Osteosarcoma (OS) is a molecularly heterogeneous, aggressive, poorly differentiated pediatric bone cancer that frequently spreads to the lung. Relatively little is known about phenotypic and epigenetic changes that promote lung metastases. To identify key drivers of metastasis, we studied human CCH-OS-D OS cells within a previously described rat acellular lung (ACL) model that preserves the native lung architecture, extracellular matrix, and capillary network. This system identified a subset of cells—termed derived circulating tumor cells (dCTCs)—that can migrate, intravasate, and spread within a bioreactor-perfused capillary network. Remarkably, dCTCs highly expressed epithelial-to-mesenchymal transition (EMT)-associated transcription factors (EMT-TFs), such as ZEB1, TWIST, and SOX9, which suggests that they undergo cellular reprogramming toward a less differentiated state by coopting the same epigenetic machinery used by carcinomas. Since YAP/TAZ and AXL tightly regulate the fate and plasticity of normal mesenchymal cells in response to microenvironmental cues, we explored whether these proteins contributed to OS metastatic potential using an isogenic pair of human OS cell lines that differ in AXL expression. We show that AXL inhibition significantly reduced the number of MG63.2 pulmonary metastases in murine models. Collectively, we present a laboratory-based method to detect and characterize a pure population of dCTCs, which provides a unique opportunity to study how OS cell fate and differentiation contributes to metastatic potential. Though the important step of clinical validation remains, our identification of AXL, ZEB1, and TWIST upregulation raises the tantalizing prospect that EMT-TF-directed therapies might expand the arsenal of therapies used to combat advanced-stage OS.

骨肉瘤 (OS) 是一种分子异质的、侵袭性的、低分化的小儿骨癌，经常扩散到肺部。关于促进肺转移的表型和表观遗传变化知之甚少。为了确定转移的关键驱动因素，我们研究了先前描述的大鼠脱细胞肺 (ACL) 模型中的人类 CCH-OS-D OS 细胞，该模型保留了天然肺结构、细胞外基质和毛细血管网络。该系统确定了一个细胞亚群——称为衍生循环肿瘤细胞 (dCTC)——可以在生物反应器灌注的毛细血管网络内迁移、渗入和扩散。值得注意的是，dCTCs 高度表达上皮间质转化 (EMT) 相关转录因子 (EMT-TFs)，例如 ZEB1、TWIST 和 SOX9，这表明它们通过采用与癌症相同的表观遗传机制进行细胞重编程，从而向分化程度较低的状态转变。由于 YAP/TAZ 和 AXL 严格调节正常间充质细胞响应微环境线索的命运和可塑性，我们使用 AXL 表达不同的一对人类 OS 细胞系的同基因对探索这些蛋白质是否有助于 OS 转移潜能。我们显示 AXL 抑制显着减少了小鼠模型中 MG63.2 肺转移的数量。总的来说，我们提出了一种基于实验室的方法来检测和表征纯 dCTC 群体，这为研究 OS 细胞命运和分化如何促成转移潜能提供了独特的机会。尽管临床验证的重要步骤仍然存在，但我们对 AXL 的识别，