利用非病毒永生化方法建立原代前列腺上皮细胞系和肿瘤细胞系

IF 4.3 2区 生物学 Q1 BIOLOGY Biological Research Pub Date : 2024-05-04 DOI:10.1186/s40659-024-00507-z
Simon Lange, Anna Kuntze, Neele Wüstmann, Theresa Reckers, Verena Humberg, Wilhelm G. Dirks, Sebastian Huss, Julia Vieler, Andres Jan Schrader, Martin Bögemann, Katrin Schlack, Christof Bernemann
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引用次数: 0

摘要

对前列腺癌的研究大多使用来自转移性疾病的细胞系,不能反映肿瘤的起始阶段或早期进展。迄今为止,还没有人描述过从原发肿瘤部位提取的癌细胞系的建立情况。根据定义,癌细胞可以无限期培养,而正常上皮细胞在体外会衰老。上皮细胞可以通过病毒整合永生化因子实现永生化。然而,病毒方法可能会受到监管和安全问题的影响,也可能会随机整合到调控遗传元件中,从而改变精确的基因表达。我们打算利用前列腺癌患者的手术标本:(i) 证明癌症细胞系的建立;(ii) 对前列腺上皮细胞进行基于睡美人(SB)转座酶的非病毒永生化。对前列腺癌患者的前列腺根治术样本(n = 4)进行离体和体外培养。培养细胞时不使用病毒,也不使用基于睡美人转座酶的稳定转染永生化因子 SV40LT 和 hTERT。对建立的细胞系进行了体外和体内分析,以确定前列腺(癌)细胞的特征。未进行基因操作的初始细胞培养物在传代次数不超过 15 次时就会衰老,这表明无法成功培育出原代前列腺癌细胞系。通过使用基于 SB 转座酶的永生化因子整合,我们建立了原代前列腺细胞系。四个细胞系中有三个显示出上皮特征,但没有前列腺(癌症)特征(如雄激素受体)的表达。在体内,一种细胞系具有致瘤潜能,但没有前列腺腺癌的特征。虽然无法建立原代前列腺癌细胞系,但我们首次利用 SB 转座酶系统实现了原代前列腺细胞的永生化,从而避免了基于病毒永生化方法的调控和分子问题。虽然新获得的细胞系都没有表现出前列腺癌的特征,但在一个细胞系中观察到了肿瘤的形成。鉴于肿瘤的非前列腺腺癌特性,细胞可能发生了致癌转化,而非前列腺癌分化。不过,这些细胞系仍可作为研究前列腺癌发生和早期癌症进展的工具。
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Establishment of primary prostate epithelial and tumorigenic cell lines using a non-viral immortalization approach
Research on prostate cancer is mostly performed using cell lines derived from metastatic disease, not reflecting stages of tumor initiation or early progression. Establishment of cancer cell lines derived from the primary tumor site has not been described so far. By definition, cancer cells are able to be cultured indefinitely, whereas normal epithelial cells undergo senescence in vitro. Epithelial cells can be immortalized, accomplished by using viral integration of immortalization factors. Viral approaches, however, might be impaired by regulatory and safety issues as well as random integration into regulatory genetic elements, modifying precise gene expression. We intend to use surgical specimen of prostate cancer patients to (i) prove for establishment of cancer cell lines, and (ii) perform non-viral, Sleeping Beauty (SB) transposase-based immortalization of prostate epithelial cells. Radical prostatectomy samples of prostate cancer patients (n = 4) were dissociated and cultured in vitro. Cells were cultivated either without or after non-viral, Sleeping-Beauty transposase-based stable transfection with immortalization factors SV40LT and hTERT. Established cell lines were analyzed in vitro and in vivo for characteristics of prostate (cancer) cells. Initial cell cultures without genetic manipulation underwent senescence within ≤ 15 passages, demonstrating inability to successfully derive primary prostate cancer cell lines. By using SB transposase-based integration of immortalization factors, we were able to establish primary prostate cell lines. Three out of four cell lines displayed epithelial characteristics, however without expression of prostate (cancer) characteristics, e.g., androgen receptor. In vivo, one cell line exhibited tumorigenic potential, yet characteristics of prostate adenocarcinoma were absent. Whereas no primary prostate cancer cell line could be established, we provide for the first-time immortalization of primary prostate cells using the SB transposase system, thereby preventing regulatory and molecular issues based on viral immortalization approaches. Although, none of the newly derived cell lines demonstrated prostate cancer characteristics, tumor formation was observed in one cell line. Given the non-prostate adenocarcinoma properties of the tumor, cells have presumably undergone oncogenic transformation rather than prostate cancer differentiation. Still, these cell lines might be used as a tool for research on prostate cancer initiation and early cancer progression.
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来源期刊
Biological Research
Biological Research 生物-生物学
CiteScore
10.10
自引率
0.00%
发文量
33
审稿时长
>12 weeks
期刊介绍: Biological Research is an open access, peer-reviewed journal that encompasses diverse fields of experimental biology, such as biochemistry, bioinformatics, biotechnology, cell biology, cancer, chemical biology, developmental biology, evolutionary biology, genetics, genomics, immunology, marine biology, microbiology, molecular biology, neuroscience, plant biology, physiology, stem cell research, structural biology and systems biology.
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