Background: Often, neoadjuvant therapy, which relies on the induction of double-strand breaks (DSBs), is used prior to surgery to shrink tumors by inducing cancer cell apoptosis. However, recent studies have suggested that this treatment may also induce a fluctuating state between senescence and stemness in PA-1 embryonal carcinoma cells, potentially affecting therapeutic outcomes. Thus, the respective epigenetic pathways are up or downregulated over a time period of days. These fluctuations go hand in hand with changes in spatial DNA organization.Methods: By means of Single-Molecule Localization Microscopy in combination with mathematical evaluation tools for pointillist data sets, we investigated the organization of euchromatin and heterochromatin at the nanoscale on the third and fifth day after etoposide treatment.Results: Using fluorescently labeled antibodies against H3K9me3 (heterochromatin tri-methylation sites) and H3K4me3 (euchromatin tri-methylation sites), we found that the induction of DSBs led to the de-condensation of heterochromatin and compaction of euchromatin, with a peak effect on day 3 after the treatment. On day 3, we also observed the co-localization of euchromatin and heterochromatin, which have marks that usually occur in exclusive low-overlapping network-like compartments. The evaluation of the SMLM data using topological tools (persistent homology and persistent imaging) and principal component analysis, as well as the confocal microscopy analysis of H3K9me3- and H3K4me3-stained PA-1 cells, supported the findings that distinct shifts in euchromatin and heterochromatin organization took place in a subpopulation of these cells during the days after the treatment. Furthermore, by means of flow cytometry, it was shown that the rearrangements in chromatin organization coincided with the simultaneous upregulation of the stemness promotors OCT4A and SOX2 and senescence promotors p21Cip1 and p27.Conclusions: Our findings suggest potential applications to improve cancer therapy by inhibiting chromatin remodeling and preventing therapy-induced senescence.
背景：通常，在手术前会采用依赖诱导DNA双链断裂（DSBs）的新辅助疗法，通过诱导癌细胞凋亡来缩小肿瘤。然而，近期研究表明，该疗法可能还会在PA-1胚胎性癌细胞中诱导衰老与干性之间的波动状态，从而可能影响治疗效果。因此，相应的表观遗传通路会在数天内发生上调或下调，这些波动与空间DNA结构的变化密切相关。 方法：通过单分子定位显微镜技术结合点彩数据集数学评估工具，我们研究了依托泊苷处理后第三天和第五天常染色质与异染色质在纳米尺度的组织结构。 结果：使用针对H3K9me3（异染色质三甲基化位点）和H3K4me3（常染色质三甲基化位点）的荧光标记抗体，我们发现DSBs的诱导导致异染色质去凝聚和常染色质压缩，并在处理后第三天达到峰值效应。第三天我们还观察到常染色质与异染色质的共定位现象——这两种通常存在于互斥低重叠网络状区室的标记出现了空间交集。通过拓扑学工具（持续同调与持续成像）和主成分分析对SMLM数据的评估，以及对H3K9me3和H3K4me3染色PA-1细胞的共聚焦显微镜分析，均证实治疗后数天内细胞亚群中发生了显著的染色质结构转变。此外，流式细胞术显示染色质结构的重排与干性促进因子OCT4A、SOX2以及衰老促进因子p21Cip1、p27的同步上调具有时间关联性。 结论：我们的研究结果表明，通过抑制染色质重塑和防止治疗诱导的衰老，可能为改善癌症治疗提供潜在应用方向。
肿瘤(癌症)患者之家