title: The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines.
abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present in esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression was enhanced, suggesting that hypoxia had been induced. Comparison of cancer drug resistance using cisplatin and doxorubicin in 3-D-cultured esophageal cancer cells showed that cancer drug resistance had increased. These results indicate that 3-D culture of esophageal squamous cell carcinoma lines is a useful method for inducing cancer stem cells.
authors.affiliation: Department of Esophageal & Gastroenterological Surgery, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Tokyo 113-8421, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Fujiwara
authors.forename: Kazunori
authors.initials: K
authors.lastname: Kato
authors.forename: Shigeo
authors.initials: S
authors.lastname: Nohara
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Iwanuma
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kajiyama
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000235:genetics; D000954:Antigens, Surface / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018929:Cell Culture Techniques / Q000379:methods*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D000071317:Nanog Homeobox Protein; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D016601:RNA-Binding Proteins / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics; D018874:Spheroids, Cellular / Q000378:metabolism* / Q000473:pathology
chemical_list: D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D018398:Homeodomain Proteins; C000715187:Lin28A protein, human; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D016601:RNA-Binding Proteins; D055748:SOXB1 Transcription Factors; D004317:Doxorubicin; D000444:Aldehyde Dehydrogenase; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; D002945:Cisplatin