Gene Expression Profiling Provides Insights into the Moleculor Mechanism of FGFR2+ Fibrocyte-to-Cancer Associated Fibroblast Differentiation in Esophageal Squamous Cell Carcinoma

To obtain an accurate overview of changes occurring during fibrocyte-to-cancer associated fibroblast differentiation in human esophageal squamous cell carcinoma (ESCC), we isolated circulating FGFR2+ fibrocytes and their paired FGFR2+ cancer associated fibroblasts (CAFs) from ESCC patients and compared their gene expression profiles by high-throughput RNA Sequencing. On average, about 55.08 million reads were obtained after eliminating low quality reads and 52.76 million reads (95.79%) were aligned to the human mRNA reference sequences. Transcripts corresponding to a total of 1,016 and 409 genes were found to be upregulated or downregulated at least two-fold in the combined set of two CAF sample pools, respectively. Furthermore, we identified the potential master regulators of CAF-specific extracelluar factors by motif enrichment analysis and addressed the molecular mechanisms underlying CAF differentiation. Overall design: Whole transcriptome profiles of ESCC-derived FGFR2+ fibrocytes and CAFs were generated by deep sequencing using Illumina GAIIx. FGFR2+ fibrocytes were isolated from blood samples of 9 ESCC patients, while, FGFR2+ CAFs were isolated from marched ESCC tissues. Sample pooling strategy was employed to reduce the effects of individual variation. Samples (total RNA) were pooled into two groups: group 1 combined 5 samples of fibrocytes (Fbc-1) and corresponding paired CAFs (CAF-1), while group 2 combined 4 samples of fibrocytes (Fbc-2) and marched CAFs (CAF-2).

为了精准解析人类食管鳞状细胞癌（human esophageal squamous cell carcinoma, ESCC）中纤维细胞向癌症相关成纤维细胞（cancer associated fibroblast, CAF）分化过程中的分子变化，我们从ESCC患者体内分离出循环FGFR2阳性纤维细胞，以及其配对的FGFR2阳性CAFs，并通过高通量RNA测序（high-throughput RNA Sequencing）比较二者的基因表达谱。经质控去除低质量读数后，平均获得约5508万条原始读数，其中5276万条（占比95.79%）可比对至人类mRNA参考序列。在两个CAF样本混合池的合并数据集中，分别有1016个和409个基因的转录本呈现至少2倍的上调或下调表达。此外，我们通过基序富集分析（motif enrichment analysis）鉴定了CAF特异性细胞外因子的潜在主调控因子，并阐明了CAF分化的潜在分子机制。本研究的整体实验设计如下：采用Illumina GAIIx测序平台进行深度测序，获取ESCC来源的FGFR2阳性纤维细胞与CAFs的全转录组表达谱。FGFR2阳性纤维细胞分离自9名ESCC患者的血液样本，而FGFR2阳性CAFs则分离自匹配的ESCC组织样本。为降低个体差异的影响，本研究采用样本混合策略：将总RNA样本分为两组，第一组混合5份纤维细胞样本（Fbc-1）及其对应的配对CAF样本（CAF-1），第二组混合4份纤维细胞样本（Fbc-2）及匹配的CAF样本（CAF-2）。