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

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.

细胞-基质黏附减弱在肿瘤转移过程中发挥关键作用。既往研究表明，组织学观察中存在漂浮细胞簇的肺癌更易发生转移。本研究旨在探究长期三维低黏附培养的肿瘤细胞是否可获得高转移潜能，并利用这些细胞解析肿瘤转移的潜在分子机制。本研究选取两株KRAS突变的肺腺癌细胞系A549与H441，在超低吸附培养皿中进行培养并筛选，最终获得的细胞被命名为FL（即floating，漂浮）亚系。将肿瘤细胞经心内注射接种至NOD/SCID小鼠体内，通过荧光素酶成像与组织病理学方法评估肿瘤转移情况。体外实验检测细胞（分别在贴壁或悬浮培养条件下）的增殖、迁移与侵袭能力，并通过全基因组分析筛选FL亚系中关键的分子改变。将亲本细胞接种至低吸附培养皿后，初期会形成圆形细胞球且增殖活性受限；随着培养时间延长，细胞逐渐形成体积更小、生长缓慢的细胞球，最终在培养3~4个月后，获得可在悬浮培养中恢复显著增殖活性的FL亚系。在普通培养皿中，FL亚系细胞可重新贴壁，且形态较亲本细胞更呈梭形。FL亚系与亲本细胞系在贴壁条件下的增殖、迁移与侵袭能力无显著差异，但FL细胞在体外悬浮培养条件下的增殖活性显著升高，且体内转移能力更强。全基因组分析显示，A549-FL与H441-FL细胞分别发生了上皮间质转化（epithelial-mesenchymal transition, EMT）与c-Myc扩增，二者均为潜在的转移介导机制。在A549-FL细胞中，通过慢病毒介导的ZEB1敲低可显著抑制FL细胞的增殖活性；在H441-FL细胞中，通过慢病毒敲低c-Myc或使用药理学抑制剂JQ1抑制c-Myc活性，同样可显著降低FL细胞的增殖能力。长期三维低黏附培养技术有望成为研究细胞-基质黏附减弱介导的肿瘤转移机制的有效手段。