Multicellular Tumor Spheroids Combined with Mass Spectrometric Histone Analysis To Evaluate Epigenetic Drugs.
Multicellular Tumor Spheroids Combined with Mass Spectrometric Histone Analysis To Evaluate Epigenetic Drugs.
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DOI：
10.1021/acs.analchem.6b03602
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发表时间：
2017-03-07
影响因子：
7.4
通讯作者：
Hummon AB
中科院分区：
文献类型：
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作者：
Feist PE;Sidoli S;Liu X;Schroll MM;Rahmy S;Fujiwara R;Garcia BA;Hummon AB
多细胞肿瘤球体（MCTS）是有价值的体外肿瘤模型，经常用于评估治疗的渗透和功效。在这项研究中，我们评估了 HCT116 结肠癌 MCTS 各层表观遗传标记的潜在差异，即组蛋白翻译后修饰 (PTM)。细胞在琼脂糖包被的 96 孔板中生长，形成可重复的 1mm 直径 MCTS。 MCTS 被分成三个径向同心部分，生成含有来自核心、中层和外层的细胞的样本。使用基于质谱 (MS) 的蛋白质组学和 EpiProfile，我们定量了数百种不同修饰形式的组蛋白肽；通过结合所有实验的结果，我们量化了 258 种不同修饰肽的丰度，发现它们跨层的相对丰度存在显着差异。在这些差异中，我们在外层中检测到比核心中更高含量的抑制标记 H3K27me3。然后，我们评估了 UNC1999 治疗后 MCTS 的表观遗传反应，UNC1999 是一种针对催化 H3K27me3 的酶的药物，即 zeste 1 的多梳抑制复合物 2 (PRC2) 亚基增强子 (EZH1) 和 zeste 2 的增强子 (EZH2)。 UNC1999 治疗导致不同持续时间的药物治疗下 MCTS 直径存在显着差异。使用基质辅助激光解吸/电离 (MALDI) 成像，我们确定药物渗透到整个 MCTS。蛋白质组学分析显示，与未处理的样品相比，H3K27me3 的丰度有所下降，正如预期的那样。有趣的是，我们观察到持续药物治疗 13 天以上的 MCTS 与生长开始时仅治疗 4 天的 MCTS 的生长曲线相当。因此，我们证明基于 MS 的蛋白质组学可以定义 3D 培养物亚毫米层中组蛋白 PTM 模式的显着差异。此外，我们表明我们的模型适用于监测药物治疗后组蛋白 PTM 的药物定位和调节。
Multicellular tumor spheroids (MCTS) are valuable in vitro tumor models frequently used to evaluate the penetration and efficacy of therapeutics. In this study, we evaluated potential differences in epigenetic markers, i.e. histone post-translational modifications (PTMs), in the layers of the HCT116 colon carcinoma MCTS. Cells were grown in agarose-coated 96 well plates, forming reproducible 1mm diameter MCTS. The MCTS were fractionated into three radially concentric portions, generating samples containing cells from the core, the mid and the external layers. Using mass spectrometry (MS) based proteomics and EpiProfile, we quantified hundreds of histone peptides in different modified forms; by combining the results of all experiments we quantified the abundance of 258 differently modified peptides, finding significant differences in their relative abundance across layers. Among these differences, we detected higher amounts of the repressive mark H3K27me3 in the external layers as compared to the core. We then evaluated the epigenetic response of MCTS following UNC1999 treatment, a drug targeting the enzymes that catalyze H3K27me3, namely the polycomb repressive complex 2 (PRC2) subunits enhancer of zeste 1 (EZH1) and enhancer of zeste 2 (EZH2). UNC1999 treatment resulted in significant differences in MCTS diameter under drug treatment of varying duration. Using matrix-assisted laser desorption/ionization (MALDI) imaging we determined that the drug penetrates the entire MCTS. Proteomic analysis revealed a decrease in abundance of H3K27me3 as compared to untreated sample, as expected. Interestingly, we observed a comparable growth curve for MCTS under constant drug treatment over 13 days with those treated for only four days at the beginning of their growth. We thus demonstrate that MS based proteomics can define significant differences in histone PTM patterns in sub-millimetric layers of 3D cultures. Moreover, we show that our model is suitable for monitoring drug localization and regulation of histone PTMs after drug treatment.