Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Summary

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Abstract

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes ^1-3.

The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of α-smooth muscle actin (α-SMA) expression^4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues ⁶. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.

Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis ^7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.

In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance ¹¹.

We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Protocol

1. Isolation of primary cultured human normal mammary fibroblasts

Experimental procedures for isolating primary cultured human normal mammary fibroblasts are outlined in Fig. 1A.

1. Prepare the cell dissociation buffer, as described previously¹²: Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal calf serum (FCS), penicillin-streptomycin (200 units/ml), collagenase type I (1 mg/ml) and hyaluronidase (125 units/ml).
2. Wash the breast tissue dissected from a reduction mammoplasty (~0.5 gram) several times in phosphate buffered saline (PBS). Mince the tissue into small fragments (<1.5 mm³) using ste....

Discussion

The lack of CAF-specific markers and the level of heterogeneity observed amongst CAFs render the characterisation of this cell type a challenge in itself. Studying CAFs in vitro has also been hindered by the additional complication that these cells senesce and stop proliferating when cultured for a long period. Our previous attempt to directly immortalise primary CAFs using a retroviral hTERT cDNA construct was unsuccessful. Therefore, to further investigate the tumour promoting properties of these cells, we dev.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Invitrogen	61965-026
Fetal calf serum	GIBCO, by Life Technologies	10270
Penicillin-streptomycin	Invitrogen	15140-122
Collagenase type I	Sigma-Aldrich	C0130-1G
hyaluronidase	Sigma-Aldrich	H4272
Vimentin (V9) antibody	Novocastra Laboratories	NCL-L- VIM-V9
Tenascin C (BC-8) antibody			a gift from Dr. Luciano Zardi
α-SMA-Cy3 (1A4) antibody	Sigma-Aldrich	C6198
Prolyl-4-hydroxylase	Dako	M0877
(5B5) antibody
Collagen type1 1A antibody	Sigma-Aldrich	HPA011795
Pan-cytokeratin antibody	Sigma-Aldrich	C5992
Fibronectin antibody	BD Biosciences	610077
S100A4/FSP-1 (fibroblast- specific protein-1) antibody	Dako	A5114
Fibroblast surface protein (clone 1B10) antibody	Abcam	ab11333
MSCV-IRES-GFP construct			Request to the the authors
pBabe-puro construct			Purchase from Addgene
Puromycin	Sigma-Aldrich	P8833
DAPI	Sigma-Aldrich	D9564
15 ml conical tube	Corning	430766
Nude mouse	Taconic	NCRNU-F	Female NCr nude
C3H/10T1/2 cells	ATCC		CCL-226