多模态基因显像研究乏氧调控的转移基因在肺癌大分割放疗后转移中的作用

Hypofractionated radiotherapy (HFRT) significantly improved the local control in early-stage non-small-cell lung cancer. However, metastases remain the first cause of death. It is indicated that hypoxia play an important role in metastases after HFRT since distant metastases could be decreased by improving tumor oxygenation or selectively killing hypoxic cancer cells. Our study found that HFRT decreased tumor blood perfusion, increased tumor hypoxia, and then activated tumor hypoxia response shortly after treatments. We hypothesized that hypoxia promote metastases through hypoxia response up-regulating metastasis related genes. An established novel lung cancer model containing stably transfected Luciferase reporter gene (M38-Luc) and hypoxia response dual reporter gene (9HRE-TK/eGFP) will be used in this study. Tumor hypoxia and hypoxia response will be monitored using PET imaging with 18F-FETNIM and 18F-FHBG (TK substrate), respectively, and tumor growth and metastases by bioluminescence imaging. This study is proposed to: 1) identify the causal effect of tumor hypoxia and tumor hypoxia response on distant metastases after HFRT by oxygen intervention (95% or 10% O2 breathing), and proteasome inhibitor Bortezomib which inhibits hypoxia response, respectively; 2) screen the hypoxia regulated dominant metastasis genes (DG) using flow cytometry hypoxic cell sorting and functional gene-chip analysis; 3) explore the potential of decreasing metastases by combining DG inhibitors with HFRT. This study will reveal the metastatic mechanism after HFRT from the view of tumor hypoxia and hypoxia response, and provide the scientific evidence for reducing metastases and improve outcome for HFRT.

大分割放疗(HFRT)显著提高早期非小细胞肺癌的局控率,但转移仍是患者死亡首因。改善乏氧或靶向杀伤乏氧肿瘤细胞可降低HFRT后转移的发生,提示乏氧在其中起重要作用。我们发现HFRT后早期肿瘤微循环血流降低、乏氧增加、激活乏氧应答,推测乏氧诱导肿瘤细胞乏氧应答,上调转移基因,促进转移。拟采用已建立的稳定表达荧光素酶报告基因(M38-Luc)和乏氧应答双报告基因(9HRE-TK/eGFP)的肺癌模型,PET显像监测肿瘤乏氧和乏氧应答,荧光素酶显像监测转移,通过①HFRT联合高氧/低氧吸入改变肿瘤氧供，或硼替佐米阻断乏氧应答，明确乏氧和乏氧应答与转移的因果关系;②流式技术分选HFRT后乏氧肿瘤细胞,行转移功能基因组芯片分析、筛选乏氧调控关键转移基因(DG);③体内外联合应用DG拮抗剂,探讨靶向阻断DG、降低转移的可能性。本项目将为揭示HFRT后转移发生的机制、降低转移提高整体疗效提供科学依据。

为深入研究放射性对乏氧微环境及其调控基因的影响，本项目开展下列研究。利用A549人肺腺癌裸鼠皮下移植瘤模型，经不同剂量X照射后，采用Pimonidazole荧光免疫组化染色检测肿瘤乏氧、Hoechst33342荧光染料检测肿瘤血供，探讨X射线照射对肿瘤乏氧及血供的影响,利用乏氧途径及肿瘤转移DNA芯片技术，探讨经照射后乏氧调控基因调控的改变；结果显示，大剂量（8Gy）局部照射后 6小时，肿瘤内乏氧较对照组改善，但照射后24小时乏氧加重，大剂量局部照射后6小时，提取RNA芯片分析，大多数乏氧调控基因mRNA水平呈下调状态，转移相关基因mRNA水平也成下调状态。利用瘤床预照射，成功建立了人食管鳞癌放疗复发裸鼠移植瘤模型，荧光免疫组化分析显示复发肿瘤乏氧程 度高，对放射线抵抗，而对乏氧增敏剂-甘氨双唑钠的增敏效果更明显。利用western blot、流式细胞分析等检测蛋白酶体抑制剂-硼替佐米对人结肠癌HT-29细胞乏氧及血管生长因子表达的调控作用，利用裸鼠移植瘤多参数免疫荧光染色，发现1）在组织学水平揭示了治疗前肿瘤初始乏氧微环境在硼替佐米治疗实体肿瘤中的关键作用-乏氧选择性肿瘤细胞和抗血管内皮细胞杀伤作用，证实硼替佐米治疗后早期即可诱导处于乏氧微环境的肿瘤血管内皮细胞凋亡，伴凋亡局部区域微循环血流降低/消失。通过系列18F-FDG PET/CT显像，比较同一患者个体内放疗后局部复发与野外转移病灶的葡萄糖代谢水平和化疗敏感性的差异，结果显示，局部复发病灶PET所示葡萄糖代谢低，对化疗疗效差，从而通过PET糖代谢分子现象观察到了放疗后局部复发肿瘤的生物学特征及临床特征，为放疗后复发肿瘤临床疗效评价及深入的分子机制研究打下了基础。本课题共资助发表SCI收录论文9篇，总影响因子24.41。