Cancer associated fibroblasts

Opinion

‘Cancer associated fibroblasts’ – more than meets the eye Shalom Madar*, Ido Goldstein*, and Varda Rotter Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 76100, Israel

Cancer associated fibroblasts (CAFs) are a subpopulation of cells that reside within the tumor microenvironment and promotes the transformation process by encouraging tumor growth, angiogenesis, inflammation, and metastasis. CAF-specific proteins serve as both prognostic markers and targets for anticancer drugs. With the growing interest in CAFs, several controversial issues have been raised, including the genomic landscape of these cells, the identity of specific markers, and their cell of origin. Here, we tackle these debated issues and put forward a new definition for ‘CAF’ as a cell ‘state’ rather than a cell type. We hope this conceptualization can resolve the ongoing discrepancies revolving around CAF research and aid in designing better anti-cancer treatment strategies. The tumor microenvironment and cancer associated fibroblasts The tumor microenvironment (see Glossary) can be crudely defined as the sum of all the non-transformed elements residing within or in the vicinity of a tumor, including, for example, nearby tissue, vasculature, and immune cells. One well-studied and populous component of the tumor microenvironment is cancer associated fibroblasts (CAFs), a subpopulation of the tumor–stroma milieu that in many cases dictates tumor outcome [1]. Normal fibroblasts are elongated cells residing in connective tissues and are responsible for the synthesis and turnover of the extracellular matrix (ECM). Fibroblasts control and support normal tissue homeostasis and participate in a plethora of biological processes such as wound healing and senescence. Unlike normal fibroblasts, CAFs either reside within the tumor margins or infiltrate the tumor mass, and facilitate the transformation process [2]. Other than their location, the exact definition of CAFs is elusive for several reasons. First, given that these cells are genetically stable and not the cause of disease, their definition seems to depend on the presence of the adjacent tumor. Moreover, CAFs share several markers with other inhabitants of the stroma, such as myofibroblasts, myoepithelial cells, muscle cells, mesenchymal stem cells (MSCs), and endothelia [3]. Thus, identifying and isolating the CAF population has proven to be an arduous task. Another debated issue is the origin Corresponding authors: Madar, S. ([email protected]); Rotter, V. ([email protected]) * These authors are equal contributors. Keywords: stroma; microenvironment; CAFs; inflammation; mutation; stem cells. 1471-4914/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2013.05.004

of CAFs; many origins have been proposed, ranging from normal fibroblasts and MSCs to trans-differentiated epithelial and endothelial cells [1,4]. These challenges are discussed in greater detail below, and by exploring the facts gathered to date, we reconstruct and present a novel definition of CAFs – ‘CAF state’ – which we believe will help to resolve some of the debate surrounding these cells. CAF phenotype Intrinsic CAF phenotype The phenotype of CAFs isolated from malignant tissues is distinct from that of normal fibroblasts, including a more rapid proliferation rate, enhanced collagen production, secretion of growth factors and other ECM modulators, and the activation of unique expression programs [5–7]. In multiple instances, CAFs have been shown to proliferate faster than normal fibroblasts. For example, CAFs extracted from oral carcinoma exhibit an increased proliferation rate and mitosis ability compared with normal fibroblasts [8], and similarly, CAFs from cutaneous squamous cell carcinoma display enhanced proliferation and migration compared with their normal counterparts [9]. Extrinsic CAF phenotype – tumor enhancing effects Apart from an increased division rate, the gold standard for identifying CAFs is their capacity to promote cancer progression in vivo, usually when coinjected with tumor cells or when recruited to the tumor site [10–12]. Interestingly, irradiated fibroblasts, or even the media in which they were grown, are sufficient to enhance the tumorigenicity of breast cancer grafts, suggesting that a transient effect induced by CAF-secreted factors is at play [13]. Among the secreted factors that mediate the tumor promoting effect of CAFs is CXCL12 (SDF1-a), a chemokine that can induce angiogenesis and enhance the proliferative capacity of cancer cells [14]. Tumor growth factor b (TGF-b) signaling in fibroblasts has also been reported to modulate Glossary Cancer associated fibroblasts (CAFs): fibroblasts residing within the tumor mass with the ability to promote tumorigenic features. Epithelial-to-mesenchymal transition (EMT): a differentiation process which converts cells of epithelial origin into mesenchymal cells. EMT occurs in normal development and wound healing and is harnessed by cancer cells mainly as a means to acquire migratory capacity – a vital step towards metastasis. Microenvironment/tumor stroma: all the non-transformed components in the vicinity of the tumor. These elements include cells of the immune system (such as macrophages and lymphocytes), blood vessel cells, fibroblasts, myofibroblasts, mesenchymal stem cells, adipocytes, and the ECM. Trends in Molecular Medicine, August 2013, Vol. 19, No. 8

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Opinion the oncogenic potential of adjacent epithelia [15], a connection between these two secreted factors has been observed: CXCL12 secreted from CAFs can act via TGF-bregulated CXCR4 in adjacent epithelial cells [16]. The epithelial cells, in turn, activate the AKT pathway and elicit tumorigenesis. Furthermore, overexpression of TGFb and hepatocyte growth factor (HGF) in stromal cells, followed by their coinjection with breast organoids, results in an increased tumor incidence compared with coinjection of organoids and normal fibroblasts [17]. The ECM modulators matrix metalloproteinases (MMPs) are also governed by the tumor stroma. CAFs obtained from breast cancer patients display elevated levels of MMP1 compared with normal fibroblasts, whereas the latter upregulate MMP1 expression when subjected to soluble breast cancer factors [18]. Moreover, MMP1 originating from the stroma has been reported to augment the migration and invasion of breast cancer cells [19]. CAFs can promote tumor drug resistance, for example, CAFs bypass anti-vascular endothelial growth factor (antiVEGF) treatment by activating the platelet-derived growth factor C (PDGF-C) pathway [20]. CAFs were also shown to confer various cancer cell lines with resistance to RAF inhibitors through HGF secretion [21]. Ablation of fibroblast activation protein (FAP)+ stromal cells in mice hampers tumor resistance to therapeutic vaccination [22]. Finally, inhibition of hedgehog signaling in the stroma of mice enabled gemcitabine antitumor effect in a pancreatic cancer model [23]. Recently Luga et al. revealed a novel form of communication between CAFs and tumor cells, whereas CAFs exosomes mobilize autocrine Wnt–PCP signaling to promote breast cancer invasiveness [24]. On a broader scope, by altering the ECM composition CAFs can influence tumor metastasis (Box 1). Finally, CAFs secrete proinflammatory cytokines as they partake in cancer-related inflammation (Box 2). The origin of CAFs Intuitively, CAFs are grasped as normal fibroblasts that have been altered as a result of continuous exposure to cancer cells. Indeed, CAFs often share several similarities with normal fibroblasts [25]. ‘Cell plasticity’ is a term describing the ability of cells to alter their differentiation status [26], and numerous studies suggest that several cell types of both mesenchymal and epithelial origin display some degree of plasticity in the vicinity of tumors and provide an alternative source for CAFs. In general, MSCs assume several roles during cancer progression, such as differentiating into blood vessel supporting cells [27] or recruiting endothelial precursor cells [14]. Recent reports support the notion that MSCs are also a possible source for CAFs. In gastrointestinal cancer female patients who received bone marrow transplants from male donors, a significant proportion of a smooth muscle actin (aSMA) expressing CAFs were of donor origin, most likely recruited from the donor bone marrow [28]. Using a mouse model, Ishii et al. reported that transplanted bone marrow cells can be detected in the margins of a grafted tumor, where they differentiate into endothelial cells and myofibroblasts [29]. Similar mouse models 448

Trends in Molecular Medicine August 2013, Vol. 19, No. 8

Box 1. The involvement of CAFs in metastasis formation The tumor–stroma interface marks a physical barrier that contains the tumor and prevents its spread. During the course of malignancy this barrier is breached by cancer cells that escape the tumor mass and are now free to roam the bloodstream and colonize in a distant site. Several reports allude to the contribution of CAFs to the metastatic process. Olaso et al. documented the presence of intrametastatic aSMA-expressing cells appearing at the early stages of hepatic metastasis derived from B-16 melanoma cells in a mouse model [90]. Labeled CAFs were also reported to be recruited by liver metastases in vivo [10]. The elevated presence of CAFs in metastatic human specimens was also evident by immunostaining in several types of cancer [91]. In another study, melanoma cells obtained from different stages of the disease were co-cultured with normal fibroblasts. The growth rate of early stage cells was attenuated by fibroblasts, whereas metastatic cells were not affected by their presence, indicating a selective pressure imposed by fibroblasts, one that selects for outgrowing metastatic clones [92]. Both tumor cells and hematopoietic cells ‘educate’ CAFs in a secondary site to form a niche for metastasis [93,94]. In these sites, CAFs ‘feed’ the secondary tumor with secreted factors which support its growth [95]. CAFs are inherently equipped with motility and migratory capacities that can be exploited by tumor cells. In a 3D ‘organotypic’ invasion assay, carcinoma cells utilized CAF characteristics in order to invade without the need to undergo EMT [96]. Another study suggests that under harsh hypoxic conditions, tumor cells elevate CXCR4 which allows them to migrate towards a gradient of CAFinduced CXCL12 and escape to a normoxic environment at a distant site [97]. Overall, CAFs seem to be abundant at metastatic tumor sites and promote the transition of in situ tumors towards malignancy by affecting the rate-limiting steps of the process.

revealed that labeled bone marrow cells introduced into tumor-bearing mice constitute 20–25% of the stromal fibroblasts in the vicinity of the tumor [30,31]. CAFs expressing high levels of CD90 (a common MSC marker) were isolated and, despite being tumor-supportive, failed to express other known MSC markers, except for CD24 [32]. Nevertheless, it is plausible that the markers CD90 and CD24 are remnants of formerly undifferentiated MSCs. Finally, human MSCs treated with conditioned media of breast cancer cells exhibit CAF-like behavior including elevated expression of aSMA and CXCL12 and tumor-supportive capabilities in vitro and in vivo [33]. Another plausible source of CAFs is smooth muscle cells (SMCs). Normal prostate stroma is enriched with SMCs expressing high levels of aSMA [34]. This population is thought to disappear during prostate cancer progression and be replaced by CAFs [35]. Prostatic SMCs express high levels of androgen receptor (AR) and are androgen-responsive. By contrast, Wikstrom et al. documented the downregulation of AR in the prostate tumor stroma and therefore hypothesized that SMCs might be the source of CAFs [36]. Myofibroblasts (a term that is sometimes used interchangeably with CAFs) are considered to be ‘activated’ fibroblasts that assume an active role in wound healing as well as in scaring and fibrotic lesions. SchmittGra¨ff et al. suggested that myofibroblasts might acquire a CAF phenotype when the proper stimuli are present [37]. CAFs may originate from recruited and differentiated circulating fibrocytes. Invasive ductal carcinomas of the breast were found to contain fewer fibrocytes but more differentiated aSMA+ fibroblasts than in situ lesions, indicating a possible differentiation of fibrocytes into CAFs within the breast stroma [38].

Opinion Box 2. CAFs and cancer-related inflammation The link between cancer and inflammation was first postulated by Virchow in 1863 who maintained that inflammation predisposes individuals to cancer. Indeed, studies revealed that persistent inflammation lasting over a decade is a major risk factor in various cancers [98]. Recently, several reports have documented a link between CAFs and cancer-related inflammation in which a common pattern emerges. First, the tumor cells secrete CAF-activating factors such as IL-1 and TNF-a [25,73–76]. Following this activation, CAFs initiate a proinflammatory response that includes, among others, IL6 and IL-8 [75,76]. These secreted factors may affect tumor growth in a direct manner or induce inflammation by recruiting components of the immune system [30,73,74]. Interestingly, both normal fibroblasts and CAFs are capable of evoking a proinflammatory response [99]. Accordingly, CAFs produced from liver metastases and normal liver fibroblasts are both able to induce IL-6 [100]. Combined, these results suggest that normal fibroblasts possess the capacity to induce a proinflammatory response, and this capacity is retained after their transition to CAFs, as it becomes beneficial for the evolving tumor.

Cells of epithelial origin are also candidates as suppliers for the CAF population. Carcinomas are the most frequent type of cancer, and although they originate from an epithelial lineage, they might also contribute to the CAF pool. For example, breast cancer cells from a biopsy specimen that were suspected to have undergone an epithelial-tomesenchymal transition (EMT) were extracted and further characterized in vitro. These cells share a specific chromosomal aberration with the adjacent tumor cells and express residual levels of keratin, confirming their epithelial origin. By contrast, they exhibit fibroblast morphology, express high levels of aSMA, and although they cannot form tumors, they increase the size of tumors from a coinjected breast cancer cell line in nude mice [39]. In another study, GFP-labeled cancer cells were shown to provide 25% of the adjacent CAF population [40]. Endothelial cells are yet another emerging source for CAFs. Zeisberg et al. reported that endothelial cells treated with TGF-b exhibit CAF morphology and elevated fibroblast-specific protein-1 (FSP1) expression, coupled with decreased expression of the endothelial marker CD31. This phenomenon is also evident in a mouse model [41]. Two recent studies have disclosed that pericytes give rise to scar-forming stromal cells at injury sites [42,43]. This implies that perivascular cells, often found in the vicinity of tumor sites, are yet another source for CAF precursors; however, future studies should test this hypothesis. Recently, adipose tissue derived stem cells were reported to differentiate into CAF-like cells following exposure to conditioned media from breast cancer cell lines [44]. Common CAF markers and gene signatures In general, normal fibroblasts display vast heterogeneity manifested by differences in appearance, behavior, and gene expression (reviewed in [45]). On top of the heterogeneity found within the fibroblasts, other stromal residents resemble fibroblasts, especially with regard to the expression of ‘specific’ markers. Thus, the heterogeneity of the stromal compartment, combined with the presence of


multiple subpopulations of fibroblasts, make it difficult to properly isolate and define CAFs based on the expression of specific markers. Nevertheless, CAF markers are widely described in the literature. Among the downregulated markers are caveolin-1 [46] and laminin [47], whereas the CAF markers most frequently reported to be upregulated include aSMA [48], vimentin [8], and FSP1 [49]. By contrast, Sugimoto et al. used a mouse model of cancer to compare six markers belonging to several mesenchymal groups that inhabit the stroma. aSMA, plateletderived growth factor receptor b (PDGFRb), and chondroitin sulfate proteoglycan 4 overlapped with each other in identifying fibroblasts, myofibroblasts, and vascular related cells; vimentin and type I collagen were also not specific to fibroblasts [49]. This work questions the specificity of these commonly accepted CAF markers. Moreover, aSMA was reported to be downregulated rather than upregulated in the stromal compartment of prostate cancer [50]. These findings emphasize the heterogeneity of the stromal compartment and lead us to surmise there is no known unequivocal marker to distinguish between mesenchymal subpopulations. Despite the findings mentioned above indicating aSMA elevation is both cancer-type specific and a general marker for mesenchymal cells, it is wrongfully accepted as the definitive marker for all CAFs [3,51]. Several studies have identified a ‘CAF gene expression profile’ [52–54] and even ‘CAF signatures’ that are predictive of tumor outcome [55,56]. Closer examination of these studies reveals that on the specific gene level, the differences outnumber the commonalities. However, if we zoom out of the gene level and examine gene families, different types and stages of cancers yield similar groups of genes – cell adhesion, immune response, and ECM modulation [52–54]. This is exactly what we would expect to find from different cell types under similar conditions – converging into performing the same tasks in different ways (i.e., genes). A novel definition for the term CAFs The inconsistency of CAF markers/gene signatures, which represents the heterogeneity of the stromal compartment and the ample reservoir for their cell of origin, suggest that the tumor and its microenvironment exhibit a considerable degree of plasticity that is not restricted to a specific germ layer. This situation warrants a new definition for the term ‘CAFs’. In the field of stem cell research, a similar controversy arose as to the exact definition of ‘stem cell’. An elegant solution for this dispute defined ‘stemness’ as a state in the life cycle of a cell [26]. By the same token, ‘CAFs’ should be defined as the dynamic state of fibroblast-like cells found in the vicinity of the tumor that promote its progression – for brevity a ‘CAF state’. But how do cells maintain their ‘CAF state’ for long periods of time? If we perceive the tumor microenvironment as an evolving element, it might be useful to borrow evolution based concepts to resolve this conundrum. Accordingly, three possible mechanisms come to mind: genetic mutations, epigenetic alterations, and persistent environmental effects. 449

Opinion Genetic mutations – the genomic landscape of CAFs A huge debate is being held over the genomic landscape of stromal cells. Generally speaking, the stromal compartment surrounding tumors is perceived as genetically stable, especially when compared with the rapidly dividing tumor cells that constitute most of the tumor mass. Nevertheless, some studies have described genomic modifications of cancer associated stroma, including amplifications, loss of heterozygosity (LOH), and smaller mutations. After injecting human prostate cancer cells into nude mice, Pathak et al. found mouse stromal cells harboring multiple copies of mouse chromosome 15 in the vicinity of the tumors [57]. Microdissection of stromal and cancer cells from prostate tumors, followed by PCR, revealed LOH of chromosome 8p in all of the tested tumors and in three out of nine mesenchymal samples [58]. LOH of p53 was also documented in microdissected stromal cells of breast and colon cancers [59]. Furthermore, microdissected stromal and epithelial tissues of 11 breast samples exhibited LOH in specific DNA loci, both in tumor and stromal cells. Importantly, several such incidents of LOH were exclusively detected in the stroma and, strikingly, some were evident in distant stromal cells [60], suggesting that the stromal cell population is heterogeneous and distinct from cancer cells and may consist of genetically unstable clones. Finally, additional studies have described p53 and PTEN mutations in the stromal compartment of breast cancer patients [59,61,62]. In overwhelming contrast to this, other reports portray the stromal population as genetically stable. Accordingly, no karyotypic abnormalities were found in the CAFs of oral carcinoma [8]. CAFs isolated from pancreatic cancer do not exhibit somatic copy variation or immunohistological evidence for p53 mutations [63]. Allinen et al. thoroughly examined breast CAFs and discovered that despite dramatic gene expression changes in CAFs compared with normal fibroblasts, no genetic alterations are evident [64]. In another study, breast CAFs extracted from 25 tumors produced only one sample that exhibited copy number variation and p53 mutation [65]. Similar results have been obtained in ovarian cancer samples [66]. What could be the reason for such an enormous discrepancy arising in the literature regarding the genomic landscape of CAFs? Bearing in mind the ‘CAF state’ hypothesis presented above, we should consider the possibility that the CAF population does not necessarily descend from normal fibroblasts. The genetically abnormal stroma might represent a subpopulation of carcinoma cells that underwent EMT. For example, 38 genes showed similar genetic alterations (either gain or loss of genetic material) in stromal and epithelial compartments of 11 ovarian tumor pairs [67], suggesting that these two cell populations originated from the same mother cell despite the different morphological appearance. Moreover, chromogenic in situ hybridization of breast cancer samples revealed that the genetic features detected in tumor cells were also present in a minority of fibroblast-like cells near the epithelial clusters, indicating that some of these cells are clonal and derived from the epithelial tumor cells [68]. Fukino et al. found more correlations between clinicopathological features and the LOH/allelic imbalance in the stroma than 450


in the epithelium of breast cancer samples [69], yet no ‘stromal-like’ tumors have been reported in this study. The fact that stromal cells multiply in a much lower rate render them less prone to the major selective forces imposed on highly prolific cancer cells. If, indeed, stromal cells were equally genetically instable, we would expect to find aggressive CAF clones giving rise to sarcoma-like tumors alongside carcinomas. As this is clearly not the case, we presume that the genetic alterations in CAFs are more sparse and subtle. In our opinion, it is more plausible that the reported ‘stromal’ populations that exhibit distinct mutations are merely subgroups of cancer cells which underwent EMT. Epigenetic alterations An alternative mechanism to CAF genomic aberrations is epigenetic alterations. A recent interesting study from the Witte group revealed that overexpression of an epigenetic regulator in the stroma leads to increased Wnt/b-catenin signaling which results in neoplastic lesions of adjacent epithelial cells [70]. These findings coincide with previous reports on aberrant Wnt/b-catenin signaling in CAFs [71]. Further evidence supporting the CAF epigenetic regulation notion are thoroughly reviewed by Rudnick and Kuperwasser [72]. Persistent environmental effects Another way by which CAFs might sustain their ‘CAF state’ is the constant presence of signals originating from tumor cells. As mentioned in Box 2, tumors are known to secrete CAF-activating factors such as interleukin-1 (IL-1) and tumor necrosis factor a (TNF-a) [25,73–76]. Epidermal growth factor (EGF) released from breast cancer cells ‘educate’ CAFs to enhance the production and secretion of leptin, eventually leading to tumor progression [77]. A recent study from our laboratory revealed that tumor cells induce ATF3 elevation in adjacent stromal cells [5]. Moreover, when ATF3 was overexpressed in three distinct types of CAFs (produced from prostate, breast, and lung cancers), the result was a very similar gene expression program, and a capacity to promote tumor growth. Abundance of a tumor signal could be the result of a specific mutation in the tumor itself [78]. The concept of ‘extended phenotype’ was first put forward by Richard Dawkins – suggesting that a gene in an organism might alter its environment [79]. Thus, if we accept the notion that CAFs are genetically stable, we can attribute the ‘CAF state’ to an ‘extended phenotype’ of tumor mutations. Box 3. Outstanding questions Does a stromal subpopulation consisting of a high mutation rate exist, or is it a cancerous subpopulation with a mesenchymal appearance? Would stromal-targeting therapies be sufficient to cure cancer or would they need to be coadministered with tumor-targeting drugs? Are there any other stromal subpopulations which exist as ‘states’ and not as cell types? If CAFs are indeed genetically stable, what are the epigenetic mechanisms that dictate their unique gene expression and behavior? What cell surface elements govern the tumor–stroma interaction?

Opinion


Although the secreted factors involved in the tumor– stroma interaction are widely studied and mentioned throughout this review, the cell surface determinants that govern this interaction are an interesting yet currently understudied angle (Box 3). A recent study reported on the intercellular transfer of Ras oncoproteins from melanoma to adjacent T cells [80]. Future studies should be dedicated to exploring this type of interaction. Concluding remarks and future perspectives The tumor microenvironment has become an important pillar in the cancer research field with research on CAFs being one of its main focuses. However, several persistent discrepancies have accumulated and warrant taking a new perspective. In this review, we suggest to conceive CAF traits (namely, mesenchymal appearance and tumor promoting ability) as a ‘state’ rather than a cell type. ‘CAF state’ might be exhibited by cells of different origins. But how could this type of information be translated into clinical practice? In recent years, the targets of several drugs are stromal components rather than the tumor itself [51]. These drugs target blood vessel growth, fibroblast-specific proteins, and secreted ECM residents. Now let us consider a tumor containing ‘CAF state’ cells (depicted in Figure 1). To tailor the proper stromal-related drugs to the right patient, we first need to establish the identity of these cells. This could be done based on the expression of a specific marker (note that a tumor might consist of several stromal subpopulations). In the next step, we should administer drug(s) that target the predominant cell type or rather a combination of drugs targeting more than one population in the stromal compartment.

For example, FAP is an integral membrane serine protease, often upregulated in fibroblasts [81]. Several FAP-related drugs have been developed, among them anti-FAP antibodies [82,83] and FAP activity inhibitors [84,85]. Endothelial cells might transform into CAF-resembling cells, as described above. These cells often express VEGF and thus could be targeted with VEGF-antagonizing agents [51]. MSCs are another considerable source for CAFs. Although MSCs share specific markers with other cell types, some markers such as GD2 are considered unique identifiers of human MSCs [86]. Recently, it was reported that GD3S is highly expressed in GD2 positive stem cells and that GD3S inhibition reduces tumor formation in vivo [87]. Although the authors referred to cancer initiating stem cells in their report, these findings could also be valid for stromal MSCs. Finally, tumor cells resembling CAFs are the outcome of an EMT process and therefore would most likely respond to anticancer drugs targeting features such as a high mutation rate occurring in tumor suppressor genes (e.g., p53) and oncogenes (e.g., Myc). Should that be the case, the proper drugs to be administered are tumor suppressor reactivators or oncogene inhibitors. On a different note, several studies and clinical trials have used modified MSCs and CAFs for antitumor drug delivery with various degrees of success and safety [88,89]. It would be interesting to investigate whether the use of other ‘CAF state’ cells coming from different origins could yield improved results. By combining our knowledge regarding stromal cell markers and the recent advancements in stromal-based anticancer therapy, the personalized therapy concept could also be extended to the tumor microenvironment, thus providing a more comprehensive way to treat cancer. Acknowledgments

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We wish to thank Professor Dov Zipori for inspiration and guidance. His ‘stem state’ concept has led to the birth of this review. We acknowledge and appreciate further work that was done in this field but could not be included owing to space limitations.

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Figure 1. Tailor-made therapy based on cancer associated fibroblast (CAF) cell of origin. CAF-resembling cells (inner circle) could be the descendants of several cell types (outer circle). These cells are identified by specific markers (purple trapezoids) and eventually might be targeted by anticancer drugs (pink trapezoids).

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Journal of Visualized Experiments

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Video Article

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts Urszula M. Polanska1,* , Ahmet Acar1,* , Akira Orimo1,2 1CR-UK 2Atopy

Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, University of Manchester Research Center, Juntendo University

*These authors contributed equally

Correspondence to: Akira Orimo at [email protected] URL: http://www.jove.com/details.php?id=3201 DOI: 10.3791/3201 Keywords: Medicine, Issue 56, cancer, stromal myofibroblasts, experimentally generated carcinoma-associated fibroblasts (exp-CAFs), fibroblast, human mammary carcinomas, tumour xenografts, Date Published: 10/25/2011 Citation: 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).

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) 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.

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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 (