Differential Activity of MAPK signalling Defines Fibroblast Subtypes in Pancreatic Cancer (RNA-Seq). Differential Activity of MAPK signalling Defines Fibroblast Subtypes in Pancreatic Cancer (RNA-Seq)

Fibroblast heterogeneity is increasingly recognised across cancer conditions. Given their important contribution to disease progression, mapping fibroblasts’ heterogeneity is critical to devise effective anti-cancer therapies. Cancer-associated fibroblasts (CAFs) represent the most abundant cell population in pancreatic ductal adenocarcinoma (PDAC). Whether CAF phenotypes are differently specified by PDAC cell lineages remains to be elucidated. Here, we reveal an important role for the MAPK signalling pathway in defining PDAC CAF phenotypes. We show that epithelial MAPK activity promotes the myofibroblastic differentiation of CAFs by sustaining the expression and secretion of TGF-β1. We integrate single-cell profiling of post-perturbation transcriptional responses from mouse models with cellular and spatial profiles of human tissues to define a MAPKhigh CAF (mapCAF) phenotype. We show that this phenotype associates with basal-like tumour cells and reduced frequency of CD8+ T cells. In addition to elevated MAPK activity, this mapCAF phenotype is characterized by TGF-b signalling, hypoxia responsive signatures, and immunoregulatory gene programs. Furthermore, the mapCAF signature is enriched in myofibroblastic CAFs from various cancer conditions and correlates with reduced response to immune checkpoint inhibition in melanoma Altogether, our data expand our knowledge on CAF phenotype heterogeneity and reveal a potential strategy for targeting of myofibroblastic CAFs in vivo. Overall design: To identify molecular features predictive of cellular dependency on MAPK signalling, we treated 6 human cell lines and 5 patient-derived organoids (PDOs) with increasing doses of the MEK1/2 inhibitor trametinib (MEKi). To understand whether transcriptional changes from the scRNA-seq experiment could be observed in vitro. we used at first mPSCs exposed to conditioned media from both basal-like and classical cancer cell lines. And subsequently, mPSCs pre-treated for two days with TGF-β1 to attenuate the polarization towards iCAFs and exposed for 24 hours to either basal-like or classical CM. We performed gene expression profile with RNA-seq

成纤维细胞异质性（fibroblast heterogeneity）在多种癌症类型中日益受到学界关注。鉴于其在疾病进展中的关键作用，解析成纤维细胞异质性对于开发高效抗癌治疗方案至关重要。癌症相关成纤维细胞（Cancer-associated fibroblasts, CAFs）是胰腺导管腺癌（Pancreatic Ductal Adenocarcinoma, PDAC）中丰度最高的细胞群体。胰腺导管腺癌细胞谱系是否会差异化塑造CAF表型，这一问题仍有待阐明。本研究揭示了MAPK信号通路在调控PDAC来源CAF表型塑造中的核心作用。我们发现上皮细胞的MAPK活性通过维持转化生长因子β1（Transforming Growth Factor-β1, TGF-β1）的表达与分泌，促进CAFs向肌成纤维细胞分化。我们整合了小鼠模型扰动后转录应答的单细胞测序数据，以及人类组织的细胞与空间转录组特征，从而定义了高MAPK活性CAF（MAPKhigh CAF, mapCAF）表型。我们证实该表型与基底样肿瘤细胞密切相关，并伴随CD8阳性T细胞浸润频率降低。除MAPK活性升高外，该mapCAF表型还具有TGF-β信号通路激活、缺氧应答特征以及免疫调节基因程序等标志性特征。此外，mapCAF特征基因集在多种癌症类型的肌成纤维细胞CAFs中富集，且与黑色素瘤患者对免疫检查点阻断治疗的应答率降低呈显著相关。综上，本研究拓展了我们对CAF表型异质性的认知，并揭示了一种在体内靶向肌成纤维细胞CAFs的潜在治疗策略。

实验整体设计：为鉴定预测细胞对MAPK信号通路依赖的分子特征，我们用梯度浓度的MEK1/2抑制剂曲美替尼（trametinib, MEKi）处理了6株人类细胞系与5例患者来源类器官（Patient-derived Organoids, PDOs）。为验证单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）实验所得的转录变化可在体外复现，我们首先将mPSCs置于基底样与经典型癌细胞系的条件培养基（conditioned media, CM）中培养。随后，我们用TGF-β1预处理mPSCs两天以削弱其向炎症相关CAFs（inflammatory CAFs, iCAFs）极化的趋势，再将其分别暴露于基底样或经典型癌细胞系的条件培养基CM中培养24小时。我们通过RNA测序（RNA-seq）完成了基因表达谱分析。