Transcriptional profiling of distinct fibroblast populations in the pancreatic tumor microenvironment. Transcriptional profiling of distinct fibroblast populations in the pancreatic tumor microenvironment

Pancreatic stellate cells are thought to be the predominant source of cancer-associated fibroblasts (CAFs) in pancreatic cancer. We developed a mouse model which allows us to track and analyze stellate cells and stellate cell-derived CAFs in vivo during pancreatic tumorigenesis for the first time. We find that stellate cells in fact give rise to a minority of all CAFs. Here, we have used lineage reporters to isolate stellate cell-derived and non-stellate cell-derived CAFs and compared them by RNA-seq. Overall design: Rosa-mTmG; Fabp4-Cre mice at 8 weeks of age were injected with FC1199 murine pancreatic cancer cells orthotopically. After 4 weeks, tumors were harvested, single cell suspensions generated, and FACS performed to isolate GFP+ (stellate cell-derived) and Tomato+ (non-stellate cell-derived) CAFs. RNA was isolated and RNA-seq performed on both cell populations.

既往认为胰腺星状细胞（pancreatic stellate cells）是胰腺癌中癌相关成纤维细胞（cancer-associated fibroblasts, CAFs）的主要来源。本研究首次构建了可在胰腺肿瘤发生过程中活体追踪并分析星状细胞及其分化来源癌相关成纤维细胞的小鼠模型。研究发现，实际上星状细胞仅能分化得到全部癌相关成纤维细胞中的一小部分。本研究借助谱系示踪标记物，分离得到星状细胞来源与非星状细胞来源的癌相关成纤维细胞，并通过RNA测序（RNA-seq）对两组细胞进行比较分析。实验整体设计如下：选取8周龄的Rosa-mTmG; Fabp4-Cre小鼠，原位注射FC1199小鼠胰腺癌细胞。4周后处死小鼠并获取肿瘤组织，制备单细胞悬液，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离得到绿色荧光蛋白（GFP）阳性（星状细胞来源）与Tomato阳性（非星状细胞来源）的癌相关成纤维细胞。提取两组细胞的总RNA并开展RNA测序（RNA-seq）。