Establishment of highly metastatic KRAS mutant lung cancer cell sublines in long-term three-dimensional low attachment cultures.
Establishment of highly metastatic KRAS mutant lung cancer cell sublines in long-term three-dimensional low attachment cultures.
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DOI：
10.1371/journal.pone.0181342
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Niki T
中科院分区：
文献类型：
--
作者：
Nakano T;Kanai Y;Amano Y;Yoshimoto T;Matsubara D;Shibano T;Tamura T;Oguni S;Katashiba S;Ito T;Murakami Y;Fukayama M;Murakami T;Endo S;Niki T
细胞-基质粘附的减少与转移密切相关。以往的研究表明，组织学上有漂浮细胞团的肺癌更容易发生转移。在本研究中，我们研究了长期三维低附着培养中的癌细胞是否具有高转移潜力;然后将这些细胞用于研究转移的潜在机制。在超低贴壁培养皿上培养和选择两种KRAS突变的腺癌细胞系（A549和H441），并将所得细胞定义为FL（用于漂浮）亚系。通过心内注射将癌细胞接种到NOD/SCID小鼠中，并使用基于胰蛋白酶的成像和组织病理学评估转移。测定体外细胞生长（贴壁或悬浮培养）、迁移和侵袭。进行全基因组分析以鉴定FL亚系中的关键分子改变。在低结合培养皿上分离后，亲本细胞最初形成具有有限生长活性的圆形球状体。然而，随着培养时间的推移，细胞逐渐形成较小的球状体，生长缓慢，3-4个月后，我们获得了FL亚系，在悬浮培养中恢复了显著的生长潜力。在普通培养皿上，FL细胞重新附着，并表现出比亲本细胞更梭形的形态。FL亚系和亲本细胞系之间的细胞生长与附着、迁移或侵袭没有显著差异;然而，FL细胞在体外悬浮条件下表现出显著增加的生长潜力和更强的体内转移能力。基因组分析确定上皮间质转化（EMT）和c-Myc扩增A549-FL和H441-FL细胞，分别作为转移的候选机制。FL细胞的生长潜力被A549-FL细胞中的慢病毒ZEB 1敲低和H441-FL细胞中通过慢病毒敲低或药理学抑制剂JQ 1抑制c-Myc显著抑制。长期的三维低附着培养可能成为一种有用的方法，用于研究细胞-基质粘附减少介导的转移机制。
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.
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影响因子：
7.7
作者：
Cappellen, David;Schlange, Thomas;Hynes, Nancy E.
通讯作者：
Hynes, Nancy E.
EMT，癌症干细胞和耐药性：癌症战争中新兴的邪恶轴。
影响因子：
8
作者：
Singh, A.;Settleman, J.
通讯作者：
Settleman, J.
DOI：
10.1073/pnas.1734137100
发表时间：
2003-09-02
影响因子：
11.1
作者：
Ohira, T;Gemmill, RM;Drabkin, HA
通讯作者：
Drabkin, HA
影响因子：
3.3
作者：
Sanchez-Mora, Nora;Cebollero Presmanes, Maria;Alvarez-Fernandez, Ernitio
通讯作者：
Alvarez-Fernandez, Ernitio
DOI：
10.1073/pnas.1216363109
发表时间：
2012-11-20
影响因子：
11.1
作者：
Lockwood, William W.;Zejnullahu, Kreshnik;Varmus, Harold
通讯作者：
Varmus, Harold