Department of Radiation Oncology, Weill Cornell Medical College.
Department of Radiation Oncology, Weill Cornell Medical College; Sandra and Edward Meyer Cancer Center; Department of Medicine, Weill Cornell Medical College.
J Vis Exp. 2024 Apr 5(206). doi: 10.3791/66680.
Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.
放射治疗（RT）是现代临床癌症管理的主要方法之一。然而，并非所有癌症类型对辐射都同样敏感，通常（但不总是）是因为恶性细胞修复电离射线引起的氧化 DNA 损伤的能力存在差异。几十年来，集落形成测定法一直被用于评估培养的癌细胞对电离辐射的敏感性，主要是因为受照射的癌细胞通常以延迟的方式死亡，这很难用短期流式细胞术或显微镜辅助技术进行量化。不幸的是，集落形成测定法不能直接用于更复杂的肿瘤模型，例如患者来源的肿瘤类器官（PDTO）。事实上，辐照已建立的 PDTO 不一定会使其作为多细胞单位的生长受到抑制，除非其干细胞样区室被完全消除。此外，辐照 PDTO 衍生的单细胞悬浮液可能无法正确反映在已建立的 PDTO 中恶性细胞对 RT 的敏感性。在这里，我们详细介绍了一种常规集落形成测定法的改编，该方法涉及将已建立的 PDTO 暴露于电离辐射，然后进行单细胞解离、在合适的培养条件下重新接种和实时成像。未辐照的（对照）PDTO 衍生的干细胞样细胞以 PDTO 特异性效率重新形成生长的 PDTO，这一效率会受到辐射的负面影响，呈剂量依赖性。在这些条件下，PDTO 形成效率和生长速率可以作为在延时图像上收集的时间依赖性放射敏感性的度量来定量，直到对照 PDTO 达到预设的空间占有率。
