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Differential knockdown of TGF-β ligands in a three-dimensional co-culture tumor- stromal interaction model of lung cancer

Masafumi Horie1, Akira Saito12*, Satoshi Noguchi1, Yoko Yamaguchi3, Mitsuhiro Ohshima4, Yasuyuki Morishita5, Hiroshi I Suzuki5, Tadashi Kohyama16 and Takahide Nagase1

Author Affiliations

1 Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

2 Division for Health Service Promotion, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

3 Department of Biochemistry, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan

4 Department of Biochemistry, Ohu University School of Pharmaceutical Sciences, Misumido 31-1, Tomitamachi, Koriyama, Fukushima 963-8611, Japan

5 Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

6 The Fourth Department of Internal Medicine, Teikyo University School of Medicine University Hospital, Mizonokuchi, 3-8-3 Mizonokuchi, Takatsu-ku, Kawasaki, Kanagawa 213-8507, Japan

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BMC Cancer 2014, 14:580  doi:10.1186/1471-2407-14-580

Published: 9 August 2014



Transforming growth factor (TGF)-β plays a pivotal role in cancer progression through regulating cancer cell proliferation, invasion, and remodeling of the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are the predominant type of stromal cell, in which TGF-β signaling is activated. Among the strategies for TGF-β signaling inhibition, RNA interference (RNAi) targeting of TGF-β ligands is emerging as a promising tool. Although preclinical studies support the efficacy of this therapeutic strategy, its effect on the tumor microenvironment in vivo remains unknown. In addition, differential effects due to knockdown of various TGF-β ligand isoforms have not been examined. Therefore, an experimental model that recapitulates tumor–stromal interaction is required for validation of therapeutic agents.


We have previously established a three-dimensional co-culture model of lung cancer, and demonstrated the functional role of co-cultured fibroblasts in enhancing cancer cell invasion and differentiation. Here, we employed this model to examine how knockdown of TGF-β ligands affects the behavior of different cell types. We developed lentivirus vectors carrying artificial microRNAs against human TGF-β1 and TGF-β2, and tested their effects in lung cancer cells and fibroblasts.


Lentiviral vectors potently and selectively suppressed the expression of TGF-β ligands, and showed anti-proliferative effects on these cells. Furthermore, knockdown of TGF-β ligands attenuated fibroblast-mediated collagen gel contraction, and diminished lung cancer cell invasion in three-dimensional co-culture. We also observed differential effects by targeting different TGF-β isoforms in lung cancer cells and fibroblasts.


Our findings support the notion that RNAi-mediated targeting of TGF-β ligands may be beneficial for lung cancer treatment via its action on both cancer and stromal cells. This study further demonstrates the usefulness of this three-dimensional co-culture model to examine the effect of therapeutic agents on tumor–stromal interaction.

RNA interference; MicroRNA; Lentivirus vector; TGF-β; Three-dimensional co-culture; Gel contraction assay