title: Three-dimensional culture promotes reconstitution of the tumor-specific hypoxic microenvironment under TGFβ stimulation.
abstract: In vitro tumor growth in a three-dimensional (3D) architecture has been demonstrated to play an important role in biology not only for developmental organogenesis and carcinogenesis, but also for analyses on reconstitution and maintenance in a variety of biological environments surrounding the cells. In addition to providing architectural similarity to living organisms, 3D culture with a radial flow bioreactor (RFB) can also closely mimic the living hypoxic microenvironment under which specific organogenesis or carcinogenesis occurs. The findings of the present study under the RFB culture conditions show that cancer cells underwent a shift from aerobic to hypoxic energy metabolism, in addition to protein expression to maintain the 3D structure. In RFB-cultured cells, protein stability of hypoxia-inducible factor 1 (HIF1) α, a subunit of HIF1, was increased without upregulation of its mRNA. Under these conditions, PHD2, HIF-prolyl-4-hydroxy-lase 2 and a HIF1 downstream enzyme, were stabilized without affecting the mRNA levels via downregulation of FK506-binding protein 8. PHD2 accumulation, which occurred concomitant with HIF1 stabilization, may have compensated for the lack of oxygen under hypoxic conditions to regulate the HIF levels. 3D-culture-induced overexpression of carbonic anhydrase (another representative HIF downstream enzyme) was found to occur independently of cell density in RFB--cultured cells, suggesting that the RFB provided an adequately hypoxic microenvironment for the cultured cells. From these results, it was hypothesized that the key factors are regulatory molecules, which stabilize and degrade HIF molecules, thereby activating the HIF1 pathway under a hypoxic milieu.
authors.affiliation: Department of Biochemistry, Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan. marushima@jikei.ac.jp
authors.forename: Hideki
authors.initials: H
authors.lastname: Marushima
authors.forename: Shun-Ichi
authors.initials: S
authors.lastname: Shibata
authors.forename: Tadashi
authors.initials: T
authors.lastname: Asakura
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Matsuura
authors.forename: Haruka
authors.initials: H
authors.lastname: Maehashi
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ishii
authors.forename: Homare
authors.initials: H
authors.lastname: Eda
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Aoki
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Iida
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Morikawa
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ohkawa
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008819:Mice, Nude; D018874:Spheroids, Cellular; D000077195:Squamous Cell Carcinoma of Head and Neck; D015533:Transcriptional Activation / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D059016:Tumor Microenvironment / Q000187:drug effects* / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
chemical_list: D000951:Antigens, Neoplasm; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016212:Transforming Growth Factor beta; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases