Cancer Res Treat.  2019 Jan;51(1):252-266. 10.4143/crt.2017.613.

Tumor-Associated Macrophages Derived TGF-β‒Induced Epithelial to Mesenchymal Transition in Colorectal Cancer Cells through Smad2,3-4/Snail Signaling Pathway

• Cai J
• Xia L
• Li J
• Ni S
• Song H
• Wu X

• KMID: 2437617
• DOI: http://doi.org/10.4143/crt.2017.613

Abstract

PURPOSE
We investigated the role of tumor-associated macrophages (TAMs) on the epithelial to mesenchymal transition (EMT) of colorectal cancer cells and determined the potential mechanism involved in the metastatic process.
MATERIALS AND METHODS
In this study, flow cytometry was used to detect the expression of target proteins. We used transwell assay to evaluate the migration of cancer cells under specific conditions. Using real-time polymerase chain reaction, we examined the expressions of cytokines and EMT-related markers in mRNA level. Animal assay was performed for analysis in vivo and hematoxylin and eosin was used to visualize the effect of TAMs on tumor metastasis. We also used immunohistochemistry and Western blotting to detect the expression of target proteins.
RESULTS
Here, we observed enrichment of TAMs in colorectal tumor tissues, resulting in high metastasis in clinical therapy. Moreover, those TAMs could facilitate the EMT progression of colorectal cancer cells, which is induced by the transforming growth factor-β (TGF-β) derived from TAMs, leading to the invasion and migration of cancer cells.
CONCLUSION
Our results demonstrated that TAMs contributed the EMT progression through a TGF-β/Smad2,3-4/Snail signaling pathway, and disrupting this pathway with TGF-β receptor inhibitor could suppress metastasis, readjusting our focus to the connection of TAMs and cancer metastasis.

Keyword

Tumor associated macrophages; Epithelial to mesenchymal transition; Transforming growth factor β; Colorectal neoplasms

MeSH Terms

Animals
Blotting, Western
Colorectal Neoplasms*
Cytokines
Eosine Yellowish-(YS)
Flow Cytometry
Hematoxylin
Immunohistochemistry
Macrophages*
Neoplasm Metastasis
Real-Time Polymerase Chain Reaction
RNA, Messenger
Cytokines
Eosine Yellowish-(YS)
Hematoxylin
RNA, Messenger

Figure

• 

  Fig. 1. Tumor-associated macrophages (TAMs) facilitate colorectal cancer cells metastasis. (A) Quantification of macrophages in tumor tissues of non-metastatic colorectal cancer patients (patients/N) (n=10) and metastatic colorectal cancer patients (patients/M) (n=10). (B) Immunohistochemistry and intensity analysis (n=10) for CD68 to detect the macrophages in tumor tissues of non-metastatic colorectal cancer patients (patients/N) and metastatic colorectal cancer patients (patients/M). Scale bar=100 μm. (C) The lung weight of tumor-bearing mice treated with phosphate buffered saline (PBS) or TAMs (n=10). (D) The lung node number of tumor-bearing mice treated with PBS or TAMs (n=10). (E) H&E staining of a representative lung of tumor-bearing mice treated with PBS or TAMs. Scale bar=200 μm. (F) The lung weight of tumor-bearing mice treated with PBS or clodronate liposomes to deplete macrophages (n=10). (G) The pulmonary tumor nodules of tumor-bearing mice treated with PBS or clodronate liposomes (n=10). (H) H&E staining of a representative lung of tumor-bearing mice treated with PBS or clodronate liposomes. Scale bar=200 μm. The data was presented as the mean±standard error of mean from three independent experiments. *p < 0.05.

• 

  Fig. 2. Tumor-associated macrophages (TAMs) promote colorectal cancer cells metastasis via secretion of transforming growth factor-β (TGF-β). (A) Relative migrant cell number in 104 CT26 and HCT116 cells treated with phosphate buffered saline (PBS) or TAMs cultural supernatant for 24 hours (n=3). (B) The wound-healing assay for evaluating the migration of CT26 and HCT116 cells treated with PBS or TAMs cultural supernatant for 24 hours. Scale bars=100 μm. (C) The relative expression of TGF-β, interleukin (IL)-10, vascular endothelial growth factor (VEGF), CCL18, CCL21, and CXCL21 in peritoneal macrophages or TAMs from tumor tissues of mice (n=3). (D) The relative expression of TGF-βR, IL-10R, vascular endothelial growth factor receptor (VEGFR), PITPNM3, CCR7, and CXCR4 in CT26 treated with TAMs or not (n=3). (E) The content of TGF-β in tumor tissues from non-metastatic or metastatic patients was detected by enzyme-linked immunosorbent assay (n=10). (F) Immunohistochemistry for TGF-β in tumor tissues of non-metastatic colorectal cancer patients (patients/N) and metastatic colorectal cancer patients (patients/M). Scale bar=100 μm. (G) The peritoneal macrophages or TAMs from tumor tissues of mice were isolated to analyze the expression of TGF-β by flow cytometry (n=3). (H) Relative migrant cell number in 104 CT26 cells (n=3) treated with PBS, IL-10, TGF-β, VEGF, CCL18, CCL21, and CXCL12 for 24 hours. (I) The lung weight of tumor-bearing mice treated with PBS, TGFβ, TAMs cultural supernatant, and TAMs cultural supernatant combined with TGF-β neutralizing antibody (n=10). (J) The pulmonary tumor nodules of tumor-bearing mice treated with PBS, TGF-β, TAMs cultural supernatant, and TAMs cultural supernatant combined with TGF-β neutralizing antibody (n=10). (K) H&E staining of a representative lung of tumor-bearing mice treated with PBS, TGF-β, TAMs cultural supernatant, and TAMs cultural supernatant combined with TGF-β neutralizing antibody. Scale bar=200 μm. The data was presented as the mean±SEM from three independent experiments. *p < 0.05, **p < 0.01; ns, not statistically significant.

• 

  Fig. 3. Transforming growth factor-β (TGF-β) induces the colorectal cancer cells metastasis through the epithelial to mesenchymal transition progress. (A) Imagesof CT26 and HCT116 cells treated with phosphate buffered saline (PBS), TGF-β (5 ng/mL), tumor-associated macrophages (TAMs) cultural supernatant and TAMs cultural supernatant combined with TGF-β neutralizing antibody (10 ng/mL) for 24 hours. Scale bar=30 μm. (B) Relative E-cadherin expression of CT26 and HCT116 cells treated with PBS, TGF-β (5 ng/mL), TAMs cultural supernatant, and TAMs cultural supernatant combined with TGF-β neutralizing antibody (10 ng/mL) for 24 hours (n=3). (C) Relative vimentin expression of CT26 and HCT116 cells treated with PBS, TGF-β (5 ng/mL), TAMs cultural supernatant, and TAMs cultural supernatant combined with TGF-β neutralizing antibody (10 ng/mL) for 24 hours (n=3). (D) Western blotting of E-cadherin and vimentin in CT26 and HCT116 cells treated with PBS, TGF-β (5 ng/mL), TAMs cultural supernatant, and TAMs cultural supernatant combined with TGF-β neutralizing antibody (10 ng/mL) for24 hours (E) Immunofluorescence of E-cadherin and vimentin in CT26 and HCT116 cells treated with PBS, TGF-β (5 ng/mL), TAMs cultural supernatant, and TAMs cultural supernatant combined with TGF-β neutralizing antibody (10 ng/mL). Scale bar=10 μm. (F) Immunofluorescence of E-cadherin andvimentin in tumor tissues of non-metastatic colorectal cancer patients (patients/N) andmetastatic colorectal cancer patients (patients/M). Scalebar=20 μm. The data was presented as the mean±standard error of mean from three independent experiments. *p< 0.05, **p< 0.01.

• 

  Fig. 4. Transforming growth factor-β (TGF-β) mediates the colorectal cancer cells epithelial to mesenchymal transition progress via Smad2,3-4/Snail/E-cadherin pathway. (A) Western blotting of p-Smad2, p-Smad3, Smad4, Snail in CT26 cells treated with phosphate buffered saline (PBS), TGF-β (5 ng/mL), tumor-associated macrophages (TAMs) cultural supernatant, and TAMs cultural supernatant combined with TGF-β neutralizing antibody (10 ng/mL). (B) Immunofluorescence of Smad2/3 in CT26 cells treated with PBS, TGF-β (5 ng/mL), TAMs cultural supernatant, and TAMs cultural supernatant combined with TGF-β neutralizing antibody (10 ng/mL). Scale bar=10 μm. (C) Immunofluorescence of Smad4 in CT26 cells treated with PBS, TGF-β (5 ng/mL), TAMs cultural supernatant, and TAMs cultural supernatant combined with TGF-β neutralizing antibody (10 ng/mL). Scale bar=10 μm. (D) Images of CT26 cells treated with PBS, TGF-β, and TGF-β combined with Snail siRNA. Scale bar=20 μm. (E) Relative migrant cell number in 104 CT26 cells treated with PBS, TGF-β, and TGF-β combined with Snail siRNA (n=3). (F) The wound-healing assay for evaluating the migration of CT26 treated with PBS, TGF-β, and TGF-β combined with Snail siRNA. Scale bar=100 μm. (G) Immunofluorescence of E-cadherin in CT26 treated with PBS, TGF-β, and TGF-β combined with Snail siRNA. Scale bar=10 μm. (H) The lung weight of tumor-bearing mice treated with PBS or LY2109761 (n=9). (I) The pulmonary tumor nodules of tumor-bearing mice treated with PBS or LY2109761 (n=9). (J) H&E staining of a representative lung of tumor-bearing mice treated with PBS or LY2109761. Scale bar=200 μm. (K) Schema of TAMs inducing the colorectal cancer cells metastasis. The data was presented as the mean±standard error of mean from three independent experiments. *p < 0.05, **p < 0.01.

Reference

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