Department of Physics and Astronomy, University of Victoria, Victoria BC V8W 3P6, Canada.
Phys Med Biol. 2011 Nov 7;56(21):6839-55. doi: 10.1088/0031-9155/56/21/006. Epub 2011 Oct 5.
This work applies noninvasive single-cell Raman spectroscopy (RS) and principal component analysis (PCA) to analyze and correlate radiation-induced biochemical changes in a panel of human tumour cell lines that vary by tissue of origin, p53 status and intrinsic radiosensitivity. Six human tumour cell lines, derived from prostate (DU145, PC3 and LNCaP), breast (MDA-MB-231 and MCF7) and lung (H460), were irradiated in vitro with single fractions (15, 30 or 50 Gy) of 6 MV photons. Remaining live cells were harvested for RS analysis at 0, 24, 48 and 72 h post-irradiation, along with unirradiated controls. Single-cell Raman spectra were acquired from 20 cells per sample utilizing a 785 nm excitation laser. All spectra (200 per cell line) were individually post-processed using established methods and the total data set for each cell line was analyzed with PCA using standard algorithms. One radiation-induced PCA component was detected for each cell line by identification of statistically significant changes in the PCA score distributions for irradiated samples, as compared to unirradiated samples, in the first 24-72 h post-irradiation. These RS response signatures arise from radiation-induced changes in cellular concentrations of aromatic amino acids, conformational protein structures and certain nucleic acid and lipid functional groups. Correlation analysis between the radiation-induced PCA components separates the cell lines into three distinct RS response categories: R1 (H460 and MCF7), R2 (MDA-MB-231 and PC3) and R3 (DU145 and LNCaP). These RS categories partially segregate according to radiosensitivity, as the R1 and R2 cell lines are radioresistant (SF(2) > 0.6) and the R3 cell lines are radiosensitive (SF(2) < 0.5). The R1 and R2 cell lines further segregate according to p53 gene status, corroborated by cell cycle analysis post-irradiation. Potential radiation-induced biochemical response mechanisms underlying our RS observations are proposed, such as (1) the regulated synthesis and degradation of structured proteins and (2) the expression of anti-apoptosis factors or other survival signals. This study demonstrates the utility of RS for noninvasive radiobiological analysis of tumour cell radiation response, and indicates the potential for future RS studies designed to investigate, monitor or predict radiation response.
本研究应用无创单细胞拉曼光谱（RS）和主成分分析（PCA）分析和关联源自不同组织、p53 状态和固有放射敏感性的人肿瘤细胞系中辐射诱导的生化变化。从前列腺（DU145、PC3 和 LNCaP）、乳腺（MDA-MB-231 和 MCF7）和肺（H460）中分离出六种人肿瘤细胞系，在体外用 6 MV 光子进行单次照射（15、30 或 50 Gy）。在照射后 0、24、48 和 72 小时，用 RS 分析收获存活细胞，并与未经照射的对照细胞进行比较。利用 785nm 激发激光，从每个样本中采集 20 个细胞的单细胞拉曼光谱。利用已建立的方法对所有 200 个细胞系的光谱进行单独后处理，并利用标准算法对每个细胞系的总数据集进行 PCA 分析。通过识别照射样品与未照射样品的 PCA 评分分布中的统计学显著变化，在照射后 24-72 小时内，检测到每个细胞系的一个诱导的 PCA 成分。这些 RS 响应特征源于细胞内芳香族氨基酸、构象蛋白结构以及某些核酸和脂质功能基团浓度的辐射诱导变化。辐射诱导的 PCA 成分之间的相关性分析将细胞系分为三个不同的 RS 响应类别：R1（H460 和 MCF7）、R2（MDA-MB-231 和 PC3）和 R3（DU145 和 LNCaP）。根据放射敏感性，这些 RS 类别部分分离，R1 和 R2 细胞系为放射抗性（SF（2）>0.6），R3 细胞系为放射敏感（SF（2）<0.5）。R1 和 R2 细胞系进一步根据 p53 基因状态分离，这与照射后细胞周期分析相符。提出了我们的 RS 观察结果的潜在辐射诱导生化反应机制，例如（1）结构蛋白的调节合成和降解和（2）抗凋亡因子或其他存活信号的表达。本研究证明了 RS 用于非侵入性肿瘤细胞放射反应的放射生物学分析的实用性，并表明了未来旨在研究、监测或预测放射反应的 RS 研究的潜力。
