Differential Activity of MAPK signalling Defines Fibroblast Subtypes in Pancreatic Cancer (Spatial Transcriptomics). Differential Activity of MAPK signalling Defines Fibroblast Subtypes in Pancreatic Cancer (Spatial Transcriptomics)

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: We performed spatial RNAseq profiling with Visium on 4 PDAC tissue samples (2 Basal-like and 2-classical) to check the presence and the spatial localization of our described mapCAF phenotype in the tissue

成纤维细胞异质性（fibroblast heterogeneity）在多种癌症中逐渐受到关注。鉴于其在疾病进展中的关键贡献，解析成纤维细胞异质性对于设计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。目前，CAF表型是否由PDAC细胞谱系差异化决定仍有待阐明。本研究揭示了丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路在定义PDAC的CAF表型中发挥的重要作用。我们证实，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的肌成纤维细胞分化。我们整合了小鼠模型经扰动后的转录反应单细胞谱，以及人类组织的细胞谱与空间谱，从而定义了MAPK高表达CAF（MAPKhigh CAF, mapCAF）表型。研究表明，该表型与基底样肿瘤细胞相关，并伴随CD8+ T细胞频率降低。除了升高的MAPK活性外，该mapCAF表型还具备TGF-β信号、缺氧应答特征以及免疫调节基因程序的特征。此外，mapCAF特征在多种癌症类型的肌成纤维细胞CAFs中富集，并与黑色素瘤患者对免疫检查点抑制剂的应答降低相关。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的潜在策略。总体实验设计：我们采用Visium平台对4例PDAC组织样本（2例基底样亚型、2例经典型亚型）开展空间RNA-seq分析，以验证本研究描述的mapCAF表型在组织中的存在及其空间定位。