b-catenin phosphorylation.

Macrophage infiltration and Wnt/b-catenin activation in mouse gastric epithelial cells by Helicobacter infection
We next examined whether Wnt/b-catenin signalling is activated by infection-associated inflammation in the mouse stomach. H. felis, a close relative of H. pylori was infected into K19-Wnt1 mouse stomach. At 8 weeks after H. felis infection, the infiltration of macrophages was found in the H. felis-infected gastric mucosa ( Figure 7A-C), whereas tissue macrophages were sparsely scattered in the non-infected mucosal area ( Figure 7G-I). Such an inflamed mucosa in the infected K19-Wnt1 mice was only detected by histological examinations, and this inflamed mucosa was distinct from the spontaneously developed dysplastic lesions ( Figure 1A). Importantly, the number of b-catenin-accumulated epithelial cells increased significantly in the infected and inflamed mucosa compared with that in the non-inflamed mucosa ( Figure 7D, J and M). These results suggest that the infection-associated inflammation promotes the Wnt/b-catenin signalling activity in gastric epithelial cells in vivo. Moreover, in the inflamed mucosa, the epithelial cell proliferation detected by Ki-67 immunostaining significantly increased ( Figure 7E, K and N). Furthermore, the number of H þ /K þ -ATPase-positive parietal cells decreased dramatically in the H. felis-infected area, thus indicating the suppression of epithelial differentiation ( Figure 7F, L and O). These results, taken together, suggest that infiltrated macrophages in response to Helicobacter infection have an important function in the activation of Wnt/b-catenin signalling, thereby enhancing the proliferation and suppression of differentiation. When Apc D716 mice were infected with H. felis, we did not find any b-catenin-accumulated dysplastic cells in the    inflamed gastric mucosa (data not shown). Therefore, it is possible that the basal activation level of Wnt/b-catenin signalling is required for its promotion.

Tumour development in H. felis-infected K19-Wnt1 mouse stomach
We finally examined whether H. felis infection contributes to gastric tumorigenesis in K19-Wnt1 mice. Importantly, K19-Wnt1 mice developed gastric tumours at 20 weeks after H. felis infection, whereas no tumours were found in the H. felis-infected wild-type mice ( Figure 8A and E). Histologically, submucosal infiltration was found in both H. felis-infected K19-Wnt1 and wild-type mouse stomach ( Figure 8B and F). Although macrophage infiltration was detected in both genotypes ( Figure 8C and G), dysplastic epithelial cells were evident in the infected K19-Wnt1 tumours but not in the wild-type mouse stomach ( Figure 8D and H). These results, taken together, suggest that infection-associated inflammation has an important function in gastric tumorigenesis through the promotion of the Wnt/b-catenin activity beyond the basal activation level.

Macrophage infiltration and cancer development, progression and stemness
The activation of Wnt/b-catenin signalling by genetic alteration, such as mutation in APC or CTNNB1, is responsible for gastrointestinal tumorigenesis. However, accumulating evidence suggests that further promotion of the Wnt/b-catenin activity has an important function in tumour growth and progression (Fodde and Brabletz, 2007). For example, increased b-catenin accumulation is found in the invasion front of colon cancer (Brabletz et al, 1998). We herein demonstrated direct evidence that the Wnt/b-catenin activity fluctuates in individual cancer cells that carry the same genetic alteration in CTNNB1. Accordingly, Wnt/b-catenin signalling can be promoted beyond the basal activated level caused by genetic alteration, and such promotion may have an important function in both tumour development and progression.
We have previously shown the induction of COX-2/PGE 2 pathway to have an important function in the intestinal tumorigenesis that is triggered by Wnt/b-catenin activation (Oshima et al, 1996;Sonoshita et al, 2001). The simultaneous induction of both Wnt and PGE 2 pathways is also responsible for gastric tumour development in mouse models (Oshima et al, 2006). Although PGE 2 may have multiple functions for tumorigenesis, we previously found an increased PGE 2 level to result in macrophage accumulation in the gastric mucosa of K19-C2mE mice (Oshima et al, 2004). Accordingly, it is possible that macrophage infiltration in dysplastic lesions of K19-Wnt1 mice is caused by the spontaneous induction of COX-2/PGE 2 pathway. We herein show that Wnt/b-catenin signalling is promoted by macrophage-derived TNF-a, which contributes to gastric tumorigenesis. It is therefore possible that the induction of the COX-2/PGE 2 pathway promotes Wnt/b-catenin signalling through the enhancement of macrophage infiltration, which is responsible for tumour development.
On the other hand, Wnt/b-catenin signalling is not activated in the stomach of either K19-C2mE mice or H. felisinfected Apc D716 mice, thus indicating that the induction of the COX-2/PGE 2 pathway and subsequent macrophage infiltration are not sufficient for the promotion of Wnt/b-catenin in gastric epithelial cells. It is conceivable that the basal activation of the Wnt/b-catenin pathway by genetic alteration is required for the promotion of Wnt/b-catenin signalling.
The activation of Wnt/b-catenin pathway is also suggested to be important for stemness. Wnt/b-catenin signalling is required for the maintenance of normal intestinal stem cells (Korinek et al, 1998), and the increase in the Wnt/b-catenin activity contributes to the maintenance of stemness in ES cells (Kielman et al, 2002). Accordingly, it is possible that the increased Wnt/b-catenin activity is also important for the maintenance of cancer stem cells. Therefore, infiltrated macrophages in tumour tissues may be an important component of niche for cancer stem cells by promoting Wnt/b-catenin signalling through TNF-a pathway. This hypothesis is supported by the results that macrophages are required for the maintenance of progenitor cells in the intestinal crypt (Pull et al, 2005).

Possible pathways other than TNF-a for the promotion of the Wnt/b-catenin signalling activity
It has been shown that inflammatory cytokines, TNF-a, IL-1b, IL-6 or IL-11, have an important function in gastric tumorigenesis (Howlett et al, 2005;Fox and Wang, 2007). Among these cytokines, TNF-a is the most potent Wnt/b-catenin-promoting factor, although IL-1b slightly increases Wnt signalling in AGS cells. However, the increased level of the Wnt/b-catenin activity by CM-LPS ( þ ) is significantly higher than that by TNF-a alone in both cell lines (Figure 4), thus suggesting the effect of other macrophagederived factors on Wnt/b-catenin promotion. It has been reported that hepatocyte growth factor activates Wnt/ b-catenin signalling through the tyrosine phosphorylation of b-catenin (Rasola et al, 2006), and platelet-derived growth factor also promotes Wnt/b-catenin signalling through suppression of b-catenin phosphorylation (Yang et al, 2006). It is therefore possible that these factor(s) contribute to Wnt/b-catenin promotion together with TNF-a in gastric tumorigenesis.
Recently, using a colon tumour mouse model, TNF R1 signalling in bone marrow-derived cells has been shown to indirectly enhance epithelial Wnt/b-catenin signalling (Popivanova et al, 2008). Accordingly, it is possible that TNF-a signalling, not only in epithelial cells but also in stromal cells, may therefore contribute to the promotion of Wnt/b-catenin signalling in cancer cells.

Promotion of Wnt/b-catenin signalling in cancer cells that retain wild-type CTNNB1
We show here that TNF-a stimulation promotes the Wnt/b-catenin signalling activity through the suppression of b-catenin phosphorylation by GSK3b. Therefore, the expression of wild-type b-catenin is required for the promotion of Wnt/b-catenin signalling in epithelial cells as well as cancer cells. Because Kato-III cells have a multicopy of the wild-type CTNNB1 (Suriano et al, 2005), TNF-a stimulation suppresses phosphorylation of these b-catenin, thus leading to the enhancement of Wnt/b-catenin signalling. On the other hand, AGS cells possess mutation in CTNNB1 close to the phosphorylation site of GSK3b (Supplementary Figure  2; Caca et al, 1999), which causes the activation of the basal Wnt/b-catenin signalling. It is therefore possible that TNF-a stimulation suppresses phosphorylation of wild-type b-catenin expressed from retained intact CTNNB1, which contributes to the promotion of the total Wnt/b-catenin activity of AGS cells. In human gastric cancer, a CTNNB1 mutation is found in about 30% of the Wnt-activated cases (Clements et al, 2002). However, either a somatic mutation or downregulation of E-cadherin (Cheng et al, 2005), secreted frizzled-related proteins (Nojima et al, 2007), or b-TrCP, a component of ubiquitin ligase complex (Kim et al, 2007) is found in gastric cancer, which leads to the basal activation of Wnt/b-catenin signalling without mutation in CTNNB1. These results, taken together, suggest that wild-type b-catenin expressed in gastric cancer cells is stabilized by inflammatory macrophages, thus resulting in the promotion of Wnt/b-catenin signalling, which contributes to cancer development, progression and the maintenance of cancer stem cells.
Accordingly, the present results suggest that the suppression of macrophage infiltration and its activation by antiinflammatory drugs or inhibitors for PGE 2 pathway is, therefore, a possible strategy for chemoprevention against gastric cancer.