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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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The differentiation potential and tumorigenic capacity of rectospheres in vitro. (a) Example of tumor spheres generated from a human rectal cancer sample that were passaged twice and differentiated cell. Primary spheres: spheres directly generated from human tumor tissues. Secondary spheres: spheres representing the second passage of primary spheres. Bars=500 μM. Differentiated cells: spheroids were induced by 20% fetal bovine serum. One representative of four independent spheroid cultures is shown. Bars=50 μM. (b) Spheres were cultured in SCM and analyzed by immunofluorescence to detect the differentiation marker CK20 at different time points. Bars=50 μM. (c) Immunofluorescent analysis using CK20 (red), CDX2 (red), CK7 (red), and DAPI (4,6-diamidino-2-phenylindole; blue) stains. One representative image of three different tumors is shown. (d) Bmi1 and Lgr5 expressed on cells from rectospheres (left panels) and rectosphere-derived differentiation progeny (right panels). Nuclei were counterstained by DAPI (blue). One representative experiment of three different tumors is shown. Bars=25 μM. (e) The example shows that spheres cultured in SFM, but not in SCM, recapitulate tumors in nude mice. (f) Tumorigenic potential of tumor spheres after subcutaneous injection. Size of subcutaneous rectal carcinoma tumors generated from 103 sphere cells and 1 × 106 differentiated cells. Data are mean±S.D. of two independent experiments, each performed with cells from different donors (*P<0.01; patients 9 and 15)
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fig1: The differentiation potential and tumorigenic capacity of rectospheres in vitro. (a) Example of tumor spheres generated from a human rectal cancer sample that were passaged twice and differentiated cell. Primary spheres: spheres directly generated from human tumor tissues. Secondary spheres: spheres representing the second passage of primary spheres. Bars=500 μM. Differentiated cells: spheroids were induced by 20% fetal bovine serum. One representative of four independent spheroid cultures is shown. Bars=50 μM. (b) Spheres were cultured in SCM and analyzed by immunofluorescence to detect the differentiation marker CK20 at different time points. Bars=50 μM. (c) Immunofluorescent analysis using CK20 (red), CDX2 (red), CK7 (red), and DAPI (4,6-diamidino-2-phenylindole; blue) stains. One representative image of three different tumors is shown. (d) Bmi1 and Lgr5 expressed on cells from rectospheres (left panels) and rectosphere-derived differentiation progeny (right panels). Nuclei were counterstained by DAPI (blue). One representative experiment of three different tumors is shown. Bars=25 μM. (e) The example shows that spheres cultured in SFM, but not in SCM, recapitulate tumors in nude mice. (f) Tumorigenic potential of tumor spheres after subcutaneous injection. Size of subcutaneous rectal carcinoma tumors generated from 103 sphere cells and 1 × 106 differentiated cells. Data are mean±S.D. of two independent experiments, each performed with cells from different donors (*P<0.01; patients 9 and 15)

Mentions: A self-renewal capability is a major property of stem cells, and spheroid formation is a self-renewal index. Culture under serum-free conditions has been used to expand stem-like spheroid cells from several primary solid tumors. Primary cells derived from serum-free culture more accurately mirror the original genotype and tumor morphology of the cells derived directly from endogenous human tumor tissues.6, 9, 15 Thus we first enriched the CICs as rectospheres from human primary rectal adenocarcinoma cells that were isolated from samples surgically removed from 30 human patients who had not received neoadjuvant chemoradiotherapy (Supplementary Table S1). Rectal cancer cells were cultured in a serum-free medium (SFM) supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After 3–4 weeks, a small fraction of tumor cells formed spheres (first rectospheres; Figure 1a). The SFM was replaced with 20% fetal bovine serum-containing medium (SCM) for the differentiation of spheres.15 The spherical cells gradually aggregated into clusters of polygonal cells and exhibited the typical epithelial-like cell morphology in culture together with expression of cytokeratin 20 (CK20) when restricted to differentiated cells in intestinal epithelium (Figures 1a and b). The expression of CDX2, cytokeratin 7 (CK7), and CK20 was detected in both serum cultured and rectosphere cells. The majority of cells within the rectospheres were positive for CDX2 and negative for CK7 and CK20. The serum-induced differentiated cells expressed CDX2 and CK20, but not CK7 (Figure 1c), which is a typical pattern of rectal cancer cells.6, 14 Moreover, the intestinal stem cell markers, Bmi1 and Lgr5, were positively expressed in the majority of spherical cells but not in serum-cultured cells (Figure 1d). Incubation of cells in SFM is therefore a useful approach for expanding malignant rectospheres.


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)

The differentiation potential and tumorigenic capacity of rectospheres in vitro. (a) Example of tumor spheres generated from a human rectal cancer sample that were passaged twice and differentiated cell. Primary spheres: spheres directly generated from human tumor tissues. Secondary spheres: spheres representing the second passage of primary spheres. Bars=500 μM. Differentiated cells: spheroids were induced by 20% fetal bovine serum. One representative of four independent spheroid cultures is shown. Bars=50 μM. (b) Spheres were cultured in SCM and analyzed by immunofluorescence to detect the differentiation marker CK20 at different time points. Bars=50 μM. (c) Immunofluorescent analysis using CK20 (red), CDX2 (red), CK7 (red), and DAPI (4,6-diamidino-2-phenylindole; blue) stains. One representative image of three different tumors is shown. (d) Bmi1 and Lgr5 expressed on cells from rectospheres (left panels) and rectosphere-derived differentiation progeny (right panels). Nuclei were counterstained by DAPI (blue). One representative experiment of three different tumors is shown. Bars=25 μM. (e) The example shows that spheres cultured in SFM, but not in SCM, recapitulate tumors in nude mice. (f) Tumorigenic potential of tumor spheres after subcutaneous injection. Size of subcutaneous rectal carcinoma tumors generated from 103 sphere cells and 1 × 106 differentiated cells. Data are mean±S.D. of two independent experiments, each performed with cells from different donors (*P<0.01; patients 9 and 15)
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: The differentiation potential and tumorigenic capacity of rectospheres in vitro. (a) Example of tumor spheres generated from a human rectal cancer sample that were passaged twice and differentiated cell. Primary spheres: spheres directly generated from human tumor tissues. Secondary spheres: spheres representing the second passage of primary spheres. Bars=500 μM. Differentiated cells: spheroids were induced by 20% fetal bovine serum. One representative of four independent spheroid cultures is shown. Bars=50 μM. (b) Spheres were cultured in SCM and analyzed by immunofluorescence to detect the differentiation marker CK20 at different time points. Bars=50 μM. (c) Immunofluorescent analysis using CK20 (red), CDX2 (red), CK7 (red), and DAPI (4,6-diamidino-2-phenylindole; blue) stains. One representative image of three different tumors is shown. (d) Bmi1 and Lgr5 expressed on cells from rectospheres (left panels) and rectosphere-derived differentiation progeny (right panels). Nuclei were counterstained by DAPI (blue). One representative experiment of three different tumors is shown. Bars=25 μM. (e) The example shows that spheres cultured in SFM, but not in SCM, recapitulate tumors in nude mice. (f) Tumorigenic potential of tumor spheres after subcutaneous injection. Size of subcutaneous rectal carcinoma tumors generated from 103 sphere cells and 1 × 106 differentiated cells. Data are mean±S.D. of two independent experiments, each performed with cells from different donors (*P<0.01; patients 9 and 15)
Mentions: A self-renewal capability is a major property of stem cells, and spheroid formation is a self-renewal index. Culture under serum-free conditions has been used to expand stem-like spheroid cells from several primary solid tumors. Primary cells derived from serum-free culture more accurately mirror the original genotype and tumor morphology of the cells derived directly from endogenous human tumor tissues.6, 9, 15 Thus we first enriched the CICs as rectospheres from human primary rectal adenocarcinoma cells that were isolated from samples surgically removed from 30 human patients who had not received neoadjuvant chemoradiotherapy (Supplementary Table S1). Rectal cancer cells were cultured in a serum-free medium (SFM) supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After 3–4 weeks, a small fraction of tumor cells formed spheres (first rectospheres; Figure 1a). The SFM was replaced with 20% fetal bovine serum-containing medium (SCM) for the differentiation of spheres.15 The spherical cells gradually aggregated into clusters of polygonal cells and exhibited the typical epithelial-like cell morphology in culture together with expression of cytokeratin 20 (CK20) when restricted to differentiated cells in intestinal epithelium (Figures 1a and b). The expression of CDX2, cytokeratin 7 (CK7), and CK20 was detected in both serum cultured and rectosphere cells. The majority of cells within the rectospheres were positive for CDX2 and negative for CK7 and CK20. The serum-induced differentiated cells expressed CDX2 and CK20, but not CK7 (Figure 1c), which is a typical pattern of rectal cancer cells.6, 14 Moreover, the intestinal stem cell markers, Bmi1 and Lgr5, were positively expressed in the majority of spherical cells but not in serum-cultured cells (Figure 1d). Incubation of cells in SFM is therefore a useful approach for expanding malignant rectospheres.

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.

Show MeSH
Related in: MedlinePlus