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

Abstract

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.

PGC1α Suppresses Prostate Cancer Cell Invasion through ERRα Transcriptional Control

“…S3E-S3G). On the other hand, MYC silencing with a validated shRNA (33,34) recapitulated the phenotype of Pgc1a expression in cell area, p-MLC2, and invasive growth ( Supplementary Fig. S3H-S3L).…”

Targeting PML in triple negative breast cancer elicits growth suppression and senescence

“…4d). To ascertain if MYC silencing recapitulated the effect of PML inhibition, we used a validated shRNA targeting this oncogene (sh42) [42,43] and confirmed that MYC silencing resulted in increased p27 levels ( Fig. 4e and Supplementary Fig.…”

Screening for 15 pathogenic viruses in human cell lines registered at the JCRB Cell Bank: characterization of in vitro human cells by viral infection

“…The absence of the RNA and reverse transcriptase in these cell lines confirmed that the cell lines are negative for HIV-1 and HIV-2. The luciferase gene was introduced into these cell lines by the lentiviral vector [ 33 ], which may be associated with the detection by PCR. Because HIV-1 and HIV-2 are highly pathogenic and must be handled under biosafety level 3 (BSL3), such cell lines are not received and distributed from our JCRB Cell Bank.…”