The Clonogenic Survival Assay (CSA) is a fundamental tool employed to assess cell survival and proliferative potential in cancer research. Despite its importance, CSA faces limitations, primarily its time- and labor-intensive nature and its binary output. To overcome these limitations and enhance CSA’s utility, several approaches have been developed, focusing on increasing the throughput. However, achieving both high-content and high-throughput analyses simultaneously has remained a challenge. In this paper, we introduce LeGO-CSA, an extension of the classical CSA that employs the imaging of cell nuclei barcoded with fluorescent lentiviral gene ontology markers, enabling both high-content and high-throughput analysis. To validate our approach, we contrasted it with results from a classical assay and conducted a proof-of-concept screen of small-molecule inhibitors targeting various pathways relevant to cancer treatment. Notably, our results indicate that the classical CSA may underestimate clonogenicity and unveil intriguing aspects of clonal cell growth. We demonstrate the potential of LeGO-CSA to offer a robust approach for assessing cell survival and proliferation with enhanced precision and throughput, with promising implications for accelerating drug discovery and contributing to a more comprehensive understanding of cellular behavior in cancer.
克隆形成存活实验是评估癌细胞存活与增殖潜能的基石性工具。尽管其重要性不言而喻，该实验方法仍存在明显局限，主要体现在耗时费力且仅能提供二元化结果。为突破这些限制并提升其实用价值，学界已开发出多种聚焦于提高通量的改进方案，但如何同时实现高内涵与高通量分析始终是技术难点。本文提出的LeGO-CSA方法，作为经典克隆形成实验的拓展方案，通过采用荧光慢病毒基因本体标记的细胞核成像技术，成功实现了高内涵与高通量的并行分析。为验证该方法，我们将其与传统实验结果进行对比，并开展了针对癌症治疗相关通路的小分子抑制剂概念验证筛选。值得注意的是，研究结果显示经典克隆形成实验可能低估细胞克隆形成能力，同时揭示了克隆细胞生长的若干新特征。本研究证实LeGO-CSA能够为细胞存活与增殖评估提供兼具高精度与高通量的可靠方案，在加速药物研发进程及深化对癌细胞行为机制理解方面展现出显著潜力。
High-Content and High-Throughput Clonogenic Survival Assay Using Fluorescence Barcoding
肿瘤(癌症)患者之家