Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts

Activated fibroblasts are associated with many different tumors. Myofibroblasts, activated fibroblasts, and perivascular mesenchymal cells such as pericytes play a role in cancer progression. Many studies suggest that myofibroblasts facilitate tumor growth and cancer progression. The source for myofibroblasts and other activated fibroblasts within the tumors is still debated. Although de novo activation of quiescent fibroblasts into alpha-smooth muscle actin (alpha SMA)-positive myofibroblasts is one likely source, epithelial to mesenchymal transition and bone marrow recruitment are also evolving as possible mechanisms for the emergence of a heterogeneous population of carcinoma-associated fibroblasts. Here, we show that transforming growth factor-beta1 could induce proliferating endothelial cells to undergo a phenotypic conversion into fibroblast-like cells. Such endothelial to mesenchymal transition (EndMT) is associated with the emergence of mesenchymal marker fibroblast-specific protein-1 (FSP1) and down-regulation of CD31/PECAM. Additionally, we show EndMT in tumors using the B16F10 melanoma model and the Rip-Tag2 spontaneous pancreatic carcinoma model. Crossing Tie2-Cre mice with R26Rosa-lox-Stop-lox-LacZ mice allows for irreversible tagging of endothelial cells. We provide unequivocal evidence for EndMT at the invasive front of the tumors in these transgenic mice. Collectively, our results show that EndMT is a unique mechanism for the accumulation of carcinoma-associated fibroblasts and suggest that antiangiogenic treatment of tumors may have a direct effect in decreasing activated fibroblasts that likely facilitate cancer progression.     

Carcinoma-associated fibroblast-like differentiation of human mesenchymal stem cells

Carcinoma-associated fibroblasts (CAF) have recently been implicated in important aspects of epithelial solid tumor biology, such as neoplastic progression, tumor growth, angiogenesis, and metastasis. However, neither the source of CAFs nor the differences between CAFs and fibroblasts from nonneoplastic tissue have been well defined. In this study, we show that human bone marrow-derived mesenchymal stem cells (hMSCs) exposed to tumor-conditioned medium (TCM) over a prolonged period of time assume a CAF-like myofibroblastic phenotype. More importantly, these cells exhibit functional properties of CAFs, including sustained expression of stromal-derived factor-1 (SDF-1) and the ability to promote tumor cell growth both in vitro and in an in vivo coimplantation model, and expression of myofibroblast markers, including alpha-smooth muscle actin and fibroblast surface protein. hMSCs induced to differentiate to a myofibroblast-like phenotype using 5-azacytidine do not promote tumor cell growth as efficiently as hMSCs cultured in TCM nor do they show increased SDF-1 expression. Furthermore, gene expression profiling revealed similarities between TCM-exposed hMSCs and CAFs. Taken together, these data suggest that hMSCs are a source of CAFs and can be used in the modeling of tumor-stroma interactions. To our knowledge, this is the first report showing that hMSCs become activated and resemble carcinoma-associated myofibroblasts on prolonged exposure to conditioned medium from MDAMB231 human breast cancer cells.     

Cancer‐associated fibroblasts and epithelial‐mesenchymal transition in metastatic oral tongue squamous cell carcinoma

We examined cancer‐associated fibroblasts (CAFs) and a panel of immunohistochemical markers of epithelial‐mesenchymal transition (EMT) in 19 pair‐matched oral tongue squamous cell carcinoma (SCC) and metastatic tumors to regional lymph nodes (RLNs). α‐Smooth muscle actin (α‐SMA) was studied to identify CAFs. EMT was studied with syndecan‐1, Cadherin‐11, fibroblast‐specific protein (FSP)‐1, s ecreted p rotein a cidic and r ich in c ysteine (SPARC) and Twist. Triple immunostaining in RLNs was used to highlight the carcinoma cells (E‐cadherin and Ki‐67) and their relationship to the CAFs (α‐SMA). We found that metastatic RLNs hosted CAFs similarly as in pair‐matched primary tumors. Expression of EMT markers is common in both primary and metastatic tumors. We demonstrate that metastatic carcinoma cells (Ki‐67 positive) downregulate E‐cadherin expression at the periphery of cancer islands, where they are in direct contact with CAFs. The supporting connective tissue microenvironment also commonly expresses syndecan‐1, Cadherin‐11, FSP‐1, and SPARC. In conclusion, CAFs are common to both primary and metastatic SCC. We hypothesize that CAFs not only promote tumor invasion but also facilitate metastases, either by cometastasizing and/or being recruited to lymph nodes. Evidence of EMT is common within primary tumors and metastatic SCC and may be further modulated by CAFs.     

Bone marrow-derived myofibroblasts contribute to the mesenchymal stem cell niche and promote tumor growth

Carcinoma-associated fibroblasts (CAFs) that express α-smooth muscle actin (αSMA) contribute to cancer progression, but their precise origin and role are unclear. Using mouse models of inflammation-induced gastric cancer, we show that at least 20% of CAFs originate from bone marrow (BM) and derive from mesenchymal stem cells (MSCs). αSMA+ myofibroblasts (MFs) are niche cells normally present in BM and increase markedly during cancer progression. MSC-derived CAFs that are recruited to the dysplastic stomach express IL-6, Wnt5α and BMP4, show DNA hypomethylation, and promote tumor growth. Moreover, CAFs are generated from MSCs and are recruited to the tumor in a TGF-β- and SDF-1α-dependent manner. Therefore, carcinogenesis involves expansion and relocation of BM-niche cells to the tumor to create a niche to sustain cancer progression.     

肺癌相关成纤维细胞的研究进展

癌相关成纤维细胞(cancer-associated fibroblasts,CAFs)是肿瘤组织中被癌细胞激活的成纤维细胞,具有成肌纤维细胞的特性。CAFs表型恶性转换与肿瘤演进密切相关,同时CAFs是肿瘤微环境的主要组织成分。本文主要从以下方面结合肺癌进行综述:①CAFs的来源及其形态学特征,CAFs异质性及其表型时、空转换的临床意义;②CAFs的研究现状及临床应用中的问题;③CAFs与肺癌侵袭与转移的关系;④CAFs对肺癌预后及其治疗的影响。     

Myofibroblasts in the stroma of colonic tumors

An ultrastructural examination of 18 colonic carcinomas detected myofibroblasts in 13 tumors. An inverse correlation was established between the level of myofibroblasts and the number of inflammatory cells. A multi-layered vascular basal membrane was found in the capillary vessels of 5 carcinomas. Myofibroblasts were invariably accompanied by altered smooth muscle cells corresponding to pericytes with smooth muscle traits. The findings suggest that myofibroblasts may develop from fibroblasts, smooth muscle cells of the intestinal wall and cells of vascular structures.     

Breast carcinoma-associated fibroblasts and their counterparts display neoplastic-specific changes

It has become clear that the initiation and progression of carcinomas depend not only on alterations in epithelial cells, but also on changes in their microenvironment. To identify these changes, we have undertaken cellular and molecular characterization of carcinoma-associated fibroblasts (CAF) and their tumor counterpart fibroblasts (TCF) isolated from 12 breast cancer patients. Normal breast fibroblasts (NBF) from plastic surgery were used as normal control. We present evidence that both CAFs and TCFs are myofibroblasts and show tumor-associated features. Indeed, the p53/p21 response pathway to gamma-rays was defective in 70% CAFs, whereas it was normal in all the TCF and NBF cells. In addition, the basal levels of the p53 and p21 proteins were significantly low in 83% of CAFs and modulated in the majority of TCFs compared with NBFs. Interestingly, both TCFs and CAFs expressed high levels of the cancer marker survivin and consequently exhibited high resistance to cisplatin and UV light. Moreover, most CAFs were positive for the proliferation marker Ki-67 and exhibited high proliferation rate compared with NBFs and TCFs. However, proliferating cell nuclear antigen was highly expressed in both CAFs and TCFs. Using the two-dimensional gel electrophoresis technique, we have also shown that CAF, TCF, and NBF cells present different proteome profiles, with many proteins differentially expressed between these cells. Taken together these results indicate that different genetic alterations can occur in breast CAFs and their corresponding adjacent counterparts, showing the important role that stroma could play in breast carcinogenesis and treatment.     

Tumour-stroma interaction: cancer-associated fibroblasts as novel targets in anti-cancer therapy?

Stroma cells, together with extracellular matrix components, provide the microenvironment that is pivotal for cancer cell growth, invasion and metastatic progression. Characteristic stroma alterations accompany or even precede the malignant conversion of epithelial cells. Crucial in this process are fibroblasts, also termed myofibroblasts or cancer-associated fibroblasts (CAFs) that are located in the vicinity of the neoplastic epithelial cells. They are able to modify the phenotype of the epithelial cells by direct cell-to-cell contacts, through soluble factors or by modification of extracellular matrix components. Seminal functional studies in various cancer types, including breast, colon, prostate and lung cancer, have confirmed the concept that fibroblasts can determine the fate of the epithelial cell, since they are able to promote malignant conversion as well as to revert tumour cells to a normal phenotype. This review focuses on characteristic changes of fibroblasts in cancer and provides the experimental background elucidating functional properties of CAFs in the carcinogenic process. A possible implication in lung carcinogenesis is emphasised. Finally, a laser-capture- and microarray-based approach is presented, which comprehensively characterises carcinoma-associated fibroblasts in their in vivo environment for the identification of potential targets for anti-cancer therapy.     

Mesenchymal stem cells regulate epithelial–mesenchymal transition and tumor progression of pancreatic cancer cells

Cancer‐associated fibroblasts contribute to cancer progression that is caused by epithelial–mesenchymal transition (EMT). Recently, mesenchymal stem cells (MSCs) were found to be the major candidate involved in the development of tumor‐promoting cancer stroma. Here we report that α‐smooth muscle actin‐positive myofibroblast‐like cells originating from MSCs contribute to inducing EMT in side population cells of pancreatic cancer. More importantly, MSC‐derived myofibroblasts function to maintain tumor‐initiating stem cell‐like characteristics, including augmenting expression levels of various stemness‐associated genes, enhancing sphere‐ forming activity, promoting tumor formation in a mouse xenograft model, and showing resistance to anticancer drugs. Furthermore, both γ‐secretase inhibitor and siRNA directed against Jagged‐1 attenuated MSC‐associated E‐cadherin suppression and sphere formation in pancreatic cancer side population cells. Thus, our results suggest that MSC‐derived myofibroblasts play important roles in regulating EMT and tumor‐initiating stem cell‐like properties of pancreatic cancer cells through an intermediating Notch signal.     

Cancer associated fibroblasts stimulated by transforming growth factor beta1 (TGF-β1) increase invasion rate of tumor cells: a population study

Cancer associated fibroblasts (CAFs) are believed to promote tumor growth and progression. Our objective was to measure the effect of TGF-beta1 on fibroblasts isolated from invasive breast cancer patients. Fibroblasts were isolated from tissue obtained at surgery from patients with invasive breast cancer (CAF; n = 28) or normal reduction mammoplasty patients (normal; n = 10). Myofibroblast activation was measured by counting cells immunostained for smooth muscle alpha actin (ACTA2) in cultures +/- TGF-beta 1. Conditioned media (CM) was collected for invasion assays and RNA was isolated from cultures incubated in media +/- TGF-beta1 for 24 h. Q-PCR was used to measure expression of cyclin D1, fibronectin, laminin, collagen I, urokinase, stromelysin-1, and ACTA2 genes. Invasion rate was measured in chambers plated with MDA-MB-231 cells and exposed to CM in the bottom chamber; the number of cells that invaded into the bottom chamber was counted. Wilcox Rank Sum tests were used to evaluate differences in CAFs and normal fibroblasts and the effect of TGF-beta 1. There was no difference in percent myofibroblasts or invasion rate between normal and CAF cultures. However, TGF-beta1 significantly increased the percent of myofibroblasts (P < 0.01) and invasion rate (P = 0.02) in CAF cultures. Stromelysin-1 expression was significantly higher in normal versus CAF cultures (P < 0.01). TGF-beta 1 significantly increased ACTA2 expression in both normal and CAF cultures (P < 0.01). Expression of fibronectin and laminin was significantly increased by TGF-beta in CAF cultures (P < 0.01). CAFs were measurably different from normal fibroblasts in response to TGF-beta 1, suggesting that TGF-beta stimulates changes in CAFs that foster tumor invasion.     

胃癌细胞促进网膜脂肪干细胞分化的机制研究

  目的  本实验主要研究在胃癌条件培养基（conditioned medium,CM）诱导下网膜脂肪干细胞（omental-adipose stromal cells,O-ASCs）是否能分化为癌相关成纤维细胞（carcinoma-associated fibroblasts,CAFs）,及ERK信号通路在其中的作用。  方法  通过诱导分化成骨、成脂及流式细胞鉴定O-ASCs,将O-ASCs与MGC803和SGC7901 CM共培养,通过RT-PCR和Western-blot检测O-ASCs细胞CAFs标志物α-SMA、FSP-1、vimentin,旁分泌因子VEGFA、TGFβ-1、FAP、SDF-1的表达水平。将O-ASCs分为对照组,SGC7901-CM实验组,SGC7901-CM+U0126处理组,12 h后收集细胞。Western blot检测O-ASCs细胞CAFs标志物α-SMA、FSP-1及ERK1/2、p-ERK1/2的表达水平。  结果  经鉴定原代培养出的细胞为O-ASCs,在SGC7901 CM和MGC803 CM作用下,CAFs标志物α-SMA、FSP-1、vimentin及旁分泌因子SDF-1、VEGFA、TGFβ-1、FAP表达均有明显增加（P < 0.05）。与对照组比较SGC7901-CM组α-SMA、FSP-1、p-ERK1/2表达明显增加（P < 0.05）,ERK表达未见明显变化（P > 0.05）。SGC7901-CM+U0126组与SGC7901-CM组比较,α-SMA、FSP-1及p-ERK1/2的蛋白表达水平明显降低（P < 0.05）,ERK表达变化无统计学意义（P > 0.05）。  结论  O-ASCs通过分化为CAFs及旁分泌作用参与胃癌腹膜转移,ERK信号通路在该过程中发挥了重要作用。      

Fibroblasts produce brain-derived neurotrophic factor and induce mesenchymal transition of oral tumor cells

Fibroblasts (Fibs) contribution to neoplastic progression, tumor growth, angiogenesis, and metastasis has been recently reported by several research groups. In this study it was investigated if fibroblasts are the source of brain-derived neurotrophic factor (BDNF), which plays a crucial role in the progression of oral squamous cell carcinoma. In a novel in vitro system oral Fibs were cultured with SCC-25 lingual squamous cell carcinoma cells for 7days. Factors related with this interaction were investigated by quantitative PCR and western blot. In the co-culture, fibroblasts were converted to carcinoma-associated fibroblasts (CAFs), which in return initiated epithelial-mesenchymal transition (EMT) of SCC-25 cells. The induced CAFs produced increased levels of BDNF, which interacted with the increased-expressed neurothrophin receptor B (TrkB) on EMT-converted SCC-25 cells. Possible regulatory factors of BDNF expression (tumor necrosis factor-α and interleukin-1-β) were detected both in CAFs and EMT-tumor cells. In CAFs: IL-1β-, in SCC-25 cells TNF-α-gene-expression was significantly increased in co-culture conditions. Activated fibroblasts (CAFs) and mesenchymal transitioned tumor cells might use the BDNF-TrkB axis and its regulation to harmonize their interaction in the process of tumor progression.     

Altered TGF-β signaling in a subpopulation of human stromal cells promotes prostatic carcinogenesis

Carcinoma-associated fibroblasts (CAF) play a critical role in malignant progression. Loss of TGF-β receptor II (TGFβR2) in the prostate stroma is correlated with prostatic tumorigenesis. To determine the mechanisms by which stromal heterogeneity because of loss of TGFβR2 might contribute to cancer progression, we attenuated transforming growth factor beta (TGF-β) signaling in a subpopulation of immortalized human prostate fibroblasts in a model of tumor progression. In a tissue recombination model, loss of TGFβR2 function in 50% of the stromal cell population resulted in malignant transformation of the nontumorigenic human prostate epithelial cell line BPH1. Mixing fibroblasts expressing the empty vector and dominant negative TGFβR2 increased the expression of markers of myofibroblast differentiation [coexpression of vimentin and alpha smooth muscle actin (αSMA)] through elevation of TGF-β1 and activation of the Akt pathway. In combination, these two populations of stromal cells recapitulated the tumor inductive activity of CAFs. TGFβR2 activity in mixed stromal cell populations cultured in vitro caused secretion of factors that are known to promote tumor progression, including TGF-β1, SDF1/CXCL12, and members of the fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) families. In vivo, tissue recombination of fibroblasts overexpressing TGF-β1 and SDF1/CXCL12 not only induced transformation of BPH1 cells, but also promoted a robust growth of highly invasive cells, similar to effects produced by CAFs. While the precise nature and/or origin of the particular stromal cell populations in vivo remain unknown, these findings strongly link heterogeneity in TGF-β signaling to tumor promotion by tumor stromal cells.     

Upregulation of cancer-associated myofibroblasts by TGF-β from scirrhous gastric carcinoma cells

Background:

Myofibroblasts in the cancer microenvironment have recently been implicated in tumour growth and metastasis of gastric cancer. However, the mechanisms responsible for the regulation of myofibroblasts in cancer-associated fibroblasts (CAFs) remain unclear. This study was performed to clarify the mechanisms for regulation of myofibroblasts in gastric cancer microenvironment.

Methods:

Two CAFs (CaF-29 and CaF-33) from the tumoural gastric wall and a normal fibroblast (NF-29) from the nontumoural gastric wall, 4 human gastric cancer cell lines from scirrhous gastric cancer (OCUM-2MD3 and OCUM-12), and non-scirrhous gastric cancer (MKN-45 and MKN-74) were used. Immunofluorescence microscopy by triple-immunofluorescence labelling (α-SMA, vimentin, and DAPI) was performed to determine the presence of α-SMA-positive myofibroblasts. Real-time RT–PCR was performed to examine α-SMA mRNA expression.

Results:

Immunofluorescence microscopy showed that the frequency of myofibroblasts in CaF-29 was greater than that in NF-29. The number of myofibroblasts in gastric fibroblasts gradually decreased with serial passages. Transforming growth factor-β (TGF-β) significantly increased the α-SMA expression level of CAFs. Conditioned medium from OCUM-2MD3 or OCUM-12 cells upregulated the α-SMA expression level of CAFs, but that from MKN-45 or MKN-74 cells did not. The α-SMA upregulation effect of conditioned medium from OCUM-2MD3 or OCUM-12 cells was significantly decreased by an anti-TGF-β antibody or Smad2 siRNA.

Conclusion:

Transforming growth factor-β from scirrhous gastric carcinoma cells upregulates the number of myofibroblasts in CAFs.     

癌症相关成纤维细胞诱导的上皮间质转化增强肿瘤细胞干性的研究进展

在肿瘤细胞干性的获得和维持方面,肿瘤微环境扮演着十分重要的角色。作为其中数量最多的基质细胞,癌症相关成纤维细胞(cancer-associated fibroblasts,CAFs)可通过旁分泌信号和改变基质硬度的方式,诱导邻近的肿瘤细胞发生上皮-间质转化(epithelial-mesenchymal transition,EMT),使上皮来源细胞部分或完全地获得间充质表型。当细胞处于EMT进程中的某个过渡阶段时,即可被诱导表达干细胞表型,促进肿瘤的发生与进展。本文主要回顾了CAFs、EMT和肿瘤细胞干性相互之间的关系以及相关分子机制的研究,并尝试论述CAFs诱导肿瘤细胞的EMT增强其细胞干性。     

Proteomic signatures of the desmoplastic invasion front reveal collagen type XII as a marker of myofibroblastic differentiation during colorectal cancer metastasis

Cancer-associated fibroblasts (CAFs), represent a pivotal compartment of solid cancers (desmoplasia), and are causatively implicated in cancer development and progression. CAFs are recruited by growth factors secreted by cancer cells and they present a myofibroblastic phenotype, similar to the one obtained by resident fibroblasts during wound healing. Paracrine signaling between cancer cells and CAFs results in a unique protein expression profile in areas of desmoplastic reaction, which is speculated to drive metastasis. In an attempt to decipher large-scale proteomic profiles of the cancer invasive margins, we developed an in vitro coculture model system, based on tumor-host cell interactions between colon cancer cells and CAFs. Proteomic analysis of conditioned media derived from these cocultures coupled to mass spectrometry and bioinformatic analysis was performed to uncover myofibroblastic signatures of the cancer invasion front. Our analysis resulted in the identification and generation of a desmoplastic protein dataset (DPD), consisting of 152 candidate proteins of desmoplasia. By using monoculture exclusion datasets, a secretome algorithm and gene-expression meta-analysis in DPD, we specified a 22-protein "myofibroblastic signature" with putative importance in the regulation of colorectal cancer metastasis. Of these proteins, we investigated collagen type XII by immunohistochemistry, a fibril-associated collagen with interrupted triple helices (FACIT), whose expression has not been reported in desmoplastic lesions in any type of cancer. Collagen type XII was highly expressed in desmoplastic stroma by and around alpha-smooth muscle actin (α-SMA) positive CAFs, as well as in cancer cells lining the invasion front, in a small cohort of colon cancer patients. Other stromal markers, such as collagen type III, were also expressed in stromal collagen, but not in cancer cells. In a complementary fashion, gene expression meta-analysis revealed that COL12A1 is also an upregulated gene in colorectal cancer. Our proteomic analysis identified previously documented markers of tumor invasion fronts and our DPD could serve as a pool for future investigation of the tumor microenvironment. Collagen type XII is a novel candidate marker of myofibroblasts, and/or cancer cells undergoing dedifferentiation.     

Reciprocal Interaction between Carcinoma-Associated Fibroblasts and Squamous Carcinoma Cells through Interleukin-1α Induces Cancer Progression

Crosstalk between cancer cells and carcinoma-associated fibroblasts (CAFs) has earned recognition as an interaction that plays a pivotal role in carcinogenesis. Thus, we attempted to clarify whether increase in the level of CAFs promotes cancer progression by proportionally enhancing the interaction between cancer cells and CAFs. We first analyzed clinical correlation between the levels of fibroblasts and cancer progression and found that the level of CAFs made a noticeable difference on the prognosis of patients with oral squamous cell carcinoma (OSCC). In vivo animal study also demonstrated that tumor volume depended on the dose of CAFs that was co-injected with OSCC cells. The same tendency was observed in an in vitro study. We also found that interleukin-1α (IL-1α) secreted from OSCC cells had dual effects on CAFs: IL-1α not only promoted the proliferation of CAFs but also upregulated the secretion of cytokines in CAFs such as CCL7, CXCL1, and IL-8. The induction activity of cytokine secretion by IL-1α surpassed that of proliferation in OSCC cells. In summary, we unraveled an important interactive mechanism of carcinogenesis: IL-1α released from carcinoma stimulates the proliferation of CAFs and the simultaneous increase in cytokine secretion from CAFs promotes cancer progression in human OSCC. On the basis of these findings, we propose that the level of CAFs is eligible for being selected as a prognostic factor that will be useful in routine diagnosis. We also propose that blockage of reciprocal interaction between cancer cells and CAFs will provide an insight for developing novel chemotherapeutic strategy.     

PRL-3 overexpression in epithelial cells is induced by surrounding stromal fibroblasts

We isolate and culture carcinoma-associated fibroblasts (CAFs) from primary tumour (CAFpt), CAFs from corresponding synchronous liver metastasis (CAFlm) as well as normal colonic fibroblasts (NCF) from the same patient. From these cultures, conditioned media (CM) was obtained. Culture of a wide panel of colorectal and pancreatic cell lines in CM from CAFlm resulted in overexpression of mRNA PRL-3 and higher overexpression in CAFs than in non-activated fibroblasts. Moreover PRL-3 mRNA expression correlates with expression of α-SMA and deposition of collagen fibrils in the stroma. We demonstrate that products secreted by CAFs trigger PRL-3 overexpression in cancer cells. Identification of these factors may contribute to new stroma-targeted therapies for desmoplastic tumours.