Establishment of highly metastatic KRAS mutant lung cancer cell sublines in long-term three-dimensional low attachment cultures.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Cell Culture Techniques*/instrumentation ; Cell Line, Tumor*/pathology ; Cell Line, Tumor*/physiology ; Neoplasm Metastasis*/genetics ; Neoplasm Metastasis*/pathology ; Neoplasm Metastasis*/physiopathology; Adenocarcinoma/*pathology ; Lung Neoplasms/*pathology ; Proto-Oncogene Proteins p21(ras)/*genetics; A549 Cells ; Adenocarcinoma/genetics ; Adenocarcinoma/physiopathology ; Adenocarcinoma/secondary ; Animals ; Apoptosis/physiology ; Cell Adhesion ; Cell Movement ; Cell Proliferation ; Female ; Genes, myc ; Humans ; Lung Neoplasms/genetics ; Lung Neoplasms/physiopathology ; Mice, Inbred NOD ; Mice, SCID ; Mutation ; Neoplasm Transplantation ; Organic Cation Transport Proteins/metabolism ; Spheroids, Cellular/pathology ; Zinc Finger E-box-Binding Homeobox 1/genetics ; Zinc Finger E-box-Binding Homeobox 1/metabolism

Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3-4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo. A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells. Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion.

Oncogene. 2007 Oct 25;26(49):6979-88. (PMID: 17486063)
Proc Natl Acad Sci U S A. 2005 Oct 25;102(43):15545-50. (PMID: 16199517)
Cancer Lett. 2010 Oct 28;296(2):216-24. (PMID: 20452118)
Cancer Res. 2010 Oct 1;70(19):7360-4. (PMID: 20823151)
Oncogene. 2010 Aug 26;29(34):4741-51. (PMID: 20531305)
Clin Transl Med. 2014 Jul 02;3:17. (PMID: 25050175)
Cell. 2004 Jun 25;117(7):927-39. (PMID: 15210113)
Histopathology. 2016 Feb;68(3):356-66. (PMID: 26109197)
Cell. 1995 May 5;81(3):323-30. (PMID: 7736585)
J Thorac Oncol. 2015 Sep;10(9):1243-60. (PMID: 26291008)
Clin Cancer Res. 1999 Aug;5(8):1966-75. (PMID: 10473073)
Proc Am Thorac Soc. 2009 Apr 15;6(2):201-5. (PMID: 19349489)
Oncogene. 2011 Sep 1;30(35):3735-44. (PMID: 21478908)
J Thorac Oncol. 2016 May;11(5):613-38. (PMID: 27013409)
Cancer Inform. 2014 Feb 12;13(Suppl 1):1-16. (PMID: 24558298)
Blood. 2003 Feb 15;101(4):1220-35. (PMID: 12393483)
Proc Natl Acad Sci U S A. 2003 Sep 2;100(18):10429-34. (PMID: 12937339)
Cancer Res. 2006 Oct 1;66(19):9543-56. (PMID: 17018611)
Cancer. 2009 Nov 15;115(22):5218-27. (PMID: 19672942)
Cancer. 2012 Jun 1;118(11):2889-99. (PMID: 22020674)
Cancer Res. 2010 Jul 15;70(14):5649-69. (PMID: 20610625)
CA Cancer J Clin. 2013 Jan;63(1):11-30. (PMID: 23335087)
J Natl Cancer Inst. 2013 Aug 21;105(16):1212-20. (PMID: 23926067)
Biochim Biophys Acta. 2013 Dec;1833(12):3481-98. (PMID: 23830918)
Cell. 2012 Mar 30;149(1):214-31. (PMID: 22464331)
EMBO Rep. 2007 Jan;8(1):70-6. (PMID: 17159920)
Clin Cancer Res. 2013 Nov 15;19(22):6183-92. (PMID: 24045185)
Hum Pathol. 2008 Mar;39(3):324-30. (PMID: 18261622)
Oncotarget. 2015 Sep 8;6(26):22905-17. (PMID: 26090868)
Proc Natl Acad Sci U S A. 2012 Nov 20;109(47):19408-13. (PMID: 23129625)
CA Cancer J Clin. 2011 Mar-Apr;61(2):69-90. (PMID: 21296855)
Br J Cancer. 2005 Jan 17;92(1):131-9. (PMID: 15597105)
J Clin Oncol. 2010 Feb 10;28(5):744-52. (PMID: 20038723)
Nat Rev Cancer. 2002 Jun;2(6):442-54. (PMID: 12189386)

0 (KRAS protein, human)
0 (Organic Cation Transport Proteins)
0 (ZEB1 protein, human)
0 (Zinc Finger E-box-Binding Homeobox 1)
0 (solute carrier family 22 (organic cation transporter), member 3)
EC 3.6.5.2 (Proto-Oncogene Proteins p21(ras))

Date Created: 20170809 Date Completed: 20171004 Latest Revision: 20220330

20240829

PMC5546599

10.1371/journal.pone.0181342

28786996