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. 2017 Sep 26;7:12317. doi: 10.1038/s41598-017-12017-y

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The induction of lung cancer stem-like cells and their characteristics. (A) A comparison of the sphere formation ability. (n = 3, *P < 0.05, Bonferroni test). (B) Dome-shaped colonies appeared in OSK-A549 cells at 10 to 15 days after the transduction of OSK. (C) Pictures of the colonies taken during passaging (left panels) and at 2 days after passaging (right panels). Spindle-shaped colonies cells appeared around the colonies after passaging. (D) The passaged colonies grew larger and gave rise to various cell phenotypes; most of the cells were spindle-shaped. (E) The cellular morphology of the OSK-A549-Colony cells (left panel), and OSK-A549-SN cells (right panel). After trypsinizing the OSK-A549-Colony cells for approximately 6 minutes, only the spindle-shaped cells around the colonies were detached; we collected them as supernatant cells (SN cells). (F) Chemoresistance among the A549, OSK-A549-Colony, and OSK-A549-SN cells following 3 days of cisplatin (0, 2, 10 µM) treatment. (n = 3, **P < 0.01; repeated measures ANOVA). (G) The cell cycle was analyzed by flow cytometry based on Ki67 and Hoechst staining. (n = 3, *P < 0.05; Dunnett’s test). (H) Immunocytochemistry of E-cadherin and Hoechst staining in the parental A549 and OSK-A549-Colony/SN cells. E-cadherin-negative cells were found around the OSK-A549-Colony cells (indicated as white arrows). (I) Phase contrast microscopy of the spheres (upper panels) and HE staining images (lower panels). Phase contrast microscopy of the OSK-A549-Colony cells showed obvious cellular aggregation, but not the parental A549 cells on the 24-well low attachment plate. (J) A comparison of the tumorigenicity in vivo between the parental A549 and OSK-A549-Colony cells (n = 10, **P < 0.01; repeated measures ANOVA).