Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Abstract

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.

Mechanical signals regulate and activate SNAIL1 protein to control the fibrogenic response of cancer-associated fibroblasts

“…In mouse PyMT breast tumor samples, only 5% of cells are fibroblastic (Sharon et al, 2013). The majority are tumor cells or inflammatory cells.…”

PTEN Loss Promotes Intratumoral Androgen Synthesis and Tumor Microenvironment Remodeling via Aberrant Activation of RUNX2 in Castration-Resistant Prostate Cancer

“…To investigate the molecular mechanisms by which ABA treatment induces TME remodeling, we examined the effect of ABA on expression of chemokine and cytokine genes in stroma of prostate tumor in the Pten -knockout mouse model. Using the method reported previously (42), we successfully isolated fibroblasts and macrophages from Pten -knockout prostate tumors in mice treated with or without ABA for a month (Supplementary Fig. S5A).…”

Hypoxia and Transforming Growth Factor β Cooperate to Induce Fibulin-5 Expression in Pancreatic Cancer

“…FACs Isolation of TAFs-Isolation of TAFs from fresh mouse PDA was done as described previously (43). Briefly, sizeable tumors were dissected from KIC mice and minced manually using a sterile razor blade.…”

“…We used PDGF receptor ␣ (PDGFR␣) as a marker to specifically select for fibroblasts (43). Bright-field images of these TAFs revealed a spindle-like morphology typical of fibroblasts (Fig.…”