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T. Shibano
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P3.02 - Biology/Pathology (ID 620)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.02-089 - Establishment of Highly Metastatic Lung Cancer Cell Sublines in Long-term Three-dimensional Low Attachment Cultures (ID 8135)
09:30 - 09:30 | Author(s): T. Shibano
- Abstract
Background:
Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. We investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential and examined the mechanisms underlying metastasis.
Method:
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. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. A whole genomic analysis was performed to identify key molecular alterations in FL sublines.
Result:
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. FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo (Fig.). 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.
Conclusion:
Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion.
