Background/Objectives: Despite the introduction of innovative therapeutics, lung cancer is still the leading cause of cancer-related death. For this reason, lung cancer still requires deep characterization to identify cellular and molecular targets that can be used to develop novel therapeutic strategies. Three-dimensional cellular models, including patient-derived organoids (PDOs), represent useful tools to study lung cancer biology and may be employed in the future as predictive tools in therapeutic decisions. However, the successful establishment of lung cancer organoids cultures that faithfully represent the respective patient tissues is still challenging due to low success rate and/or overgrowth of normal airway epithelial cells. Methods: We set up a two-step protocol that allows for establishing both short-term and long-term 3D cultures, with different characteristics and success rates. Results: Cancer tissue-originated spheroids (CTOSs) show a 100% success rate and allow for the concomitant isolation of autologous tumor infiltrating leukocytes (TILs). On the contrary, PDOs can be expanded for a medium-long term and bio-banked but retain a lower success rate and the possibility of contamination with normal airway epithelial cells. To overcome these problems, we set up an optimal medium formulation and we implemented rigorous quality controls, leading to a substantial improvement in the success rate of tumoral PDO establishment. Conclusions: Overall, this protocol guarantees flexibility and reliability, also providing useful guidelines for quality control checks to support different experimental settings. The setting up of a robust protocol for lung cancer PDO culture establishment and expansion is a key requirement for their employment both in cancer research and as predictive tools in clinical practice.
背景/目的：尽管创新疗法不断涌现，肺癌仍是癌症相关死亡的主要原因。因此，仍需对肺癌进行深入表征，以确定可用于开发新型治疗策略的细胞和分子靶点。包括患者来源类器官（PDOs）在内的三维细胞模型是研究肺癌生物学的有效工具，未来或可作为治疗决策的预测工具。然而，由于成功率低和/或正常气道上皮细胞过度生长，成功建立能真实反映患者组织特征的肺癌类器官培养体系仍具挑战性。 方法：我们建立了一种两步法方案，可同时建立具有不同特征和成功率的短期与长期三维培养体系。 结果：癌组织来源球体（CTOSs）的成功率达100%，并能同时分离自体肿瘤浸润白细胞（TILs）。相比之下，PDOs可进行中长期扩增并建立生物样本库，但成功率较低，且存在正常气道上皮细胞污染的可能性。为解决这些问题，我们优化了培养基配方并实施了严格的质量控制，使肿瘤PDOs建立成功率得到显著提升。 结论：总体而言，该方案在保证灵活性和可靠性的同时，还为质量控制提供了实用指导原则，可支持不同的实验设置。建立稳健的肺癌PDOs培养与扩增方案，是将其应用于癌症研究及临床实践预测工具的关键前提。
A Two-Step Protocol for Isolation and Maintenance of Lung Cancer Primary 3D Cultures
肿瘤(癌症)患者之家