Stromal expression of connective tissue growth factor promotes angiogenesis and prostate cancer tumorigenesis

Our previous studies have defined reactive stroma in human prostate cancer and have developed the differential reactive stroma (DRS) xenograft model to evaluate mechanisms of how reactive stroma promotes carcinoma tumorigenesis. Analysis of several normal human prostate stromal cell lines in the DRS model showed that some rapidly promoted LNCaP prostate carcinoma cell tumorigenesis and others had no effect. These differential effects were due, in part, to elevated angiogenesis and were transforming growth factor (TGF)-beta1 mediated. The present study was conducted to identify and evaluate candidate genes expressed in prostate stromal cells responsible for this differential tumor-promoting activity. Differential cDNA microarray analyses showed that connective tissue growth factor (CTGF) was expressed at low levels in nontumor-promoting prostate stromal cells and was constitutively expressed in tumor-promoting prostate stromal cells. TGF-beta1 stimulated CTGF message expression in nontumor-promoting prostate stromal cells. To evaluate the role of stromal-expressed CTGF in tumor progression, either engineered mouse prostate stromal fibroblasts expressing retroviral-introduced CTGF or 3T3 fibroblasts engineered with mifepristone-regulated CTGF were combined with LNCaP human prostate cancer cells in the DRS xenograft tumor model under different extracellular matrix conditions. Expression of CTGF in tumor-reactive stroma induced significant increases in microvessel density and xenograft tumor growth under several conditions tested. These data suggest that CTGF is a downstream mediator of TGF-beta1 action in cancer-associated reactive stroma and is likely to be one of the key regulators of angiogenesis in the tumor-reactive stromal microenvironment.     

The fibrotic and immune microenvironments as targetable drivers of metastasis

Although substantial progress has been made over the past 40 years in treating patients with cancer, effective therapies for those who are diagnosed with advanced metastatic disease are still few and far between. Cancer cells do not exist in isolation: rather, they exist within a complex microenvironment composed of stromal cells and extracellular matrix. Within this tumour microenvironment exists an interplay between the two main stromal cell subtypes, cancer-associated fibroblasts (CAFs) and immune cells, that are important in controlling metastasis. A complex network of paracrine signalling pathways between CAFs, immune cells and tumour cells are involved at multiple stages of the metastatic process, from invasion and intravasation at the primary tumour site to extravasation and colonisation in the metastatic site. Heterogeneity and plasticity within stromal cell populations also contribute to the complexity. Although many of these processes are likely to be common to a number of metastatic sites, we will describe in detail the interplay within the liver, a preferred site of metastasis for many tumours. A greater understanding of these networks provides opportunities for the design of new therapeutic approaches for targeting the metastatic disease.Subject terms: Cancer microenvironment, Mechanisms of disease     

HB-EGF and PDGF mediate reciprocal interactions of carcinoma cells with cancer-associated fibroblasts to support progression of uterine cervical cancers

Tumor stroma drives the growth and progression of cancers. A heparin-binding epidermal growth factor-like growth factor, HB-EGF, is an EGF receptor ligand that stimulates cell growth in an autocrine or paracrine fashion. While elevated expression of HB-EGF in cancer cells and its contribution to tumor progression are well documented, the effects of HB-EGF expression in the tumor stroma have not been clarified. Here, we show that HB-EGF is expressed in stromal fibroblasts where it promotes cancer cell proliferation. In uterine cervical cancers, HB-EGF was detected immunohistochemically in the stroma proximal to the cancer epithelium. Proliferation of cervical cancer cells in vitro was enhanced by coculture with fibroblasts isolated from tumor tissues of patients with cervical cancer. Inhibition of HB-EGF function or treatment with platelet-derived growth factor (PDGF) inhibitors abrogated cancer cell growth enhanced by cervical cancer-associated fibroblast (CCF) coculture. Furthermore, tumor formation in a mouse xenograft model was enhanced by cotransplantation of CCF or mouse embryonic fibroblasts, but not with embryonic fibroblasts from HB-EGF-deficient mice. Conversely, conditioned medium from cancer cells induced HB-EGF expression in CCF. Mechanistic investigations established that PDGF was the primary factor responsible. Together, our findings indicate that HB-EGF and PDGF reciprocally mediate the interaction of cancer cells with cancer-associated fibroblasts, promoting cancer cell proliferation in a paracrine manner that has implications for novel combinatorial cancer therapies.     

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.     

Wnt‐3a‐activated human fibroblasts promote human keratinocyte proliferation and matrix destruction

Aberrant Wnt regulation, detectable by nuclear translocation of beta‐catenin, is a hallmark of many cancers including skin squamous cell carcinomas (SCCs). By analyzing primary human skin SCCs, we demonstrate that nuclear beta‐catenin is not restricted to SCC cells but also detected in stromal fibroblasts, suggesting an important role for aberrant Wnt regulation also in the tumor microenvironment. When human keratinocytes and fibroblasts were treated with Wnt‐3a, fibroblasts proved to be more responsive. Accordingly, Wnt‐3a did not alter HaCaT cell functions in a cell‐autonomous manner. However, when organotypic cultures (OTCs) were treated with Wnt‐3a, HaCaT keratinocytes responded with increased proliferation. As nuclear beta‐catenin was induced only in the fibroblasts, this argued for a Wnt‐dependent, paracrine keratinocyte stimulation. Global gene expression analysis of Wnt‐3a‐stimulated fibroblasts identified genes encoding interleukin‐8 (IL‐8) and C‐C motif chemokine 2 (CCL‐2) as well as matrix metalloproteinase‐1 (MMP‐1) as Wnt‐3a targets. In agreement, we show that IL‐8 and CCL‐2 were secreted in high amounts by Wnt‐3a‐stimulated fibroblasts also in OTCs. The functional role of IL‐8 and CCL‐2 as keratinocyte growth regulators was confirmed by directly stimulating HaCaT cell proliferation in conventional cultures. Most important, neutralizing antibodies against IL‐8 and CCL‐2 abolished the Wnt‐dependent HaCaT cell hyperproliferation in OTCs. Additionally, MMP‐1 was expressed in high amounts in Wnt‐3a‐stimulated OTCs and degraded the stromal matrix. Thus, our data show that Wnt‐3a stimulates fibroblasts to secrete both keratinocyte proliferation‐inducing cytokines and stroma‐degrading metalloproteinases, thereby providing evidence for a novel Wnt deregulation in the tumor‐stroma directly contributing to skin cancer progression.     

Down-regulation of KLF5 in cancer-associated fibroblasts inhibit gastric cancer cells progression by CCL5/CCR5 axis

It was well known that cancer-associated fibroblasts (CAFs) were an essential factor in tumor progression. However, the actual mechanism of stromal fibroblasts activation and tumor promoting effects remain unclear. Here, we showed that KLF5 expression was more frequently observed in gastric cancer-associated fibroblasts compared with normal mucosal fibroblasts. Moreover, KLF5 expression in tumor stroma was closely associated with clinicopathological features such as tumor size, invasion depth, cell grade and lymph node metastasis, as well as poor prognosis in patients with gastric cancer. In addition, we further demonstrated that KLF5-regulating CAFs affect gastric cancer cells progression by CCL5 secretion and activation of CCR5. Taken together, we concluded that KLF5 expression in gastric cancer-associated fibroblasts contribute to poor survival and promote cancer cells progression by activation of CCL5/CCR5 axis, which suggesting that KLF5 in CAFs might be considered as a promising target for the treatment of gastric cancer.     

Chemotherapy induces the cancer-associated fibroblast phenotype,activating paracrine Hedgehog-GLI signalling in breast cancer cells

Cancer cells recruit normal cells such as fibroblasts to establish reactive microenvironments. Via metabolic stress, catabolism and inflammation, these cancer-associated fibroblasts set up a synergistic relationship with tumour cells, that contributes to their malignancy and resistance to therapy. Given that chemotherapy is a systemic treatment, the possibility that healthy cell damage affects the metastatic risk or the prospect of developing a second malignancy becomes relevant. Here, we demonstrate that standard chemotherapies phenotypically and metabolically transform stromal fibroblasts into cancer-associated fibroblasts, leading to the emergence of a highly glycolytic, autophagic and pro-inflammatory microenvironment. This catabolic microenvironment, in turn, activates stemness (Sonic hedgehog/GLI signalling), antioxidant response and interferon-mediated signalling, in adjacent breast cancer cells. Thus, we propose a model by which chemotherapy-induced catabolism in healthy fibroblasts constitutes a source of energy-rich nutrients and inflammatory cytokines that would activate stemness in adjacent epithelial cells, possibly triggering new tumorigenic processes. In this context, immune cell recruitment would be also stimulated to further support malignancy.     

The cyan fluorescent protein nude mouse as a host for multicolor-coded imaging models of primary and metastatic tumor microenvironments

The tumor microenvironment (TME) has an important influence on tumor progression. For example, we have discovered that passenger stromal cells are necessary for metastasis. In this report, we describe six different cyan fluorescent protein (CFP) multicolor TME nude mouse models. The six different implantation models were used to image the TME using multiple colors of fluorescent proteins: I) Red fluorescent protein (RFP)- or green fluorescent protein (GFP)-expressing HCT-116 human colon cancer cells were implanted subcutaneously in the CFP-expressing nude mice. CFP stromal elements from the subcutaneous TME were visualized interacting with the RFP- or GFP-expressing tumors. II) RFP-expressing HCT-116 cells were transplanted into the spleen of CFP nude mice, and experimental metastases were then formed in the liver. CFP stromal elements from the liver TME were visualized interacting with the RFP-expressing tumor. III) RFP-expressing HCT-116 cancer cells were transplanted in the tail vein of CFP-expressing nude mice, forming experimental metastases in the lung. CFP stromal elements from the lung were visualized interacting with the RFP-expressing tumor. IV) In order to visualize two different tumors in the TME, GFP-expressing and RFP-expressing HCT-116 cancer cells were co-implanted subcutaneously in CFP-expressing nude mice. A 3-color TME was formed subcutaneously in the CFP mouse, and CFP stromal elements were visualized interacting with the RFP- and GFP-expressing tumors. V) In order to have two different colors of stromal elements, GFP-expressing HCT-116 cells were initially injected subcutaneously in RFP-expressing nude mice. After 14 days, the tumor, which consisted of GFP cancer cells and RFP stromal cells derived from the RFP nude mouse, was harvested and transplanted into the CFP nude mouse. CFP stromal cells invaded the growing transplanted tumor containing GFP cancer cells and RFP stroma. VI) Mouse mammary tumor (MMT) cells expressing GFP in the nucleus and RFP in the cytoplasm were implanted in the spleen of a CFP nude mouse. Cancer cells were imaged in the liver 3 days after cell injection. The dual-color dividing MMT cells and CFP hepatocytes, as well as CFP non-parenchymal cells of the liver were imaged interacting with the 2-color cancer cells. CFP-expressing host cancer-associated fibroblasts (CAFs) were predominantly observed in the TME models developed in the CFP nude mouse. Thus, the CFP nude mouse adds another color to the pallet of the TME, allowing multiple types of color-coded cancer and stromal cells to be imaged simultaneously. The multi-colored models described in this report provide new opportunities to study the cellular interactions in the live primary and metastatic TME.     

Ovarian cancer microenvironment: implications for cancer dissemination and chemoresistance acquisition

Ovarian adenocarcinoma is characterized by a late detection, dissemination of cancer cells into the whole peritoneum, and the frequent acquisition of chemoresistance. If these particularities can be explained in part by intrinsic properties of ovarian cancer cells, an increased number of studies show the importance of the tumor microenvironment in tumor progression. Ovarian cancer cells can regulate the composition of their stroma in promoting the formation of ascitic fluid, rich in cytokines and bioactive lipids, and in stimulating the differentiation of stromal cells into a pro-tumoral phenotype. In return, cancer-associated fibroblasts, cancer-associated mesenchymal stem cells, tumor-associated macrophages, or other peritoneal cells, such as adipocytes and mesothelial cells can regulate tumor growth, angiogenesis, dissemination, and chemoresistance. This review focuses on the current knowledge about the roles of stromal cells and the associated secreted factors on tumor progression. We also summarize the different studies showing that targeting the microenvironment represents a great potential for improving the prognosis of patients with ovarian adenocarcinoma.     

Stromal deletion of the APC tumor suppressor in mice triggers development of endometrial cancer

The contribution of the stromal microenvironment to the progression of endometrial cancer has not been well explored. We have conditionally expressed a mutant allele of adenomatous polyposis coli (APC(cKO)) in murine uterine stroma cells to study its effect on uterine development and function. In addition to metrorrhagia, the mice develop complex atypical endometrial gland hyperplasia that progresses to endometrial carcinoma in situ and endometrial adenocarcinoma as evidenced by myometrial invasion. Stromal cells subjacent to the carcinoma cells express alpha-smooth muscle actin (αSMA) with fewer cells expressing platelet-derived growth factor α compared with normal stromal cells, suggesting that the mutant stromal cells have acquired a more myofibroblastic phenotype, which have been described as cancer-associated fibroblasts and have been shown to induce carcinogenesis in other organ systems. Analyses of human endometrial cancer specimens showed substantial αSMA expression in the stroma compared with normal endometrial stroma cells. We also show that APC(cKO) mutant uteri and human endometrial cancer have decreased stromal levels of transforming growth factor β and bone morphogenetic protein activities and that the mutant uteri failed to respond to exogenous estradiol stimulation. The mutant stroma cells also had higher levels of vascular endothelial growth factor and stromal derived factor signaling components and diminished expression of estrogen receptor α and progesterone receptor, which is common in advanced stages of human endometrial cancer and is an indicator of poor prognosis. Our results indicate that de novo mutation or loss of heterozygosity in stromal APC is sufficient to induce endometrial hyperplasia and endometrial carcinogenesis by mechanisms that are consistent with unopposed estrogen signaling in the endometrial epithelium.     

Lung cancer secreted microvesicles: Underappreciated modulators of microenvironment in expanding tumors

Microvesicles (MVs) are shed from cell membranes of several cell types and have an important function in cell‐to‐cell communication. Exponentially growing lung cancer cells secrete large quantities of MVs and we were interested in their role in tumor progression. We observed that both human and murine lung cancer cell lines secrete more MVs in response to non‐apoptotic doses of hypoxia and irradiation. These tumor‐derived (t)MVs activate and chemoattract stroma fibroblasts and endothelial cells. Furthermore, they induce expression of several pro‐angiopoietic factors in stromal cells such as IL‐8, VEGF, LIF, OSM, IL‐11 and MMP‐9. We also noticed that conditioned media harvested from stroma cells stimulated by tMVs enhanced the metastatic potential of both human and murine lung cancer cells in vivo. Thus, we postulated that tMVs are underappreciated constituents of the tumor microenvironment and play a pivotal role in tumor progression, metastasis and angiogenesis. © 2009 UICC     

Expression of activated platelet-derived growth factor receptor in stromal cells of human colon carcinomas is associated with metastatic potential

Platelet-derived growth factor receptor (PDGF-R) expression has been reported in a variety of cancers, including colorectal, breast, lung, ovarian and pancreatic cancers, but the role of PDGF-R expression in the development and progression of colon carcinoma has not yet been elucidated. The purpose of this study was to examine the expression of PDGF and PDGF-R in human colon carcinomas. The expression of PDGF, PDGF-R and phosphorylated PDGF-R (p-PDGF-R) was examined by immunofluorescence in 12 surgical specimens of colon carcinoma and in human colon carcinoma cells growing in the subcutis (ectopic site) and the cecal wall (orthotopic site) of nude mice. In most surgical specimens, tumor cells expressed PDGF-A and -B subunits, without corresponding levels of PDGF-Ralpha and PDGF-Rbeta. PDGF-Rbeta was predominantly expressed by tumor-associated stromal cells and pericytes of tumor vasculature. The expression of PDGF-Rbeta in the stroma was associated with advanced stage disease. Under culture conditions, human colon carcinoma cell lines expressed PDGF-A and -B, but not PDGF-R. In orthotopic tumors, the KM12 cells (Duke's stage B) expressed PDGF-A and -B, but PDGF-Rbeta was expressed only by stromal cells and pericytes in the tumor vasculature. This expression of PDGF-Rbeta by stromal cells and pericytes was higher in tumors growing at the orthotopic site than in those at the ectopic site. The expression of PDGF-Rbeta in the stroma was higher in highly metastatic KM12SM tumors than in low metastatic KM12C tumors. In conclusion, the expression of PDGF-Rbeta in stromal cells is influenced by the organ-specific microenvironment and is associated with metastatic potential.     

Different patterns of stromal and cancer cell thymidine phosphorylase reactivity in non-small-cell lung cancer: impact on tumour neoangiogenesis and survival.

Angiogenesis is recognized as an important step in tumour pathogenesis that is related to invasion and metastatic spread and which consequently results in poor clinical outcome. In this study, we have examined the role of tumour stroma-activated fibroblasts and macrophage infiltration in the development of the angiogenic and metastatic phenotype in non-small-cell lung cancer (NSCLC). A total of 141 cases of early stage I-II NSCLC treated with surgery alone were analysed. The JC-70 (anti-CD31) MAb was used for the assessment of vascular grade. The P-GF.44C MAb was used to assess thymidine phosphorylase (TP) reactivity in cancer cells, stromal fibroblasts and macrophages. Cancer cell TP overexpression related to high vascular grade and to advanced T stage (P = 0.0004 and P = 0.02). Expression of TP in stromal fibroblasts also correlated with high angiogenesis (P = 0.01), but was independent of cancer cell expression. Fibroblast TP overexpression was related to abundant stroma (P = 0.003), suggesting that TP may be a marker of active stroma. Moreover, intense macrophage infiltration was associated with fibroblast TP reactivity, regardless of the amount of stroma, suggesting that macrophages may be a major contributor to TP expression in stroma. Survival analysis showed that cancer cell TP overexpression was related to poor prognosis (P = 0.005). Although stroma TP is related to angiogenesis, in the low vascular grade group it defined a group of patients with better prognosis (P = 0.02). It may be that fibroblast TP reactivity is an indirect marker of tumour infiltration by functional macrophages, which have an antitumour effect. We conclude that stromal macrophage and fibroblast TP reactivity may have an important role in non-small-cell lung cancer behaviour. Understanding the role of stromal fibroblasts and inflammatory cells and their interaction with oncoprotein expression is essential for the elucidation of lung cancer pathogenesis.     

Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling.

PURPOSE: Generation of a reactive stroma environment occurs in many human cancers and is likely to promote tumorigenesis. However, reactive stroma in human prostate cancer has not been defined. We examined stromal cell phenotype and expression of extracellular matrix components in an effort to define the reactive stroma environment and to determine its ontogeny during prostate cancer progression. EXPERIMENTAL DESIGN: Normal prostate, prostatic intraepithelial neoplasia (PIN), and prostate cancer were examined by immunohistochemistry. Tissue samples included radical prostatectomy specimens, frozen biopsy specimens, and a prostate cancer tissue microarray. A human prostate stromal cell line was used to determine whether transforming growth factor beta1 (TGF-beta1) regulates reactive stroma. RESULTS: Compared with normal prostate tissue, reactive stroma in Gleason 3 prostate cancer showed increased vimentin staining and decreased calponin staining (P < 0.001). Double-label immunohistochemistry revealed that reactive stromal cells were vimentin and smooth muscle alpha-actin positive, indicating the myofibroblast phenotype. In addition, reactive stroma cells exhibited elevated collagen I synthesis and expression of tenascin and fibroblast activation protein. Increased vimentin expression and collagen I synthesis were first observed in activated periacinar fibroblasts adjacent to PIN. Similar to previous observations in prostate cancer, TGF-beta1-staining intensity was elevated in PIN. In vitro, TGF-beta1 stimulated human prostatic fibroblasts to switch to the myofibroblast phenotype and to express tenascin. CONCLUSIONS: The stromal microenvironment in human prostate cancer is altered compared with normal stroma and exhibits features of a wound repair stroma. Reactive stroma is composed of myofibroblasts and fibroblasts stimulated to express extracellular matrix components. Reactive stroma appears to be initiated during PIN and evolve with cancer progression to effectively displace the normal fibromuscular stroma. These studies and others suggest that TGF-beta1 is a candidate regulator of reactive stroma during prostate cancer progression.