Laboratory of Radiobiology and Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Martini-Klinik, Prostate Cancer Center, University Medical Hamburg Eppendorf, Hamburg, Germany.
Int J Cancer. 2019 Apr 1;144(7):1685-1696. doi: 10.1002/ijc.32018. Epub 2019 Jan 5.
Here, we present a functional assay to detect the repair switch to the alternative PARP1-dependent end joining (PARP1-EJ) pathway and the associated susceptibility to PARPi-mediated radiosensitization in freshly collected tumor samples from prostate cancer (PCa) patients, thereby facilitating the selection of patients who should benefit from combined PARPi plus radiotherapy (RT) treatment. Our optimized ex-vivo approach sustains tumor slices for up to 15 days under culture conditions that maintain proliferation and oxygenation rates, as measured by EdU incorporation and pimonidazole staining, respectively. We present a robust system to analyze DSB repair using, for the first time in an ex vivo tumor slice setting, two DSB-markers simultaneously i.e. γH2AX and 53BP1. A computer-based processing method (i) controls variations in DNA content and slicing on the number of repair foci and (ii) measures the PARPi-mediated enhancement ratio on DSB foci numbers to ensure inter-patient-comparability. We validated this approach using a PC3 xenograft model with its previously described repair switch to PARP1-EJ. More importantly, we show that approximately 30% of the analyzed tumor tissue samples collected from PCa patients display a switch to PARP1-EJ, as indicated by the enhanced number of residual γH2AX/53BP1 foci exclusively after PARPi+RT. Furthermore, normal prostatic tissues show no repair switch to PARP1-EJ, indicating that this repair switch and its associated radiosensitizing effect is tumor-specific. Collectively, we present here a predictive assay for the switch to PARP1-EJ that enables individualization of anti-cancer treatment using a combination of RT and radiosensitizing anticancer agents such as PARPi in PCa.
在这里，我们提出了一种功能测定法，用于检测来自前列腺癌（PCa）患者的新鲜肿瘤样本中替代 PARP1 依赖性末端连接（PARP1-EJ）途径的修复开关，以及与 PARPi 介导的放射增敏相关的易感性，从而促进了选择应受益于 PARPi 联合放疗（RT）治疗的患者。我们优化的离体方法可在培养条件下维持肿瘤切片长达 15 天，这些条件分别通过 EdU 掺入和 pimonidazole 染色来维持增殖和氧合率。我们提出了一种强大的系统来分析 DSB 修复，这是首次在离体肿瘤切片环境中同时使用两种 DSB 标记物，即 γH2AX 和 53BP1。基于计算机的处理方法（i）控制 DNA 含量的变化，并根据修复焦点的数量控制切片，（ii）测量 PARPi 介导的 DSB 焦点数量增强比，以确保患者间的可比性。我们使用具有先前描述的 PARP1-EJ 修复开关的 PC3 异种移植模型验证了这种方法。更重要的是，我们表明，大约 30％的来自 PCa 患者的分析肿瘤组织样本显示出对 PARP1-EJ 的修复开关，这是通过仅在 PARPi+RT 后残留 γH2AX/53BP1 焦点数量增加来指示的。此外，正常的前列腺组织没有修复到 PARP1-EJ 的开关，这表明这种修复开关及其相关的放射增敏作用是肿瘤特异性的。总的来说，我们在这里提出了一种用于 PARP1-EJ 开关的预测测定法，该测定法可通过使用 RT 和放射增敏抗癌剂（如 PARPi）的组合来实现 PCa 的个体化抗癌治疗。
