PO.TB03.05 · 肿瘤生物学
界定CRK和CRKL在胶质母细胞瘤侵袭性中的作用：从细胞培养到小鼠模型
Defining the role of CRK and CRKL in glioblastoma invasiveness: from cell culture to mouse models
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
胶质母细胞瘤是同时影响成人和儿童的最具侵袭性的原发性恶性脑肿瘤，尽管采用手术、放疗和化疗等标准治疗，中位生存期仍仅约15个月。由于胶质母细胞瘤细胞高度浸润的本质，完全的手术切除受到阻碍，使肿瘤侵袭成为复发最重要的原因。CT10激酶调节因子（CRK）和CRK样蛋白（CRKL）的失调已被证实与多种癌症相关，包括胶质母细胞瘤，在其中它们的过表达与不良预后相关。我们假设CRK和CRKL在胶质母细胞瘤细胞侵袭中至关重要，且抑制它们可抑制肿瘤浸润并增强治疗疗效。为检验这一假设，我们使用siRNA转染和shRNA慢病毒转导，在胶质母细胞瘤细胞系（U118-MG和LN-229）中对CRK、CRKL或两者进行了瞬时和稳定敲低。Western blot分析证实靶蛋白水平显著降低。使用xCELLigence实时细胞分析系统，我们评估了CRK和CRKL敲低对胶质母细胞瘤细胞黏附、迁移和侵袭的影响。CRKL敲低大幅降低了全部三个过程，而CRK/CRKL联合敲低则完全消除了黏附、迁移和侵袭。这些在三个胶质母细胞瘤系中一致的结果凸显了CRK和CRKL在调控胶质母细胞瘤细胞运动性中必不可少且相互重叠的作用。为检验体内胶质母细胞瘤生长和侵袭对CRK和CRKL的依赖性，我们通过立体定向手术将表达荧光素酶和GFP的LN-229胶质母细胞瘤细胞移植到免疫缺陷（NSG）小鼠脑内。荧光素酶和GFP使我们能够通过IVIS成像监测体内肿瘤生长，并通过GFP免疫组化鉴定浸润的肿瘤细胞。对异种移植脑的组织学分析显示胶质母细胞瘤细胞弥漫性侵袭至周围脑区。目前，我们正在移植经CRK/CRKL敲低的胶质母细胞瘤细胞，以确定CRK/CRKL在体内胶质母细胞瘤侵袭中的必要性。随后，我们计划测试我们研发的CRK/CRKL拮抗肽对小鼠脑内胶质母细胞瘤侵袭的影响。我们的研究凸显了靶向CRK和CRKL作为减轻胶质母细胞瘤侵袭、改善胶质母细胞瘤标准治疗疗效的治疗策略的前景。
查看英文原文 English abstract
Glioblastoma is the most aggressive primary malignant brain tumor affecting both adults and children, with a median survival of only about 15 months despite the standard treatments of surgery, radiation, and chemotherapy. Complete surgical resection is hindered by the highly infiltrative nature of glioblastoma cells, making tumor invasion the most significant cause of recurrence. Dysregulation of CT10 regulator of kinase (CRK) and CRK-like (CRKL) proteins has been implicated in multiple cancers, including glioblastoma, in which their overexpression correlates with poor prognosis. We hypothesized that CRK and CRKL are essential in glioblastoma cell invasion and that their inhibition suppresses tumor infiltration and enhances treatment efficacy. To test this hypothesis, we performed transient and stable knockdown of CRK, CRKL, or both in glioblastoma cell lines (U118-MG and LN-229) using siRNA transfection and shRNA lentiviral transduction. Western blot analyses confirmed significant reductions in target protein levels. Using the xCELLigence real-time cell analysis system, we assessed the impact of CRK and CRKL knockdown on glioblastoma cell adhesion, migration, and invasion. CRKL knockdown substantially reduced all three processes, whereas combined CRK/CRKL knockdown completely abolished adhesion, migration, and invasion. These consistent results across three glioblastoma lines highlight essential, overlapping roles of CRK and CRKL in regulating glioblastoma cell motility. To examine the dependence of in vivo glioblastoma growth and invasion on CRK and CRKL, we transplanted LN-229 glioblastoma cells expressing luciferase and GFP into immunodeficient (NSG) mouse brains using stereotactic surgery. Luciferase and GFP enabled us to monitor in vivo tumor growth through IVIS imaging and identify infiltrating tumor cells by GFP immunohistochemistry. Histological analysis of the xenografted brains revealed diffuse invasion of glioblastoma cells into surrounding brain regions. Currently, we transplant glioblastoma cells with CRK/CRKL knockdown to determine the requirement of CRK/CRKL in glioblastoma invasion in vivo. Then, we plan to test the effects of CRK/CRKL-antagonist peptides we developed on glioblastoma invasion in the mouse brain. Our study highlights the promise of targeting CRK and CRKL as a therapeutic strategy to mitigate glioblastoma invasion and improve the efficacy of standard care for glioblastoma.
利益披露 Disclosure
P. Hettiarachchi, None..
N. Large, None.