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Cancer-initiating cells derived from human rectal adenocarcinoma tissues carry mesenchymal phenotypes and resist drug therapies.

Fan CW, Chen T, Shang YN, Gu YZ, Zhang SL, Lu R, OuYang SR, Zhou X, Li Y, Meng WT, Hu JK, Lu Y, Sun XF, Bu H, Zhou ZG, Mo XM - Cell Death Dis (2013)

Bottom Line: These R-CICs generated tumors similar to their tumor of origin when injected into immunodeficient mice, differentiated into rectal epithelial cells in vitro, and were capable of self-renewal both in vitro and in vivo.More importantly, subpopulations of R-CICs resisted both 5-fluorouracil/calcium folinate/oxaliplatin (FolFox) and cetuximab treatment, which are the most common therapeutic regimens used for patients with advanced or metastatic rectal cancer.Thus, the identification, expansion, and properties of R-CICs provide an ideal cellular model to further investigate tumor progression and determine therapeutic resistance in these patients.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Digestive Surgery, West China Hospital, Sichuan University, Chengdu, People's Republic of China [2] Medical Center of Stem Cell Biology, West China Hospital, Sichuan University, Chengdu, People's Republic of China [3] Department of Gastrointestinal Surgery, West China Hospital, Sichuan University, Chengdu, People's Republic of China.

ABSTRACT
Accumulating evidence indicates that cancer-initiating cells (CICs) are responsible for cancer initiation, relapse, and metastasis. Colorectal carcinoma (CRC) is typically classified into proximal colon, distal colon, and rectal cancer. The gradual changes in CRC molecular features within the bowel may have considerable implications in colon and rectal CICs. Unfortunately, limited information is available on CICs derived from rectal cancer, although colon CICs have been described. Here we identified rectal CICs (R-CICs) that possess differentiation potential in tumors derived from patients with rectal adenocarcinoma. The R-CICs carried both CD44 and CD54 surface markers, while R-CICs and their immediate progenies carried potential epithelial-mesenchymal transition characteristics. These R-CICs generated tumors similar to their tumor of origin when injected into immunodeficient mice, differentiated into rectal epithelial cells in vitro, and were capable of self-renewal both in vitro and in vivo. More importantly, subpopulations of R-CICs resisted both 5-fluorouracil/calcium folinate/oxaliplatin (FolFox) and cetuximab treatment, which are the most common therapeutic regimens used for patients with advanced or metastatic rectal cancer. Thus, the identification, expansion, and properties of R-CICs provide an ideal cellular model to further investigate tumor progression and determine therapeutic resistance in these patients.

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Rectal cancer sphere cells exhibit mesenchymal phenotypes. (a) Confocal images of rectospheres stained with antibodies against E-cadherin (green), EpCAM (green), fibronectin (red), vimentin (red), and α-SMA (α-smooth muscle actin; red). One representative image of two different tumors is shown. Bar=25 μM. (b) Expression levels of mRNAs encoding E-cadherin, Snail, Slug, fibronectin, vimentin, and α-SMA in different cellular subpopulations were determined by real-time reverse transcriptase-PCR. Data are mean±S.D. of three independent experiments, each performed with cells from different donors (patients 9, 11, and 15). (c) Immunoblotting of EMT-related proteins from lysates of different cell populations isolated from rectospheres of two different patients (patients 9 and 15). (d) Migration capacity of different cellular subpopulations. Data are mean±S.D. of three independent experiments, each performed with cells from different donors (*P<0.05; patients 9, 11, and 15)
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fig3: Rectal cancer sphere cells exhibit mesenchymal phenotypes. (a) Confocal images of rectospheres stained with antibodies against E-cadherin (green), EpCAM (green), fibronectin (red), vimentin (red), and α-SMA (α-smooth muscle actin; red). One representative image of two different tumors is shown. Bar=25 μM. (b) Expression levels of mRNAs encoding E-cadherin, Snail, Slug, fibronectin, vimentin, and α-SMA in different cellular subpopulations were determined by real-time reverse transcriptase-PCR. Data are mean±S.D. of three independent experiments, each performed with cells from different donors (patients 9, 11, and 15). (c) Immunoblotting of EMT-related proteins from lysates of different cell populations isolated from rectospheres of two different patients (patients 9 and 15). (d) Migration capacity of different cellular subpopulations. Data are mean±S.D. of three independent experiments, each performed with cells from different donors (*P<0.05; patients 9, 11, and 15)

Mentions: We next analyzed the relative EMT gene expression of the different cellular subpopulations or rectospheres. E-cadherin and EpCAM, which are both epithelial markers of colorectal mucosa, were highly expressed in the spheroids (Figure 3a). In addition, we detected the expression of vimentin, fibronectin, and α-SMA proteins, which are typical mesenchymal cell markers (Figure 3a).25 Moreover, specific EMT-inducing transcription factors and marker proteins, including fibronectin, vimentin, α-SMA, Snail, and Slug were also upregulated based on qRT-PCR and western blot (Figures 3b and c).25, 26 To test whether CD44+CD54+ cells possess EMT potential, a transwell migration assay was performed. The migration assay showed that CD44+CD54+ cells had a higher migratory capacity compared with other subpopulations in different patient tumors (Figure 3d). Collectively, these results indicate that CD44+CD54+ cells carry epithelial and mesenchymal phenotypes with distinct expression patterns and potential EMT characteristics.


Cancer-initiating cells derived from human rectal adenocarcinoma tissues carry mesenchymal phenotypes and resist drug therapies.

Fan CW, Chen T, Shang YN, Gu YZ, Zhang SL, Lu R, OuYang SR, Zhou X, Li Y, Meng WT, Hu JK, Lu Y, Sun XF, Bu H, Zhou ZG, Mo XM - Cell Death Dis (2013)

Rectal cancer sphere cells exhibit mesenchymal phenotypes. (a) Confocal images of rectospheres stained with antibodies against E-cadherin (green), EpCAM (green), fibronectin (red), vimentin (red), and α-SMA (α-smooth muscle actin; red). One representative image of two different tumors is shown. Bar=25 μM. (b) Expression levels of mRNAs encoding E-cadherin, Snail, Slug, fibronectin, vimentin, and α-SMA in different cellular subpopulations were determined by real-time reverse transcriptase-PCR. Data are mean±S.D. of three independent experiments, each performed with cells from different donors (patients 9, 11, and 15). (c) Immunoblotting of EMT-related proteins from lysates of different cell populations isolated from rectospheres of two different patients (patients 9 and 15). (d) Migration capacity of different cellular subpopulations. Data are mean±S.D. of three independent experiments, each performed with cells from different donors (*P<0.05; patients 9, 11, and 15)
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Rectal cancer sphere cells exhibit mesenchymal phenotypes. (a) Confocal images of rectospheres stained with antibodies against E-cadherin (green), EpCAM (green), fibronectin (red), vimentin (red), and α-SMA (α-smooth muscle actin; red). One representative image of two different tumors is shown. Bar=25 μM. (b) Expression levels of mRNAs encoding E-cadherin, Snail, Slug, fibronectin, vimentin, and α-SMA in different cellular subpopulations were determined by real-time reverse transcriptase-PCR. Data are mean±S.D. of three independent experiments, each performed with cells from different donors (patients 9, 11, and 15). (c) Immunoblotting of EMT-related proteins from lysates of different cell populations isolated from rectospheres of two different patients (patients 9 and 15). (d) Migration capacity of different cellular subpopulations. Data are mean±S.D. of three independent experiments, each performed with cells from different donors (*P<0.05; patients 9, 11, and 15)
Mentions: We next analyzed the relative EMT gene expression of the different cellular subpopulations or rectospheres. E-cadherin and EpCAM, which are both epithelial markers of colorectal mucosa, were highly expressed in the spheroids (Figure 3a). In addition, we detected the expression of vimentin, fibronectin, and α-SMA proteins, which are typical mesenchymal cell markers (Figure 3a).25 Moreover, specific EMT-inducing transcription factors and marker proteins, including fibronectin, vimentin, α-SMA, Snail, and Slug were also upregulated based on qRT-PCR and western blot (Figures 3b and c).25, 26 To test whether CD44+CD54+ cells possess EMT potential, a transwell migration assay was performed. The migration assay showed that CD44+CD54+ cells had a higher migratory capacity compared with other subpopulations in different patient tumors (Figure 3d). Collectively, these results indicate that CD44+CD54+ cells carry epithelial and mesenchymal phenotypes with distinct expression patterns and potential EMT characteristics.

Bottom Line: These R-CICs generated tumors similar to their tumor of origin when injected into immunodeficient mice, differentiated into rectal epithelial cells in vitro, and were capable of self-renewal both in vitro and in vivo.More importantly, subpopulations of R-CICs resisted both 5-fluorouracil/calcium folinate/oxaliplatin (FolFox) and cetuximab treatment, which are the most common therapeutic regimens used for patients with advanced or metastatic rectal cancer.Thus, the identification, expansion, and properties of R-CICs provide an ideal cellular model to further investigate tumor progression and determine therapeutic resistance in these patients.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Digestive Surgery, West China Hospital, Sichuan University, Chengdu, People's Republic of China [2] Medical Center of Stem Cell Biology, West China Hospital, Sichuan University, Chengdu, People's Republic of China [3] Department of Gastrointestinal Surgery, West China Hospital, Sichuan University, Chengdu, People's Republic of China.

ABSTRACT
Accumulating evidence indicates that cancer-initiating cells (CICs) are responsible for cancer initiation, relapse, and metastasis. Colorectal carcinoma (CRC) is typically classified into proximal colon, distal colon, and rectal cancer. The gradual changes in CRC molecular features within the bowel may have considerable implications in colon and rectal CICs. Unfortunately, limited information is available on CICs derived from rectal cancer, although colon CICs have been described. Here we identified rectal CICs (R-CICs) that possess differentiation potential in tumors derived from patients with rectal adenocarcinoma. The R-CICs carried both CD44 and CD54 surface markers, while R-CICs and their immediate progenies carried potential epithelial-mesenchymal transition characteristics. These R-CICs generated tumors similar to their tumor of origin when injected into immunodeficient mice, differentiated into rectal epithelial cells in vitro, and were capable of self-renewal both in vitro and in vivo. More importantly, subpopulations of R-CICs resisted both 5-fluorouracil/calcium folinate/oxaliplatin (FolFox) and cetuximab treatment, which are the most common therapeutic regimens used for patients with advanced or metastatic rectal cancer. Thus, the identification, expansion, and properties of R-CICs provide an ideal cellular model to further investigate tumor progression and determine therapeutic resistance in these patients.

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Related in: MedlinePlus