The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γH2AX), which is an indicator of DNA double-strand breaks. While γH2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γH2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γH2AX immunostained images confirmed strong correlation between SLAM image features and γH2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.

中文翻译：

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通过同步无标记自发荧光多谐波显微镜预测非小细胞肺癌类器官中的 DNA 损伤反应


DNA 损伤反应 (DDR) 是评估癌症疗法疗效的基本读数，其中许多疗法针对 DNA 相关过程。目前评估 DDR 的技术依赖于磷酸化组蛋白 H2AX (γH2AX) 的免疫染色，这是 DNA 双链断裂的指标。虽然 γH2AX 免疫染色可以提供固定细胞和组织样本中 DDR 的快照，但该方法在技术上很麻烦，因为需要时间点重复对 DDR 进行时间监测、对 3D 细胞培养样本（如类器官）进行大量的检测开发工作，以及耗时的实验方案γH2AX免疫染色及其评价。目前这项研究的目标是通过利用无标记多光子成像来减轻非小细胞肺癌 (NSCLC) 类器官测定持续时间和发育的总体负担。在这项研究中，使用同时无标记自发荧光多谐波（SLAM）显微镜来提供基于内源对比度的丰富的细胞内信息。 SLAM 显微镜能够对活样品进行成像，无需生成牺牲样品复制品，并且与传统共焦显微镜相比，改进了 3D 空间中的图像采集。无标记 SLAM 显微镜和 γH2AX 免疫染色图像之间的预测模型证实了 SLAM 图像特征和 γH2AX 信号之间的强相关性。在多种 DNA 靶向化疗药物和多种患者来源的 NSCLC 类器官系中，光学氧化还原比和三次谐波产生通道可用于稳健预测 DDR。通过 SLAM 显微镜成像可用于更快速地预测活体 3D NSCLC 类器官中的 DDR，只需最少的样本处理且无需标记。