Human gastric cancer cell line; CGH array analysis of SNU-16 parental and clones cells. Human gastric cancer cell line; CGH array analysis of SNU-16 parental and clones cells

Chromosomal instability in gastric cancer cells is associated with the amplification of oncogenes that encode receptor tyrosine kinases (RTKs), such as HER2 and FGFR2; such gene amplification varies from cell to cell and manifests as genetic heterogeneity within tumours. The intratumoural genetic heterogeneity of RTK gene amplification causes heterogeneity in RTK protein expression, which has been suggested to be associated with therapeutic resistance to RTK inhibitors; however, the underlying mechanism is not fully understood. Here, we show that extrachromosomal DNA (ecDNA) causes intratumoural genetic heterogeneity in RTKs and drug resistance due to diverse dynamic changes. We analysed the dynamics of FGFR2 and MYC ecDNA in a gastric cancer cell line after single-cell cloning. Similar to those in parental cells, the copy numbers of FGFR2 and MYC in subclones differed significantly between cells, indicating intraclonal genetic heterogeneity. Furthermore, the ecDNA composition differed between subclones, which affected FGFR2 protein expression and drug sensitivity. Interestingly, clone cells that were resistant to the FGFR2 inhibitor AZD4547 presented diverse changes in ecDNA, including chimeric ecDNA, large ecDNA and increased ecDNA numbers; these changes were associated with high expression and rephosphorylation of FGFR2. Conversely, when resistant clone cells were cultured under conditions that excluded AZD4547, the ecDNA status became similar to that of the original clone cells, and the inhibitory effect on cell growth was restored. Our results show that dynamic quantitative and qualitative changes in ecDNA can drive the intratumoural genetic heterogeneity of RTKs and resistance to RTK inhibitors. Overall design: Control vs. SNU-16 parental cells, Control vs. SNU-16 clone D7 cells, Control vs. SNU-16 clone E8 cells, Control vs. SNU-16 resitant clone D7 cells, SNU-16 parental cells vs. SNU-16 clone D7 cells, SNU-16 parental cells vs. SNU-16 clone E8, SNU-16 parental cells vs. SNU-16 resistant clone D7 cells. Biological replicates: Control vs. samples 1 replicate, SNU-16 parental cells vs. samples 1 replicate.

胃癌细胞的染色体不稳定性与编码受体酪氨酸激酶（Receptor Tyrosine Kinases, RTKs）的癌基因扩增相关，例如HER2与FGFR2；此类基因扩增存在细胞间差异，并表现为肿瘤内的遗传异质性。RTK基因扩增所介导的肿瘤内遗传异质性，会导致RTK蛋白表达的异质性，该现象被认为与RTK抑制剂的治疗耐药相关，但其潜在机制尚未完全阐明。

本研究证实，染色体外DNA（extrachromosomal DNA, ecDNA）可通过多样的动态变化，介导RTKs的肿瘤内遗传异质性并引发耐药性。我们对单细胞克隆培养后的胃癌细胞系中FGFR2与MYC的ecDNA动态变化进行了分析。与亲本细胞类似，亚克隆细胞间的FGFR2与MYC拷贝数存在显著差异，表明克隆内遗传异质性的存在。进一步研究发现，不同亚克隆的ecDNA组成存在差异，这会影响FGFR2蛋白表达与药物敏感性。

有趣的是，对FGFR2抑制剂AZD4547产生耐药的克隆细胞，其ecDNA出现了多种变化，包括嵌合ecDNA、大型ecDNA以及ecDNA数量增加；这些变化与FGFR2的高表达及重新磷酸化相关。反之，当耐药克隆细胞在不含AZD4547的培养环境中生长时，其ecDNA状态恢复至原始克隆细胞的状态，细胞生长的抑制效应也得以恢复。

本研究结果表明，ecDNA的动态定量与定性变化，可驱动RTKs的肿瘤内遗传异质性，并引发对RTK抑制剂的耐药性。

整体实验设计：对照组 vs. SNU-16亲本细胞、对照组 vs. SNU-16克隆D7细胞、对照组 vs. SNU-16克隆E8细胞、对照组 vs. SNU-16耐药克隆D7细胞、SNU-16亲本细胞 vs. SNU-16克隆D7细胞、SNU-16亲本细胞 vs. SNU-16克隆E8细胞、SNU-16亲本细胞 vs. SNU-16耐药克隆D7细胞。

生物学重复：对照组与各组样本均设置1次生物学重复；SNU-16亲本细胞与各组样本亦设置1次生物学重复。