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Exposure of tumor-associated macrophages to apoptotic MCF-7 cells promotes breast cancer growth and metastasis.

Zhou N, Zhang Y, Zhang X, Lei Z, Hu R, Li H, Mao Y, Wang X, Irwin DM, Niu G, Tan H - Int J Mol Sci (2015)

Bottom Line: Tumor-associated macrophages (TAMs) have been found to be associated with the progression and metastasis of breast cancer.Macrophages co-cultured with apoptotic MCF-7 had increased tumor growth and metastatic ability in a nude mouse transplantation assay.During this process, macrophages secreted increased amounts of interleukin 6 (IL-6) leading to increased phosphorylation of signal transducers and activators of transcription 3 (STAT3), which likely explains the increased transcription of STAT3 target genes such as TGF-β1 and HIF-1α.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Peking University, Health Science Center, Beijing 100191, China. zhouna@bjmu.edu.cn.

ABSTRACT
Tumor-associated macrophages (TAMs) have been found to be associated with the progression and metastasis of breast cancer. To clarify the mechanisms underlying the crosstalk between TAMs and cancer stem cells (CSCs) in breast cancer recurrence and metastasis, we used a co-culture model of macrophages and apoptotic human breast cancer cell line MCF-7 cells to investigate the effects of TAMs on MCF-7 in vitro and in vivo. Macrophages co-cultured with apoptotic MCF-7 had increased tumor growth and metastatic ability in a nude mouse transplantation assay. The macrophages exposed to apoptotic cells also induce an increase in the proportion of CD44+/CD24- cancer stem-like cells, as well as their proliferative ability accompanied with an increase in mucin1 (MUC1) expression. During this process, macrophages secreted increased amounts of interleukin 6 (IL-6) leading to increased phosphorylation of signal transducers and activators of transcription 3 (STAT3), which likely explains the increased transcription of STAT3 target genes such as TGF-β1 and HIF-1α. Our results indicate that when cancer cells endure chemotherapy induced apoptosis, macrophages in their microenvironment can then activate cancer stem cells to promote cancer growth and metastasis by secreting IL-6, which activates STAT3 phosphorylation to regulate the transcription of its downstream target genes.

No MeSH data available.


Related in: MedlinePlus

Changes in the expression of macrophage expressed genes in macrophages co-cultured with apoptotic MCF-7 cells. Relative amounts of mRNA of (A) IL-6; (B) STAT3; (C) TGF-β1; (D) HIF-1α mRNA was determined by real-time RT-PCR, where β-actin was used as an internal standard. Control is normal cultured macrophages while co-culture is macrophages grown with apoptotic MCF-7 cells. Results are typical of three independent experiments. Data represent means ± S.E. (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ns means no significance; (E) Analysis of the phosphorylation of STAT3 in macrophages (Control) or macrophages cocultured with apoptotic MCF-7 cells (Coculture) by Western blot. Bands were analyzed using Quantity One software with β-actin used as a loading control. Protein levels were compared to the normal group. Results are typical of three independent experiments. Data represent means ± S.E. (n = 3). *p < 0.05.
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ijms-16-11966-f004: Changes in the expression of macrophage expressed genes in macrophages co-cultured with apoptotic MCF-7 cells. Relative amounts of mRNA of (A) IL-6; (B) STAT3; (C) TGF-β1; (D) HIF-1α mRNA was determined by real-time RT-PCR, where β-actin was used as an internal standard. Control is normal cultured macrophages while co-culture is macrophages grown with apoptotic MCF-7 cells. Results are typical of three independent experiments. Data represent means ± S.E. (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ns means no significance; (E) Analysis of the phosphorylation of STAT3 in macrophages (Control) or macrophages cocultured with apoptotic MCF-7 cells (Coculture) by Western blot. Bands were analyzed using Quantity One software with β-actin used as a loading control. Protein levels were compared to the normal group. Results are typical of three independent experiments. Data represent means ± S.E. (n = 3). *p < 0.05.

Mentions: Since conditioned media generated by the co-culture of macrophages and apoptotic MCF-7 cells changes the proliferative ability of MCF-7 cells, this suggests that the macrophages secrete a protein into the conditioned media that induces proliferation. Macrophages, as immunocytes, secrete several kinds of cytokines, each of which plays different roles [6]. The cytokine interleukin 6 (IL-6) is produced by macrophages at sites of inflammation, especially acute inflammation, which occurs in cancer tumors during chemotherapy. Signal transducers and activators of transcription 3 (STAT3) is the main transcription factor through which IL-6 signals in target cells, where it regulates many genes including those that promote cancer proliferation and metastasis (e.g., TGF-β1) and angiogenesis (e.g., HIF-1α) [17]. To determine whether the IL-6 pathway had been activated in our chemotherapy microenvironment model we examined the levels of expression for the IL-6, STAT3, TGF-β1 and HIF-1α genes (Figure 4). As expected, the mRNA levels for IL-6, TGF-β1 and HIF-1α in the macrophage co-culture with apoptotic MCF-7 were much higher than in the control group (Figure 4A). However, no significant difference in the mRNA levels for STAT3 was observed between the two groups. However, since activation of the STAT3 signaling pathway is due to the level of phosphorylated STAT3, and not abundance of total STAT3, we then examined the phosphorylation status of STAT3 using a Western blot (Figure 4E). As expected from the real-time RT-PCR results (Figure 4B), no significant difference in the total protein levels of STAT3 was detected between the CoA and Mac groups, but activation of the STAT3 pathway, as assessed by the presence of a p-STAT3 band, was only detected in the macrophage co-culture with the apoptotic MCF-7 cell group, and not in the Mac group (Figure 4E). These results show that our chemotherapy microenvironment model yields macrophages that are activated and produce cytokines, such as IL-6, and thus are able to modify the proliferative and metastatic properties of cancer stem-like cells.


Exposure of tumor-associated macrophages to apoptotic MCF-7 cells promotes breast cancer growth and metastasis.

Zhou N, Zhang Y, Zhang X, Lei Z, Hu R, Li H, Mao Y, Wang X, Irwin DM, Niu G, Tan H - Int J Mol Sci (2015)

Changes in the expression of macrophage expressed genes in macrophages co-cultured with apoptotic MCF-7 cells. Relative amounts of mRNA of (A) IL-6; (B) STAT3; (C) TGF-β1; (D) HIF-1α mRNA was determined by real-time RT-PCR, where β-actin was used as an internal standard. Control is normal cultured macrophages while co-culture is macrophages grown with apoptotic MCF-7 cells. Results are typical of three independent experiments. Data represent means ± S.E. (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ns means no significance; (E) Analysis of the phosphorylation of STAT3 in macrophages (Control) or macrophages cocultured with apoptotic MCF-7 cells (Coculture) by Western blot. Bands were analyzed using Quantity One software with β-actin used as a loading control. Protein levels were compared to the normal group. Results are typical of three independent experiments. Data represent means ± S.E. (n = 3). *p < 0.05.
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Related In: Results  -  Collection

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ijms-16-11966-f004: Changes in the expression of macrophage expressed genes in macrophages co-cultured with apoptotic MCF-7 cells. Relative amounts of mRNA of (A) IL-6; (B) STAT3; (C) TGF-β1; (D) HIF-1α mRNA was determined by real-time RT-PCR, where β-actin was used as an internal standard. Control is normal cultured macrophages while co-culture is macrophages grown with apoptotic MCF-7 cells. Results are typical of three independent experiments. Data represent means ± S.E. (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ns means no significance; (E) Analysis of the phosphorylation of STAT3 in macrophages (Control) or macrophages cocultured with apoptotic MCF-7 cells (Coculture) by Western blot. Bands were analyzed using Quantity One software with β-actin used as a loading control. Protein levels were compared to the normal group. Results are typical of three independent experiments. Data represent means ± S.E. (n = 3). *p < 0.05.
Mentions: Since conditioned media generated by the co-culture of macrophages and apoptotic MCF-7 cells changes the proliferative ability of MCF-7 cells, this suggests that the macrophages secrete a protein into the conditioned media that induces proliferation. Macrophages, as immunocytes, secrete several kinds of cytokines, each of which plays different roles [6]. The cytokine interleukin 6 (IL-6) is produced by macrophages at sites of inflammation, especially acute inflammation, which occurs in cancer tumors during chemotherapy. Signal transducers and activators of transcription 3 (STAT3) is the main transcription factor through which IL-6 signals in target cells, where it regulates many genes including those that promote cancer proliferation and metastasis (e.g., TGF-β1) and angiogenesis (e.g., HIF-1α) [17]. To determine whether the IL-6 pathway had been activated in our chemotherapy microenvironment model we examined the levels of expression for the IL-6, STAT3, TGF-β1 and HIF-1α genes (Figure 4). As expected, the mRNA levels for IL-6, TGF-β1 and HIF-1α in the macrophage co-culture with apoptotic MCF-7 were much higher than in the control group (Figure 4A). However, no significant difference in the mRNA levels for STAT3 was observed between the two groups. However, since activation of the STAT3 signaling pathway is due to the level of phosphorylated STAT3, and not abundance of total STAT3, we then examined the phosphorylation status of STAT3 using a Western blot (Figure 4E). As expected from the real-time RT-PCR results (Figure 4B), no significant difference in the total protein levels of STAT3 was detected between the CoA and Mac groups, but activation of the STAT3 pathway, as assessed by the presence of a p-STAT3 band, was only detected in the macrophage co-culture with the apoptotic MCF-7 cell group, and not in the Mac group (Figure 4E). These results show that our chemotherapy microenvironment model yields macrophages that are activated and produce cytokines, such as IL-6, and thus are able to modify the proliferative and metastatic properties of cancer stem-like cells.

Bottom Line: Tumor-associated macrophages (TAMs) have been found to be associated with the progression and metastasis of breast cancer.Macrophages co-cultured with apoptotic MCF-7 had increased tumor growth and metastatic ability in a nude mouse transplantation assay.During this process, macrophages secreted increased amounts of interleukin 6 (IL-6) leading to increased phosphorylation of signal transducers and activators of transcription 3 (STAT3), which likely explains the increased transcription of STAT3 target genes such as TGF-β1 and HIF-1α.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Peking University, Health Science Center, Beijing 100191, China. zhouna@bjmu.edu.cn.

ABSTRACT
Tumor-associated macrophages (TAMs) have been found to be associated with the progression and metastasis of breast cancer. To clarify the mechanisms underlying the crosstalk between TAMs and cancer stem cells (CSCs) in breast cancer recurrence and metastasis, we used a co-culture model of macrophages and apoptotic human breast cancer cell line MCF-7 cells to investigate the effects of TAMs on MCF-7 in vitro and in vivo. Macrophages co-cultured with apoptotic MCF-7 had increased tumor growth and metastatic ability in a nude mouse transplantation assay. The macrophages exposed to apoptotic cells also induce an increase in the proportion of CD44+/CD24- cancer stem-like cells, as well as their proliferative ability accompanied with an increase in mucin1 (MUC1) expression. During this process, macrophages secreted increased amounts of interleukin 6 (IL-6) leading to increased phosphorylation of signal transducers and activators of transcription 3 (STAT3), which likely explains the increased transcription of STAT3 target genes such as TGF-β1 and HIF-1α. Our results indicate that when cancer cells endure chemotherapy induced apoptosis, macrophages in their microenvironment can then activate cancer stem cells to promote cancer growth and metastasis by secreting IL-6, which activates STAT3 phosphorylation to regulate the transcription of its downstream target genes.

No MeSH data available.


Related in: MedlinePlus