Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
Department of Pediatric Hematology and Oncology, Hannover Medical School, 30625 Hannover, Germany.
Cells. 2022 Dec 14;11(24):4045. doi: 10.3390/cells11244045.
Clonal heterogeneity in acute myeloid leukemia (AML) forms the basis for treatment failure and relapse. Attempts to decipher clonal evolution and clonal competition primarily depend on deep sequencing approaches. However, this prevents the experimental confirmation of the identified disease-relevant traits on the same cell material. Here, we describe the development and application of a complex fluorescent genetic barcoding (cFGB) lentiviral vector system for the labeling and subsequent multiplex tracking of up to 48 viable AML clones by flow cytometry. This approach allowed the visualization of longitudinal changes in the in vitro growth behavior of multiplexed color-coded AML clones for up to 137 days. Functional studies of flow cytometry-enriched clones documented their stably inherited increase in competitiveness, despite the absence of growth-promoting mutations in exome sequencing data. Transplantation of aliquots of a color-coded AML cell mix into mice revealed the initial engraftment of similar clones and their subsequent differential distribution in the animals over time. Targeted RNA-sequencing of paired pre-malignant and de novo expanded clones linked gene sets associated with Myc-targets, embryonic stem cells, and RAS signaling to the foundation of clonal expansion. These results demonstrate the potency of cFGB-mediated clonal tracking for the deconvolution of verifiable driver-mechanisms underlying clonal selection in leukemia.
急性髓系白血病 (AML) 中的克隆异质性是治疗失败和复发的基础。解析克隆进化和克隆竞争的尝试主要依赖于深度测序方法。然而，这阻止了在相同的细胞材料上对鉴定出的与疾病相关的特征进行实验验证。在这里，我们描述了一种复杂的荧光遗传条形码 (cFGB) 慢病毒载体系统的开发和应用，该系统用于通过流式细胞术对多达 48 个存活的 AML 克隆进行标记和随后的多重跟踪。这种方法允许可视化体外培养的多重彩色编码 AML 克隆的生长行为的纵向变化，长达 137 天。对流式细胞术富集克隆的功能研究证明了它们稳定遗传的竞争力增加，尽管外显子组测序数据中没有促进生长的突变。将彩色编码 AML 细胞混合物的等分试样移植到小鼠中，揭示了相似克隆的初始植入，以及随着时间的推移它们在动物中的随后差异分布。对配对的前恶性和新扩增克隆进行靶向 RNA-seq 将与 Myc 靶标、胚胎干细胞和 RAS 信号相关的基因集与克隆扩展的基础联系起来。这些结果表明，cFGB 介导的克隆跟踪对于解析白血病中克隆选择的可验证驱动机制具有强大的作用。
