Departments of Neurosurgery and Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Clin Cancer Res. 2012 Apr 1;18(7):1901-13. doi: 10.1158/1078-0432.CCR-11-3084. Epub 2012 Feb 7.
Despite the high incidence of epidermal growth factor receptor (EGFR) gene amplification and rearrangement in glioblastomas, no suitable cell line exists that preserves these alterations in vitro and is tumorigenic in immunocompromised mice. On the basis of previous observations that glioblastoma cells cultured with serum lose the EGFR amplification rapidly and that EGF can inhibit the growth of EGFR-amplified tumor cells, we hypothesized that serum-free and EGF-free culture conditions could promote maintenance of the EGFR amplification.
Cells from EGFR-amplified glioblastomas were taken into culture using neural stem cell conditions with modifications, including varying oxygen concentrations and omission of routine EGF supplementation.
High-level EGFR amplification was rapidly lost in 5 glioblastoma cultures supplemented with EGF, whereas it was preserved in cultures from the same tumors established without EGF. Cultures from 2 glioblastomas developed into pairs of cell lines, with either stable maintenance or irreversible loss of high-level EGFR amplification in the majority of cells. One EGFR-amplified cell line preserved expression of the receptor variant EGFRvIII. Cell lines with high-level EGFR amplification/EGFRvIII expression formed highly aggressive tumors in nude mice, whereas nonamplified cell lines were either nontumorigenic or grew significantly more slowly. In contrast, nonamplified cell lines proliferated faster in vitro. All cell lines responded to erlotinib, with inhibition of receptor activation and proliferation but partly different effects on downstream signaling and migration.
Isogenic glioblastoma cell lines maintaining stable differences in EGFR/EGFRvIII status can be derived by varying exposure to EGF ligand and reflect the intratumoral genetic heterogeneity.
尽管表皮生长因子受体（EGFR）基因扩增和重排在胶质母细胞瘤中发生率很高，但体外不存在保留这些改变的合适细胞系，并且在免疫功能低下的小鼠中具有致瘤性。基于先前的观察结果，即与血清培养的神经胶质瘤细胞迅速失去 EGFR 扩增，并且 EGF 可以抑制 EGFR 扩增肿瘤细胞的生长，我们假设无血清和无 EGF 的培养条件可以促进 EGFR 扩增的维持。
使用神经干细胞条件对 EGFR 扩增的胶质母细胞瘤细胞进行培养，包括改变氧浓度和常规 EGF 补充。
在 5 种用 EGF 补充的胶质母细胞瘤培养物中，高水平的 EGFR 扩增迅速丢失，而在没有 EGF 的相同肿瘤建立的培养物中则保留了扩增。来自 2 个胶质母细胞瘤的培养物发展成了一对细胞系，其中大多数细胞中高水平 EGFR 扩增的稳定维持或不可逆丢失。一个 EGFR 扩增的细胞系保留了受体变体 EGFRvIII 的表达。具有高水平 EGFR 扩增/EGFRvIII 表达的细胞系在裸鼠中形成高度侵袭性肿瘤，而非扩增细胞系则无致瘤性或生长速度明显较慢。相比之下，非扩增细胞系在体外增殖更快。所有细胞系均对厄洛替尼有反应，抑制受体激活和增殖，但对下游信号转导和迁移有部分不同的影响。
通过改变 EGF 配体的暴露，可以衍生出具有稳定 EGFR/EGFRvIII 状态差异的同基因胶质母细胞瘤细胞系，反映了肿瘤内的遗传异质性。
