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Crosstalk between Tumor Cells and Macrophages in Stroma Renders Tumor Cells as the Primary Source of MCP-1/CCL2 in Lewis Lung Carcinoma.

Yoshimura T, Liu M, Chen X, Li L, Wang JM - Front Immunol (2015)

Bottom Line: The chemokine MCP-1/CCL2 is produced by a variety of tumors and plays an important role in cancer progression.Macrophages from TNFα(-/-) mice failed to activate LLC cells and anti-TNFα neutralizing antibody abolished the effect of WT macrophages on LLC cells.Thus, disruption of tumor-stromal cell interaction may inhibit tumor progression by reducing the production of tumor-promoting proinflammatory mediators, such as MCP-1.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute , Frederick, MD , USA.

ABSTRACT
The chemokine MCP-1/CCL2 is produced by a variety of tumors and plays an important role in cancer progression. We and others previously demonstrated that the primary source of MCP-1 in several mouse tumors, including 4T1 breast cancer, M5076 sarcoma, and B16 melanoma, was stromal cells. In the present study, we identified that tumor cells were the primary source of MCP-1 in Lewis lung carcinoma (LLC), because MCP-1 mRNA was highly expressed in tumors grown in both wild type (WT) and MCP-1(-/-) mice with elevated serum MCP-1 levels. Since LLC cells isolated from tumors expressed low levels of MCP-1 in vitro, it appeared that the tumor-stromal cell interaction in a tumor microenvironment increased MCP-1 expression in LLC cells. In fact, co-culture of LLC cells with normal mouse peritoneal macrophages or normal lung cells containing macrophages increased MCP-1 expression by LLC cells. Macrophages from TNFα(-/-) mice failed to activate LLC cells and anti-TNFα neutralizing antibody abolished the effect of WT macrophages on LLC cells. When LLC cells were transplanted into TNFα(-/-) mice, the levels of MCP-1 mRNA in tumors and serum MCP-1 levels were markedly lower as compared to WT mice, and importantly, tumors grew more slowly. Taken together, our results indicate that TNFα released by tumor cell-activated macrophages is critical for increased MCP-1 production by tumors cells. Thus, disruption of tumor-stromal cell interaction may inhibit tumor progression by reducing the production of tumor-promoting proinflammatory mediators, such as MCP-1.

No MeSH data available.


Related in: MedlinePlus

The role of macrophage MyD88 and TNFα in MCP-1 mRNA expression by LLC cells. (A) One thousand LLC cells were seeded into 12-well plates. After overnight incubation at 37°C, three different numbers of PC from WT, MyD88−/− or TNF−/− mice were added to the wells. To neutralize TNFα, 2 or 10 μg of anti-mouse TNFα IgG (R&D Systems) was added with 1 × 105 PC from WT mouse. After incubation at 37°C for 5 days, total RNA was isolated and the expression of MCP-1 mRNA was examined by Northern blotting (10 μg per lane). (B) The experiment presented in A was repeated with 1 × 105 PC from WT, MyD88−/− or TNF−/− mice and the MCP-1 concentration in the culture supernatants was measured by ELISA. The results are shown as the mean ± SEM. *p < 0.0001, n = 4. (C) The expression of TNF mRNA in 4T1 tumors or LLC tumors was examined by Nanostring gene profiling. The results are shown as the mean ± SEM.
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Figure 4: The role of macrophage MyD88 and TNFα in MCP-1 mRNA expression by LLC cells. (A) One thousand LLC cells were seeded into 12-well plates. After overnight incubation at 37°C, three different numbers of PC from WT, MyD88−/− or TNF−/− mice were added to the wells. To neutralize TNFα, 2 or 10 μg of anti-mouse TNFα IgG (R&D Systems) was added with 1 × 105 PC from WT mouse. After incubation at 37°C for 5 days, total RNA was isolated and the expression of MCP-1 mRNA was examined by Northern blotting (10 μg per lane). (B) The experiment presented in A was repeated with 1 × 105 PC from WT, MyD88−/− or TNF−/− mice and the MCP-1 concentration in the culture supernatants was measured by ELISA. The results are shown as the mean ± SEM. *p < 0.0001, n = 4. (C) The expression of TNF mRNA in 4T1 tumors or LLC tumors was examined by Nanostring gene profiling. The results are shown as the mean ± SEM.

Mentions: TLR ligands activate macrophages and can be released from tumor cells. Those activated macrophages then produce proinflammatory cytokines, such as TNFα, in a tumor microenvironment. To examine whether these proinflammatory mediators are involved in promoting MCP-1 expression by LLC tumors, we co-cultured LLC cells with PC from MyD88−/− or TNFα−/− mice and examined MCP-1 mRNA expression or MCP-1 protein production by LLC cells. PC from WT mice increased MCP-1 mRNA expression by LLC cells (Figure 4A, lanes 1–4). In contrast, PC from TNFα−/− mice (Figure 4A, lanes 8–10, Figure 4B) or MyD88−/− mice (Figure 4A, lanes 5–7, Figure 4B) did not increase MCP-1 expression or production by LLC cells. Addition of an anti-TNFα neutralizing antibody in the co-culture of LLC cells and WT PC almost completely inhibited the effect of WT PC to promote MCP-1 mRNA expression or production by LLC (Figure 4A, lanes 11, 12, Figure 4B). The expression of TNFα mRNA was detected in both 4T1 and LLC tumors with significantly higher expression levels in LLC tumors (Figure 4C). Thus, TNFα is available in a LLC tumor microenvironment.


Crosstalk between Tumor Cells and Macrophages in Stroma Renders Tumor Cells as the Primary Source of MCP-1/CCL2 in Lewis Lung Carcinoma.

Yoshimura T, Liu M, Chen X, Li L, Wang JM - Front Immunol (2015)

The role of macrophage MyD88 and TNFα in MCP-1 mRNA expression by LLC cells. (A) One thousand LLC cells were seeded into 12-well plates. After overnight incubation at 37°C, three different numbers of PC from WT, MyD88−/− or TNF−/− mice were added to the wells. To neutralize TNFα, 2 or 10 μg of anti-mouse TNFα IgG (R&D Systems) was added with 1 × 105 PC from WT mouse. After incubation at 37°C for 5 days, total RNA was isolated and the expression of MCP-1 mRNA was examined by Northern blotting (10 μg per lane). (B) The experiment presented in A was repeated with 1 × 105 PC from WT, MyD88−/− or TNF−/− mice and the MCP-1 concentration in the culture supernatants was measured by ELISA. The results are shown as the mean ± SEM. *p < 0.0001, n = 4. (C) The expression of TNF mRNA in 4T1 tumors or LLC tumors was examined by Nanostring gene profiling. The results are shown as the mean ± SEM.
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Related In: Results  -  Collection

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Figure 4: The role of macrophage MyD88 and TNFα in MCP-1 mRNA expression by LLC cells. (A) One thousand LLC cells were seeded into 12-well plates. After overnight incubation at 37°C, three different numbers of PC from WT, MyD88−/− or TNF−/− mice were added to the wells. To neutralize TNFα, 2 or 10 μg of anti-mouse TNFα IgG (R&D Systems) was added with 1 × 105 PC from WT mouse. After incubation at 37°C for 5 days, total RNA was isolated and the expression of MCP-1 mRNA was examined by Northern blotting (10 μg per lane). (B) The experiment presented in A was repeated with 1 × 105 PC from WT, MyD88−/− or TNF−/− mice and the MCP-1 concentration in the culture supernatants was measured by ELISA. The results are shown as the mean ± SEM. *p < 0.0001, n = 4. (C) The expression of TNF mRNA in 4T1 tumors or LLC tumors was examined by Nanostring gene profiling. The results are shown as the mean ± SEM.
Mentions: TLR ligands activate macrophages and can be released from tumor cells. Those activated macrophages then produce proinflammatory cytokines, such as TNFα, in a tumor microenvironment. To examine whether these proinflammatory mediators are involved in promoting MCP-1 expression by LLC tumors, we co-cultured LLC cells with PC from MyD88−/− or TNFα−/− mice and examined MCP-1 mRNA expression or MCP-1 protein production by LLC cells. PC from WT mice increased MCP-1 mRNA expression by LLC cells (Figure 4A, lanes 1–4). In contrast, PC from TNFα−/− mice (Figure 4A, lanes 8–10, Figure 4B) or MyD88−/− mice (Figure 4A, lanes 5–7, Figure 4B) did not increase MCP-1 expression or production by LLC cells. Addition of an anti-TNFα neutralizing antibody in the co-culture of LLC cells and WT PC almost completely inhibited the effect of WT PC to promote MCP-1 mRNA expression or production by LLC (Figure 4A, lanes 11, 12, Figure 4B). The expression of TNFα mRNA was detected in both 4T1 and LLC tumors with significantly higher expression levels in LLC tumors (Figure 4C). Thus, TNFα is available in a LLC tumor microenvironment.

Bottom Line: The chemokine MCP-1/CCL2 is produced by a variety of tumors and plays an important role in cancer progression.Macrophages from TNFα(-/-) mice failed to activate LLC cells and anti-TNFα neutralizing antibody abolished the effect of WT macrophages on LLC cells.Thus, disruption of tumor-stromal cell interaction may inhibit tumor progression by reducing the production of tumor-promoting proinflammatory mediators, such as MCP-1.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute , Frederick, MD , USA.

ABSTRACT
The chemokine MCP-1/CCL2 is produced by a variety of tumors and plays an important role in cancer progression. We and others previously demonstrated that the primary source of MCP-1 in several mouse tumors, including 4T1 breast cancer, M5076 sarcoma, and B16 melanoma, was stromal cells. In the present study, we identified that tumor cells were the primary source of MCP-1 in Lewis lung carcinoma (LLC), because MCP-1 mRNA was highly expressed in tumors grown in both wild type (WT) and MCP-1(-/-) mice with elevated serum MCP-1 levels. Since LLC cells isolated from tumors expressed low levels of MCP-1 in vitro, it appeared that the tumor-stromal cell interaction in a tumor microenvironment increased MCP-1 expression in LLC cells. In fact, co-culture of LLC cells with normal mouse peritoneal macrophages or normal lung cells containing macrophages increased MCP-1 expression by LLC cells. Macrophages from TNFα(-/-) mice failed to activate LLC cells and anti-TNFα neutralizing antibody abolished the effect of WT macrophages on LLC cells. When LLC cells were transplanted into TNFα(-/-) mice, the levels of MCP-1 mRNA in tumors and serum MCP-1 levels were markedly lower as compared to WT mice, and importantly, tumors grew more slowly. Taken together, our results indicate that TNFα released by tumor cell-activated macrophages is critical for increased MCP-1 production by tumors cells. Thus, disruption of tumor-stromal cell interaction may inhibit tumor progression by reducing the production of tumor-promoting proinflammatory mediators, such as MCP-1.

No MeSH data available.


Related in: MedlinePlus