Shibata Shun-Ichi, Marushima Hideki, Asakura Tadashi, Matsuura Tomokazu, Eda Homare, Aoki Katsuhiko, Matsudaira Hiroshi, Ueda Kazu, Ohkawa Kiyoshi
Department of Biochemistry, Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
Int J Oncol. 2009 May;34(5):1433-48.
To confirm the usefulness of the radial flow type bioreactor (RFB) for a three-dimensional (3D) culture system, which provides a tissue architecture and molecular function mimicking the in vivo environment, molecular expression in the A431 human squamous carcinoma cell line during culture were analyzed under the physically different environments of 3D culture in the RFB, 2D culture in a monolayer as well as in nude mice. Time-dependent accumulation of autocrine transforming growth factor (TGF) beta1 was found in spent culture media obtained only from 3D cultured A431 cancer cells, which grew well with a stratified-sheet morphology. Cells in the RFB overexpressed matrix metalloproteinase 7 (MMP7) and showed an increased release of soluble 80-kDa fragments of E-cadherin into the media time-dependently, resulting in the reduction of E-cadherin protein at the cell surface without down-regulation of the mRNA. beta-Catenin and its nuclear partner, LEF1, were up-regulated and Wnt protein secretion was also accelerated. Additional up-regulation of the transcriptional factors, HMGA2 and down-stream Slug, was noted. TGFbeta1-dependent, MMP7-mediated up-regulation of beta-catenin/LEF1 signaling and TGFbeta1-activated HMGA2 pathways consequently converged with Slug overexpression, due to disassembly and further repression of E-cadherin expression, which was reproducible in the epithelial mesenchymal transition process without any manipulation. Other transcriptional factors, Notch/HEY1 and NF-kappaB, were also up-regulated in 3D-cultured cells. These signals recruited molecules related to extracellular matrix-cell remodeling and angiogenesis. Expression of several representative molecules in the 3D cultured cells was parallel with that in xenotransplanted A431 tumor tissues in nude mice. 3D culture of tumor cells in the RFB is a useful tool for cancer experimental biology and evaluation of cancer therapeutic-like systems in nude mice.
为证实径向流型生物反应器（RFB）用于三维（3D）培养系统的有效性，该系统可提供模拟体内环境的组织结构和分子功能，我们分析了在RFB中进行3D培养、单层2D培养以及在裸鼠体内等物理环境不同的条件下，A431人鳞状癌细胞系培养过程中的分子表达情况。仅从呈分层片状良好生长的3D培养A431癌细胞获得的废弃培养基中发现了自分泌转化生长因子（TGF）β1随时间的积累。RFB中的细胞过表达基质金属蛋白酶7（MMP7），并随时间依赖性地向培养基中释放增加的可溶性80 kDa E-钙黏蛋白片段，导致细胞表面E-钙黏蛋白蛋白减少，而mRNA未下调。β-连环蛋白及其核伴侣LEF1上调，Wnt蛋白分泌也加速。还注意到转录因子HMGA2和下游Slug的进一步上调。由于E-钙黏蛋白表达的解体和进一步抑制，TGFβ1依赖性、MMP7介导的β-连环蛋白/LEF1信号上调和TGFβ1激活的HMGA2途径最终与Slug过表达汇合，这在上皮-间质转化过程中无需任何操作即可重现。其他转录因子Notch/HEY1和NF-κB在3D培养细胞中也上调。这些信号募集了与细胞外基质-细胞重塑和血管生成相关的分子。3D培养细胞中几种代表性分子的表达与裸鼠体内异种移植的A431肿瘤组织中的表达平行。在RFB中对肿瘤细胞进行3D培养是癌症实验生物学和裸鼠体内癌症治疗样系统评估的有用工具。
