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

Summary

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFRα as a surface marker.

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^1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker α-SMA³. 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 microenvironment⁴. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion^5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages⁹. 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 microenvironment¹⁰. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture^11,12 . 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)^13,14. 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^9,15 . 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 sorting¹⁶. 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.

Protocol

1. Dissecting Mammary or Skin Tissue from Mice

1. Before dissecting the desired tissue from mice, prepare the following supplies and reagents:
   
   Supplies:
   
   • Surgery tools (washed with detergent and then dipped in 70% ethanol).
   • Styrofoam surface to rest the mouse on during dissection.
   • Pins or needles to hold mice during dissection.
   • Glass jar with lid for digestion, containing magnetic stir bar (autoclaved).
   • Water bath at 37 °C, containing a submersible magnetic stirrer and enough water to submerge the digestion jar, while resting on the stir plate.
   
   

Results

Using PDGFRα as a marker for fibroblasts results in isolation of highly enriched populations of tissue fibroblasts. The level of purity after sorting was 99%, as quantified by post-sort analysis (Figure 2A). Estimating the percentage of contaminating non-fibroblast cells by the relative expression of cell-specific control genes (Figure 2B) typically shows 0.1-0.6% contamination. This level of purity allows high quality transcriptome profiling of isolated fibroblasts⁹.

Discussion

While experiments performed in tissue culture can be informative and suggest functional principles that can be verified in vivo, it is known that large changes occur in gene expression of cells in culture^11,12 . In order to avoid a tissue culture step when profiling gene expression in fibroblasts, we developed a protocol that allows isolation of normal, as well as cancer-associated fibroblasts from fresh mouse or human tissue. This protocol was successfully applied with skin, pancreas and breast tissu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Gibco	41965
PBS	Biological Industries	02-023-1A
Collagenase II	Worthington	LS4176
Collagenase IV	Worthington	LS4188
Deoxyribonuclease	Worthington	LS2007
PharmLyse	BD	555899
Cell strainer 70 μm	SPL	93070
Purified anti-mouse CD16/CD32	BD Pharmingen	553142
Via probe (7AAD)	e-Bioscience	00-6993-50
Anti-mouse CD140a-PE (PDGFRa)	e-Bioscience	12-1401-81
Anti-mouse F4/80- FITC	Cederlane	CL8940F
DMEM w/o Phenol Red	Gibco	31053
Collagen Type I	BD Biosciences	354236