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The significance of the co-existence of osteopontin and tumor-associated macrophages in gastric cancer progression.

Lin CN, Wang CJ, Chao YJ, Lai MD, Shan YS - BMC Cancer (2015)

Bottom Line: M2 tumor-associated macrophages (M2-TAMs) also play an important role in cancer progression.In the co-culture study, OPN was able to attract U937 cells and skew them toward M2-TAMs through paracrine action.The M2-TAMs could increase the invasiveness of OPN+-AGS cells and the growth rate of xenograft of a mixture of co-cultured OPN+-AGS and U937 cells.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Osteopontin (OPN) can recruit macrophages to the site of inflammation and promote tumorigenesis. M2 tumor-associated macrophages (M2-TAMs) also play an important role in cancer progression. This study aimed to clarify the role of OPN and M2-TAMs co-existence in gastric cancer.

Methods: The levels of OPN and M2-TAMs were evaluated by immunohistochemical staining in 170 resected gastric cancer specimens that were collected from 1998 to 2012. M2-TAMs were identified by staining for an M2 marker, CD204. The prognostic significance and correlation between OPN and CD204 expression were analyzed. A co-culture system of OPN+-AGS and U937 cells was designed to study the effect of OPN on the skewing of macrophages toward M2-TAMs for gastric cancer progression in vitro and in vivo.

Results: Patients with high expression (>50%) of OPN or CD204 exhibited poor 5-year overall survival rates (48.61%, p = 0.0055, and 52.14%, p = 0.0498, respectively). A positive correlation was observed between OPN and CD204 expression and high co-expression of OPN and CD204 demonstrated poor 5-year overall survival rates (48.90%, p = 0.0131). In the co-culture study, OPN was able to attract U937 cells and skew them toward M2-TAMs through paracrine action. The M2-TAMs could increase the invasiveness of OPN+-AGS cells and the growth rate of xenograft of a mixture of co-cultured OPN+-AGS and U937 cells.

Conclusion: OPN can skew macrophages toward M2-TAMs during gastric cancer progression. The co-existence of OPN and infiltrating M2-TAMs correlates with disease progression and poor survival and thus can serve as a prognostic marker in gastric cancer.

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The paracrine effect of OPN skewed macrophages toward M2-TAMs in gastric cancer. (a) Dual immunofluorescence staining of OPN (green) and CD204 (red) shows co-localization (yellow) in gastric cancer specimens. The immunohistochemistry of CD204 shows that macrophages did not come in contact with tumor cells. (b) A co-culture method was designed to mimic the tumor microenvironment of gastric cancer without direct contact between cancer cells and macrophages. (c) The confocal images show that OPN contributed to the recruitment of U937 cells and skewed the cells toward M2-TAMs after treatment with conditioning medium; this phenomenon could be blocked by an OPN monoclonal antibody. Recombinant OPN was able to increase CD204 expression on macrophages. (d) The amount of CD204+TAMs was shown by flow cytometry. The CD204+-TAM phenotype increased dramatically after co-culture with OPN+AGS cells (red, open histogram). Parental cells (filled histogram), OPN-shRNA AGS cells (orange, open histogram), OPN neutralizing antibody (green, open histogram), or recombinant OPN (blue, open histogram) were compared. The TAMcli cells isolated from human gastric cancer were used as a positive control.
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Fig4: The paracrine effect of OPN skewed macrophages toward M2-TAMs in gastric cancer. (a) Dual immunofluorescence staining of OPN (green) and CD204 (red) shows co-localization (yellow) in gastric cancer specimens. The immunohistochemistry of CD204 shows that macrophages did not come in contact with tumor cells. (b) A co-culture method was designed to mimic the tumor microenvironment of gastric cancer without direct contact between cancer cells and macrophages. (c) The confocal images show that OPN contributed to the recruitment of U937 cells and skewed the cells toward M2-TAMs after treatment with conditioning medium; this phenomenon could be blocked by an OPN monoclonal antibody. Recombinant OPN was able to increase CD204 expression on macrophages. (d) The amount of CD204+TAMs was shown by flow cytometry. The CD204+-TAM phenotype increased dramatically after co-culture with OPN+AGS cells (red, open histogram). Parental cells (filled histogram), OPN-shRNA AGS cells (orange, open histogram), OPN neutralizing antibody (green, open histogram), or recombinant OPN (blue, open histogram) were compared. The TAMcli cells isolated from human gastric cancer were used as a positive control.

Mentions: The dual immunofluorescence results indicated that OPN and CD204 were co-localized even in high-expression or low-expression gastric cancer specimens. This finding suggests that OPN was bound to the surface of M2-TAMs. The IHC staining demonstrated that the macrophages were located beside the tumor cells (Figure 4a). The results implied that paracrine regulation occurs between OPN and M2-TAMs within gastric cancer. Thus, we designed a co-culture system to mimic the tumor microenvironment. To explore the chemoattractant effect of OPN on M2-TAMs infiltration, U937 (5 × 105) were cultured in the insert, and OPN+-AGS gastric cancer cells were grown on the base of a Boyden chamber (Figure 4b). After incubated for 72 hours, the condition medium was collected and added into another lower chamber. New insert containing U937 cells was put into the chamber and then incubated for 72 hours. The number of M2-TAMs in the lower chamber was significantly increased, compared with those cells treated with conditioning medium containing a monoclonal OPN antibody (Figure 4c). Furthermore, when rOPN was added to the normal medium, M2-TAMs were still observed in the base of chamber. Flow cytometry further proved that the M2-TAMs differentiation was associated with the presence of OPN (Figure 4d). Taken together, these results suggest that OPN was required for M2-TAMs infiltration in gastric cancer.Figure 4


The significance of the co-existence of osteopontin and tumor-associated macrophages in gastric cancer progression.

Lin CN, Wang CJ, Chao YJ, Lai MD, Shan YS - BMC Cancer (2015)

The paracrine effect of OPN skewed macrophages toward M2-TAMs in gastric cancer. (a) Dual immunofluorescence staining of OPN (green) and CD204 (red) shows co-localization (yellow) in gastric cancer specimens. The immunohistochemistry of CD204 shows that macrophages did not come in contact with tumor cells. (b) A co-culture method was designed to mimic the tumor microenvironment of gastric cancer without direct contact between cancer cells and macrophages. (c) The confocal images show that OPN contributed to the recruitment of U937 cells and skewed the cells toward M2-TAMs after treatment with conditioning medium; this phenomenon could be blocked by an OPN monoclonal antibody. Recombinant OPN was able to increase CD204 expression on macrophages. (d) The amount of CD204+TAMs was shown by flow cytometry. The CD204+-TAM phenotype increased dramatically after co-culture with OPN+AGS cells (red, open histogram). Parental cells (filled histogram), OPN-shRNA AGS cells (orange, open histogram), OPN neutralizing antibody (green, open histogram), or recombinant OPN (blue, open histogram) were compared. The TAMcli cells isolated from human gastric cancer were used as a positive control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384326&req=5

Fig4: The paracrine effect of OPN skewed macrophages toward M2-TAMs in gastric cancer. (a) Dual immunofluorescence staining of OPN (green) and CD204 (red) shows co-localization (yellow) in gastric cancer specimens. The immunohistochemistry of CD204 shows that macrophages did not come in contact with tumor cells. (b) A co-culture method was designed to mimic the tumor microenvironment of gastric cancer without direct contact between cancer cells and macrophages. (c) The confocal images show that OPN contributed to the recruitment of U937 cells and skewed the cells toward M2-TAMs after treatment with conditioning medium; this phenomenon could be blocked by an OPN monoclonal antibody. Recombinant OPN was able to increase CD204 expression on macrophages. (d) The amount of CD204+TAMs was shown by flow cytometry. The CD204+-TAM phenotype increased dramatically after co-culture with OPN+AGS cells (red, open histogram). Parental cells (filled histogram), OPN-shRNA AGS cells (orange, open histogram), OPN neutralizing antibody (green, open histogram), or recombinant OPN (blue, open histogram) were compared. The TAMcli cells isolated from human gastric cancer were used as a positive control.
Mentions: The dual immunofluorescence results indicated that OPN and CD204 were co-localized even in high-expression or low-expression gastric cancer specimens. This finding suggests that OPN was bound to the surface of M2-TAMs. The IHC staining demonstrated that the macrophages were located beside the tumor cells (Figure 4a). The results implied that paracrine regulation occurs between OPN and M2-TAMs within gastric cancer. Thus, we designed a co-culture system to mimic the tumor microenvironment. To explore the chemoattractant effect of OPN on M2-TAMs infiltration, U937 (5 × 105) were cultured in the insert, and OPN+-AGS gastric cancer cells were grown on the base of a Boyden chamber (Figure 4b). After incubated for 72 hours, the condition medium was collected and added into another lower chamber. New insert containing U937 cells was put into the chamber and then incubated for 72 hours. The number of M2-TAMs in the lower chamber was significantly increased, compared with those cells treated with conditioning medium containing a monoclonal OPN antibody (Figure 4c). Furthermore, when rOPN was added to the normal medium, M2-TAMs were still observed in the base of chamber. Flow cytometry further proved that the M2-TAMs differentiation was associated with the presence of OPN (Figure 4d). Taken together, these results suggest that OPN was required for M2-TAMs infiltration in gastric cancer.Figure 4

Bottom Line: M2 tumor-associated macrophages (M2-TAMs) also play an important role in cancer progression.In the co-culture study, OPN was able to attract U937 cells and skew them toward M2-TAMs through paracrine action.The M2-TAMs could increase the invasiveness of OPN+-AGS cells and the growth rate of xenograft of a mixture of co-cultured OPN+-AGS and U937 cells.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Osteopontin (OPN) can recruit macrophages to the site of inflammation and promote tumorigenesis. M2 tumor-associated macrophages (M2-TAMs) also play an important role in cancer progression. This study aimed to clarify the role of OPN and M2-TAMs co-existence in gastric cancer.

Methods: The levels of OPN and M2-TAMs were evaluated by immunohistochemical staining in 170 resected gastric cancer specimens that were collected from 1998 to 2012. M2-TAMs were identified by staining for an M2 marker, CD204. The prognostic significance and correlation between OPN and CD204 expression were analyzed. A co-culture system of OPN+-AGS and U937 cells was designed to study the effect of OPN on the skewing of macrophages toward M2-TAMs for gastric cancer progression in vitro and in vivo.

Results: Patients with high expression (>50%) of OPN or CD204 exhibited poor 5-year overall survival rates (48.61%, p = 0.0055, and 52.14%, p = 0.0498, respectively). A positive correlation was observed between OPN and CD204 expression and high co-expression of OPN and CD204 demonstrated poor 5-year overall survival rates (48.90%, p = 0.0131). In the co-culture study, OPN was able to attract U937 cells and skew them toward M2-TAMs through paracrine action. The M2-TAMs could increase the invasiveness of OPN+-AGS cells and the growth rate of xenograft of a mixture of co-cultured OPN+-AGS and U937 cells.

Conclusion: OPN can skew macrophages toward M2-TAMs during gastric cancer progression. The co-existence of OPN and infiltrating M2-TAMs correlates with disease progression and poor survival and thus can serve as a prognostic marker in gastric cancer.

Show MeSH
Related in: MedlinePlus