Prrx1 is a Master Transcription Factor of myofibroblastic Cancer-Associated Fibroblasts [Murine_Fibroblast_RNA_seq]

Although cancer associated fibroblasts (CAF) play a critical role in cancer progression, their identities are still unknown. Because cells' own identity is determined by core regulatory circuit (CRC) comprising master transcription factors (TFs), it is critical to find CAF's master TF. Therefore, we attempt to find master TF of CAF through the extensive screening of single cell RNA-seq database of cancer tissues, and discovered Prrx1, master regulator of embryonic mesenchymal cells, as candidate for master TF of CAF. Prrx1 was abundantly expressed in CAFs with significant correlation with unfavorable clinical outcomes. Furthermore, Prrx1 in fibroblasts dramatically enhanced tumor progression and metastasis in fibroblast-specific Prrx1 inducible mice. On the other hand, tumor formation was severely inhibited in conditional Prrx1 knock-down mice. Finally, we confirmed that Prrx1 is a master TF of CAF and normal fibroblast executing wound healing program using ChIP-SEQ, CRC modeling, and in vivo validation. Furthermore we also revealed that targeting Prrx1 in fibroblast alone induced complete remission of chemotherapy-resistant cancer in preclinical mouse experiments. Overall design: First, three murine fibroblasts such as mouse embryonic fibroblast, wound healing fibroblast, and MMTV CAF are obtained from C57BL/6, FVB strains. The ChIP-seq were performed in these fibroblasts using H3K27ac, H3K4me3, and Prrx1 antibody and the RNA-seq also were performed. Second, single cells of murine wound tissue obtained from BALB/c were performed using 10X platform. Third, nine ex vivo cultured human CAFs isolated from advanced colorectal cancer tissue were established. we performed H3K27ac ChIP-seq and RNA-seq from these human CAFs.

尽管癌症相关成纤维细胞（cancer associated fibroblasts, CAF）在癌症进展中发挥关键作用，但其具体身份仍未明确。由于细胞的自身身份由包含核心转录因子（master transcription factors, TFs）的核心调控回路（core regulatory circuit, CRC）所决定，因此鉴定CAF的核心转录因子至关重要。为此，我们通过对癌症组织的单细胞RNA测序（single cell RNA-seq）数据库开展大规模筛选，尝试寻找CAF的核心转录因子，并发现胚胎间充质细胞的主调控因子Prrx1可作为CAF核心转录因子的候选靶点。Prrx1在CAFs中高表达，且与不良临床结局显著相关。进一步实验显示，在成纤维细胞特异性诱导Prrx1表达的小鼠模型中，成纤维细胞内的Prrx1可显著促进肿瘤进展与转移；而在条件性敲低Prrx1的小鼠中，肿瘤形成则受到严重抑制。最后，我们通过染色质免疫沉淀测序（ChIP-SEQ）、核心调控回路建模以及体内验证实验，证实Prrx1是CAF以及执行伤口愈合程序的正常成纤维细胞的核心转录因子。此外，我们还在临床前小鼠实验中发现，仅靶向成纤维细胞中的Prrx1即可使化疗耐药性癌症实现完全缓解。

实验整体设计如下：第一，从C57BL/6、FVB品系小鼠中分离得到三种小鼠成纤维细胞，即小鼠胚胎成纤维细胞、伤口愈合成纤维细胞以及MMTV来源的癌症相关成纤维细胞；使用H3K27ac、H3K4me3抗体及Prrx1抗体对这些成纤维细胞进行ChIP-seq实验，同时开展RNA-seq检测。第二，采用10X平台对来自BALB/c小鼠的伤口组织进行单细胞测序。第三，构建9株从晚期结直肠癌组织中分离的体外培养人源CAFs，并对这些人源CAFs进行H3K27ac ChIP-seq与RNA-seq检测。