1CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, University of Manchester, 2Atopy Research Center, Juntendo University
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Polanska, U. M., Acar, A., Orimo, A. Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts. J. Vis. Exp. (56), e3201, doi:10.3791/3201 (2011).
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) expression4,5. 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.
1. Isolation of primary cultured human normal mammary fibroblasts
Experimental procedures for isolating primary cultured human normal mammary fibroblasts are outlined in Fig. 1A.
2. Generation of GFP-labelled, puromycin-resistant, immortalised human normal mammary fibroblasts
3a. Coinjection of human mammary fibroblasts with breast carcinoma cells into an immunodeficient mouse
Experimental procedures for generating exp-CAFs are illustrated in Fig. 1Ba.
3b. Injection of human mammary fibroblasts into an immunodeficient mouse
*Note that the inoculated fibroblasts will form the fibroblastic tissue but not tumour under the skin.
4a. Dissection of tumour xenograft
4b. Dissection of fibroblast xenograft
5. Preparation of primary cultured cells from xenografts
6. Isolation of puromycin-resistant cells in culture
7a. Isolation of exp-CAF2 cells in culture
To further boost the activated myofibroblastic phenotype of CAFs, mix the 42-day-old exp-CAF1 cells with MCF-7-ras cells and inject them again subcutaneously into a nude mouse for additional periods of 200 days. Dissect and dissociate the tumour xenograft into a single-cell suspension and culture the cells in a 15 cm petri dish with 10% FCS-DMEM in the presence of puromycin (1 μg/ml), as indicated in P6.1. Propagate the cells until confluent (8-10 days). Store the cells at -80°C using freezing medium. The resulting puroR cells are named 242-day-old exp-CAF2 cells (Fig. 1Ba).
7b. Extraction of control fibroblast-2 cells in culture
To generate control fibroblasts against exp-CAF2 cells, inject 42-day-old control fibroblast-1 cells alone without carcinoma cells subcutaneously into a nude mouse additionally for 200 days. Dissect and dissociate the fibroblastic tissue into a single cell suspension and culture the cells in a 15 cm petri dish with 10% FCS-DMEM in the presence of puromycin (1 μg/ml) as indicated in P6.1. Propagate the cells until confluent (3-4 weeks). Store the cells at -80°C using freezing medium. The resulting puroR cells are named control fibroblast-2 cells (Fig. 1Bb).
8. Representative Results:
Control fibroblast-2 and exp-CAF2 cells, which have been extracted from breast tumour xenografts, stained strongly positive for mesenchymal markers, including human-specific vimentin, prolyl-4-hydroxylase, collagen 1A, fibronectin, S100A4, and fibroblast surface protein (Fig. 2A)11, indicating human origin and mesenchymal nature of these cells. In contrast, the cytokeratin, a marker for epithelial cells, was not stained in these GFP+fibroblasts (Fig 2B). These findings therefore suggest that the extracted exp-CAF2 and control fibroblast-2 cells have originated from the parental human mammary fibroblasts initially introduced into mouse xenografts.
Importantly, a higher proportion of exp-CAF2 cells stained positive for α-SMA and extracellular matrix glycoprotein tenascin-C5, both of which are markers of myofibroblasts compared to 42-day-old exp-CAF1 and control fibroblast-2 cells (Fig. 2C, D)11. These data indicate that resident human mammary fibroblasts progressively evolve into CAF myofibroblasts within tumour xenografts.
Figure 1 Schematic representation of isolation of human mammary fibroblasts. A) The reduction mammoplasty tissue was minced using sterile razor blades (P1.2) and transferred into a 15 ml conical tube (P1.3). The small tissue fragments were digested in the cell dissociation buffer (P1.4) and prepared for culture in vitro (P1.5-7). To immortalise the isolated primary human mammary fibroblasts, a retroviral pMIG (MSCV-IRES-GFP) vector, expressing both hTERT and GFP, was introduced, and the resulting GFP-positive cells were sorted using flow cytometry (P2.1). A retroviral pBabe-puro vector encoding a puromycin resistance gene was then introduced into these cells. Upon the puromycin treatment, GFP-labelled (GFP+) puromycin-resistant (PuroR), immortalised human mammary fibroblasts were isolated (P2.2).
Ba) To generate exp-CAFs, GFP+puroR immortalised human mammary fibroblasts were coinjected with MCF-7-ras breast carcinoma cells subcutaneously into an immunodeficient nude mouse (P3a). The tumour xenograft was resected at 42 days after implantation (P4a) and dissociated into a single-cell suspension (P5). These cells were then cultured in vitro in the presence of puromycin to eliminate any contaminating carcinoma cells and mouse stromal cells (P6). The resulting puromycin-resistant cells were termed experimentally generated CAF1 (exp-CAF1) cells. These cells, resected 42 days post-implantation, were once again mixed with MCF-7-ras cells and implanted subcutaneously into a host mouse as before (P7a). The resulting tumour was allowed to grow for additional periods of 200 days, then dissected, dissociated, and cultured in the presence of puromycin. The isolated puromycin-resistant cells were termed exp-CAF2 cells (242-day-old).
Bb)To isolate control cells against exp-CAFs, GFP+puroR immortalized human mammary fibroblasts were injected subcutaneously into a nude mouse as pure cultures without MCF-7-ras cells (P3b). The fibroblastic tissue, dissected 42 days post-implantation (P4b), was dissociated into single-cell suspensions (P5) and puromycin-resistant cells, named control fibroblast-1 cells, were isolated as described earlier (P6). These fibroblasts were once again implanted subcutaneously into a nude mouse without MCF-7-ras cells additionally for 200 days (P7b). The fibroblastic tissue was dissected, dissociated, and cultured in the presence of puromycin. The isolated puromycin-resistant cells were termed control fibroblast-2 cells (242-day-old).
Figure 2 Exp-CAFs and the control fibroblasts originate from the parental human mammary fibroblasts. (A) Immunofluorescence analyses of control fibroblast-2 (control f.) and exp-CAF2 cells. The both cell types stain positive for mesenchymal markers (red), including human vimentin, prolyl-4-hydroxylase, collagen 1A, fibronectin, S100A4, and fibroblast surface protein. (B) In contrast, pan-cytokeratin, a marker for epithelial cells, is not detected in exp-CAF2 cells expressing GFP (green). Cell nuclei are stained with 4'-6-Diamidino-2-phenylindole (DAPI) (blue). Scale bar, 50 μm (referred from Kojima et al.11)
(C) Immunofluorescence of exp-CAF2 cells and control fibroblast-2 cells (control f.) using antibodies against α-SMA (red) or tenascin-C (TN-C) (red). Cell nuclei are stained with DAPI (blue). Scale bar, 50 μm. (D) 48% of exp-CAF2 cells stain positive for α-SMA, whereas 14% of 42-day-old exp-CAF1 and 2.5% of the control fibroblast-2 cell populations are positive for α-SMA. (referred from Kojima et al.11)
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 developed a method to generate immortalised CAFs from human mammary fibroblasts using a coimplantation tumour xenograft model. Primary human mammary fibroblasts, extracted from the reduction mammoplasty tissue, were immortalised before being injected with breast carcinoma cells into mice. The human fibroblasts were then isolated from the resulting tumour xenografts (see Fig. 1Ba for details). Importantly, the human mammary fibroblasts increasingly evolved into tumour-promoting myofibroblastic CAFs during the course of tumour progression. We indeed observed that 242-days-old exp-CAF2 cells show a far greater tumour-promoting myofibroblastic ability compared to 42- or 70-days-old exp-CAF1 cells and control fibroblast-2 cells11. Such tumour-promoting myofibroblastic ability of exp-CAF2 cells, which is also observed in CAFs prepared from breast cancer patients, depends on the establishment of autocrine signalling loops mediated by TGF-β and SDF-1 cytokines11. Neither gene transfer or cell fusion between carcinoma cells and fibroblasts, mediates the phenotypes of CAFs generated in our xenograft model11. Taken together, these observations suggest that the xenograft-derived exp-CAFs serve as a useful tool for studying CAFs present within human breast carcinomas. The described experimental protocol may also be extended for isolating various CAFs originating from other types of human carcinomas.
No conflicts of interest declared.
We thank Dr. Robert A. Weinberg (Whitehead Institute for Biomedical Research, Cambridge) for generous support and supervision of this work and Mr. Kieran Mellody (University of Manchester, Manchester) for critical editing of this manuscript. This project was supported by Research UK (CR-UK) grant number C147/A6058 (A.O.).
|Fetal calf serum||GIBCO, by Life Technologies||10270|
|Collagenase type I||Sigma-Aldrich||C0130-1G|
|Vimentin (V9) antibody||Novocastra Laboratories||
|Tenascin C (BC-8) antibody||
a gift from
|α-SMA-Cy3 (1A4) antibody||Sigma-Aldrich||C6198|
|Collagen type1 1A antibody||Sigma-Aldrich||HPA011795|
|Fibronectin antibody||BD Biosciences||610077|
S100A4/FSP-1 (fibroblast-specific protein-1) antibody
Fibroblast surface protein(clone 1B10) antibody
|MSCV-IRES-GFP construct||Request to the the authors|
|15 ml conical tube||Corning||430766|
|Nude mouse||Taconic||NCRNU-F||Female NCr nude|