通过免疫荧光测定 可视化 患者来源的卵巢癌类器官中的 DNA 损伤修复蛋白
Summary
本协议描述了在患者来源的卵巢癌类器官中评估DNA损伤修复蛋白的方法。这里包括全面的电镀和染色方法，以及详细、客观的定量程序。
Abstract
免疫荧光是使用最广泛的技术之一，用于以高灵敏度和特异性可视化靶抗原，从而可以准确鉴定和定位蛋白质、聚糖和小分子。虽然该技术在二维（2D）细胞培养中已经成熟，但对其在三维（3D）细胞模型中的使用知之甚少。卵巢癌类器官是3D肿瘤模型，概括了肿瘤细胞克隆异质性，肿瘤微环境以及细胞-细胞和细胞-基质相互作用。因此，它们在评估药物敏感性和功能生物标志物方面优于细胞系。因此，在原发性卵巢癌类器官上利用免疫荧光的能力对于理解这种癌症的生物学非常有益。目前的研究描述了免疫荧光技术，以检测高级别浆液性患者来源的卵巢癌类器官（PDO）中的DNA损伤修复蛋白。将PDO暴露于电离辐射后，对完整的类器官进行免疫荧光以评估核蛋白作为病灶。在共聚焦显微镜上使用z-stack成像收集图像，并使用自动病灶计数软件进行分析。所描述的方法允许分析DNA损伤修复蛋白的时间和特殊募集，以及这些蛋白质与细胞周期标记物的共定位。
Introduction
卵巢癌是妇科恶性肿瘤导致死亡的主要原因。大多数患者使用卡铂等 DNA 损伤药物治疗，同源重组修复 （HRR） 缺陷肿瘤患者可给予聚 （ADP-核糖） 聚合酶 （PARP） 抑制剂1，2。然而，大多数患者对这些疗法产生耐药性，并在诊断后 5 年内死亡。DNA损伤反应（DDR）失调与卵巢癌的发展以及化疗和PARP抑制剂耐药有关3。因此，研究DDR对于了解卵巢癌的病理生理学，潜在的生物标志物和新的靶向疗法至关重要。
目前评估DDR的方法利用免疫荧光（IF），因为这可以准确鉴定和定位DNA损伤蛋白和核苷酸类似物。一旦DNA中出现双链断裂（DSB），组蛋白H2AX就会迅速磷酸化，形成DNA损伤修复蛋白聚集的焦点4。这种磷酸化可以很容易地利用IF识别;事实上，ɣ-H2AX测定通常用于确认DSB5，6，7，8，9的诱导。DNA损伤的增加与DNA损伤剂10，11，12的铂敏感性和功效有关，并且ɣ-H2AX已被提议作为与其他癌症治疗中的化疗反应相关的生物标志物13。在DSB上，精通HRR的细胞执行一系列事件，导致BRCA1和BRCA2招募RAD51以取代复制蛋白A（RPA）并与DNA结合。HRR修复使用DNA模板来忠实地修复DSB。然而，当肿瘤缺乏HRR时，它们依赖于替代修复途径，例如非同源末端连接（NHEJ）。众所周知，NHEJ容易出错，并且对细胞产生高突变负担，细胞使用53BP1作为正调节因子14。这些DNA损伤蛋白都可以使用IF准确地识别为病灶。除了蛋白质染色外，IF还可用于研究叉保护和单链DNA间隙形成。具有稳定叉的能力与铂反应相关，最近，间隙测定已显示出预测对PARP抑制剂6，15，16，17的反应的潜力。因此，在引入基因组后对核苷酸类似物进行染色是研究DDR的另一种方法。
迄今为止，卵巢癌中DDR的评估主要局限于同质2D细胞系，这些细胞系不能概括体内肿瘤的克隆异质性，微环境或结构18，19。最近的研究表明，类器官在研究复杂的生物过程（如DDR机制）方面优于2D细胞系6。本方法评估PDO中的RAD51，ɣ-H2AX，53BP1，RPA和geminin。这些方法评估完整的类器官，并允许在更类似于体内肿瘤微环境中研究DDR机制。结合共聚焦显微镜和自动病灶计数，这种方法可以帮助了解卵巢癌的DDR途径并为患者制定个性化的治疗计划。
Protocol
肿瘤组织和腹水是在获得患者同意后获得的，作为圣路易斯华盛顿大学机构审查委员会 （IRB） 批准的妇科肿瘤学生物储存库的一部分。如果患者患有晚期高级别浆液性卵巢癌（HGSOC），则纳入患者。除非另有说明，否则所有程序均在室温下在工作台上进行。所有试剂均在室温下制备（除非另有说明）并储存在4°C。 1. 类器官生成 按照先前发表的报告<sup cla…
Representative Results
所提出的方案可以成功地对类器官中的核DNA损伤修复蛋白进行染色、可视化和定量。该技术用于在照射前后对PDO进行染色和评估。PDO暴露于10 Gy的辐射下，并评估以下生物标志物：ɣ-H2AX（图1），DNA损伤的标志物;RAD51（图2），HRR的标志物;53BP1，NHEJ的标志物;RPA，复制压力的标志物（图3）;和 geminin，一种 G2/S 期细胞周期标志物<sup cla…
Discussion
DNA损伤反应在卵巢癌和化疗耐药性的发展和中起着不可或缺的作用。因此，彻底了解DNA修复机制势在必行。在这里，提出了一种研究3D完整类器官中DNA损伤修复蛋白的方法。利用标志性抗体开发了一种可重复、可靠的方案，以评估 DNA 损伤、同源重组、非同源末端连接和复制应激。重要的是，这些方法使用转基因对照进行了验证，证明了这些方法的特异性和敏感性。
该方案中?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Pavel Lobachevsky博士在建立该协议方面的指导。我们还要感谢华盛顿大学圣路易斯医学院妇产科和妇科肿瘤科、华盛顿大学院长学者计划、妇科肿瘤学小组基金会和生殖科学家发展计划对这个项目的支持。
Materials
| 1x phosphate buffered saline with calcium and magnesium (PBS++) | Sigma | 14-040-133 | |
| 1x phosphate buffered saline without calcium and magnesium (PBS) | Fisher Scientific | ICN1860454 | |
| Ant-53BP1 Antibody | BD Biosciences | 612522 | diluted to 1:500 in staining buffer |
| Ant-Geminin Antibody | Abcam | ab104306 | diluted to 1:200 in staining buffer |
| Anti-Geminin Antibody | ProteinTech | 10802-1-AP | diluted to 1:400 in staining buffer |
| Anti-RAD51 Antibody | Abcam | ab133534 | diluted to 1:1000 in staining buffer |
| Anti-yH2AX Antibody | Millipore-Sigma | 05-636 | diluted to 1:500 in staining buffer |
| Ant-phospho-RPA32 (S4/S8) Antibody | Bethyl Laboratories | A300-245A-M | diluted to 1:200 in staining buffer |
| Bovine Serum Albumin (BSA) | Fisher Scientific | BP1605 100 | |
| Centrifuge; Sorvall St 16R Centrifuge | Thermo Scientific | 75004240 | |
| Confocal Microscope, Leica SP5 confocal system DMI4000 | Leica | 389584 | |
| Conical Tubes, 15 mL | Corning | 14-959-53A | |
| Countess 3 FL Automated Cell Counter (Cell Counting Machine) | Thermo Scientific | AMQAF2000 | |
| Countess Cell Counting Chamber Slides | Thermo Scientific | C10228 | |
| Cover Slip | LA Colors | Any clear nail polish will suffice | |
| Cultrex RGF Basement Membrane Extract, Type 2 | R&D Systems | 3533-010-02 | Could probably use Matrigel or other BME Matrix |
| DAPI | Thermo Scientific | R37606 | NucBlue Fixed Cell ReadyProbes Reagent, Diluted in 1x PBS |
| Glycine | Fisher Scientific | NC0756056 | |
| JCountPro | JCountPro | For access to the software, Email: jcountpro@gmail.com | |
| Microcentrifuge Tubes | Fisher Scientific | 07-000-243 | |
| Nail Polish | StatLab | SL102450 | |
| Parafomraldehyde (PFA), 2% | Electron Microscopy Sciences | 157-4 | Dilute to 4% PFA in PBS++ to obtain 2% PFA |
| Permeabilization Buffer | Made in Lab | 0.2% X-100 Triton in PBS++ | |
| Pipette | Rainin | 17014382 | |
| Pipette Tips | Rainin | 17014967 | |
| ProLong Gold Antifade Mountant | Thermo Scientific | P36930 | |
| Staining Buffer | Made in Lab | 0.5% BSA, 0.15% Glycine, 0.1% X-100 Triton in PBS++ | |
| Thermo Scientific Nunc Lab-Tek II Chamber Slide System | Thermo Scientific | 12-565-8 | |
| Triton X-100 | Sigma-Alderich | 11332481001 | |
| Trypan Blue Solution, 0.4% | Thermo Scientific | 15250061 | |
| TrypLE Express | Invitrogen | 12604013 | animal origin-free, recombinant enzyme |
| X-RAD 320 Biological Irradiator | Precision X-Ray Irradiation | X-RAD320 |
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Cite This Article
van Biljon, L., Fashemi, B., Rodriguez, J., Graham, O., Compadre, A., Fuh, K., Khabele, D., Mullen, M. Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays. J. Vis. Exp. (192), e64881, doi:10.3791/64881 (2023).