Sharon Yoray, Alon Lina, Glanz Sarah, Servais Charlotte, Erez Neta
Department of Pathology, Sackler School of Medicine, Tel Aviv University.
J Vis Exp. 2013 Jan 14(71):e4425. doi: 10.3791/4425.
Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker α-SMA. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture. To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS). Our approach relies on utilizing PDGFRα as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFRα is abundantly expressed by both normal fibroblasts and CAFs. This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to α-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.
癌症相关成纤维细胞（CAFs）是许多癌症（尤其是乳腺癌）肿瘤基质中最主要的细胞类型，它们的显著存在通常与预后不良相关。CAFs是基质成纤维细胞的一个活化亚群，其中许多表达肌成纤维细胞标志物α-SMA。CAFs起源于局部组织成纤维细胞以及募集到正在形成的肿瘤中的骨髓来源细胞，并在肿瘤微环境的影响下呈现CAF表型。研究表明，CAFs通过促进肿瘤细胞增殖、血管生成和侵袭的信号传导来促进肿瘤的起始、生长和进展。我们证明，CAFs从最早的肿瘤前阶段开始，通过介导促肿瘤炎症来增强肿瘤生长。尽管越来越多的证据表明CAFs在促进肿瘤生长中起关键作用，但由于缺乏CAF特异性标志物以及这些细胞的巨大异质性，研究CAFs一直是一项持续的挑战，许多亚型共存于肿瘤微环境中。此外，在体外研究成纤维细胞受到这样一个事实的阻碍，即它们的基因表达谱在组织培养中经常发生改变。为了解决这个问题，并允许对来自新鲜小鼠和人类组织的成纤维细胞进行无偏倚的基因表达谱分析，我们基于先前的荧光激活细胞分选（FACS）方案开发了一种方法。我们的方法依赖于利用血小板衍生生长因子受体α（PDGFRα）作为表面标志物，从新鲜小鼠和人类组织中分离成纤维细胞。正常成纤维细胞和CAFs都大量表达PDGFRα。这种方法允许分离出纯的正常成纤维细胞和CAFs群体，包括但不限于α-SMA+活化肌成纤维细胞。然后，分离出的成纤维细胞可用于表征和比较肿瘤发生过程中CAFs中发生的基因表达演变。事实上，我们和其他人报道了通过细胞分选分离的成纤维细胞的表达谱分析。该方案已成功用于从皮肤、乳腺、胰腺和肺组织中分离和分析高度富集的成纤维细胞群体。此外，我们的方法还允许培养分选后的细胞，以便进行功能实验，并避免肿瘤细胞的污染，而肿瘤细胞污染在试图培养CAFs时常常是一个很大的障碍。
