Gene expression analysis of tumors of TN and R clonal pairs in vivo. Gene expression analysis of tumors of TN and R clonal pairs in vivo

We previously had generated clonal mouse melanoma cell lines, where each clone was isolated in a RAFi/MEKi-resistant ( R ) or treatment-naïve ( TN ) state using the method CaTCH (clones: 2646TN, 2646R, 14559TN, 14559R, 13TN). We assessed the response to targeted therapy of these clones in in vivo experiments using RAFi/MEKi-treatments. Herey, we saw that, compared to the other treatment-naïve clones, 2646TN exhibited a strongly reduced response to targeted therapy. 2646TN displayed fast growth on drug after only five days, indicating an increased persistence and fast adaption to the treatment, while all other TN clones responded to the treatments and showed tumor regression for weeks before relapsing. To uncover intrinsic programs that might underlie th minimal response of 2646TN, we examined the transcriptome of untreated and short-term treated tumors comprising 2646TN, 2646R, 14559TN, 14559R, or 13TN clones . These clonal cell lines were injected subcutaneously into C57BL/6 mice and tumors were harvested either at tumor engraftment (untreated) or after 3 days of RAFi/MEKi treatment. Lviving cancer cells were sorted from the tumors by FACS and the markers CD45- and the cell line specific marker GFP+. Libraries were prepared using the Lexogen Quantseq 3' mRNA seq kit and sqeuencing was performed on an Illumina HiSeqV4 sequencer (single-read 50 read mode). Overall design: 5 different mouse melanoma cell lines FACS sorted from subcutaneously injectd tumors, each untreated and RAFi/MEKi treated, 3 tumors per cell line and condition (= 30 samples total)

我们此前已构建克隆型小鼠黑色素瘤细胞系，采用CaTCH技术从获得性RAFi/MEKi（RAF激酶抑制剂/MEK激酶抑制剂）耐药状态（耐药型，R）或治疗初治状态（初治型，TN）中分离得到各克隆（克隆编号：2646TN、2646R、14559TN、14559R、13TN）。我们通过RAFi/MEKi给药的体内实验，评估了上述克隆对靶向治疗的响应情况。研究发现，与其他初治型克隆相比，2646TN对靶向治疗的响应显著降低。2646TN仅在给药5天后即出现快速增殖，提示其对治疗的留存能力更强且适应更快；而其余所有TN克隆均对治疗产生响应，在复发前可维持数周的肿瘤消退状态。为揭示可能介导2646TN低响应性的内在调控程序，我们对包含上述5种克隆的未处理及短期处理肿瘤组织进行了转录组分析。将上述克隆型细胞系皮下接种至C57BL/6小鼠体内，分别在肿瘤定植时（未处理组）或RAFi/MEKi给药3天后收获肿瘤组织。通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting，FACS）从肿瘤组织中分离活癌细胞，分选标记为CD45阴性且细胞系特异性标记GFP阳性。采用Lexogen Quantseq 3' mRNA测序试剂盒构建文库，并在Illumina HiSeqV4测序仪上以单端50bp读长模式进行测序。实验整体设计：从皮下接种的肿瘤中通过FACS分选出5种不同的小鼠黑色素瘤细胞系，每种细胞系均设置未处理与RAFi/MEKi处理两个条件，每个条件下设置3个生物学重复（总计30个样本）