Induced Fibroblast Activation Research Articles

MicroRNA-29b inhibits supernatants from silica-treated macrophages from inducing extracellular matrix synthesis in lung fibroblasts.

Silicosis is pathologically characterized by diffused pulmonary fibrosis and abundant deposition of extracellular matrix (ECM) components. The ECM is mainly secreted by myofibroblasts which are the activated state of fibroblasts. MicroRNA-29b (miR-29b) is one of the well-known microRNAs involved in fibrosis, but its roles in silicosis have not been specified. In this study, we hypothesized that miR-29b might play a protective role in the progression of silicosis. MTT assay, qRT-PCR, immunofluorescence and western blotting were applied. The results demonstrated that the supernatants from silica-treated macrophages not only caused the proliferation of fibroblasts (NIH-3T3 and MRC-5) but were also involved in the down-regulation of miR-29b. Meanwhile they could induce fibroblast activation, increasing the expression of ECM components such as collagen1 and collagen3, in a silica dose-dependent manner. Furthermore, overexpression of miR-29b by transfecting mimics markedly reduced the expression of ECM components and inhibited ECM synthesis. These findings indicate that miR-29b inhibits the supernatants from silica-treated macrophages from inducing extracellular matrix synthesis, thus miR-29b might have a strong anti-fibrotic capacity in silicosis and serve as a potential therapeutic agent for the treatment.

Abstract 752: Fibroblast growth factor receptor 2 - HER2 transactivation: Role of fbroblasts in the acquisition and maintenance of anti-Her2 target therapies resistance in breast cancer

Abstract INTRODUCTION Mechanisms underlying tumor progression after chemotherapy are not well understood. Therapeutic treatments can favor the clonal selection of cells with unique properties and different fitness for a given microenvironment. Indeed, tumor cells can induce changes in the structure and composition of the microenvironment to support their growth and spread. Our aim is to study if clonal selection induced by target therapies like Trastuzumab and Lapatinib favors the outgrowth of cells with different microenvironmental crosstalk capability, and in particular the role of fibroblast in the selection of these particular clones. MATERIALS AND METHODS We developed different cell lines resistant to these drugs from the parental SKBR3, BT474 and MDA-MB-453 Her2+ cells lines. We determined their molecular profile and investigated the changes in the expression of selected genes codifying soluble factors known to induce stromal changes, like chemokines, cytokines, matrix remodeling-related enzymes, angiogenic and neurogenic factors. To study the role of fibroblast in the selection of the resistant clones, and the crosstalk between these two populations, we developed fibroblast immortalized lines derived from breast cancer tumors and normal breast tissue, and study the effect of the fibroblasts in resistant induction, as well as the effect of resistant clones in the fibroblast activation. RESULTS AND DISCUSION We found that factors secreted by fibroblast can support cancer growth and progression by influencing and promoting the resistant phenotype. In our proposed model, HER2 positive BC cell lines resistant to Trastuzumab and Lapatinib have an active FGFR2 signalling pathway, able to maintain HER2 signalling by receptor transactivation. This bypasses HER2 inhibition and contributes to the maintenance of the cellular addiction to HER2 pathways even when the protein is being targeted, promoting cell proliferation and survival. Moreover, resistant cell lines secreted factors induced fibroblast activation and also modulated fibroblasts tumour promoting factors secretion pattern. As a result, after exposure to resistant cell lines secreted factors, fibroblasts become more efficient on promoting BC cell lines resistance. Similarly, the supernatant derived from fibroblasts isolated from HER2+ tumours was able to induce the activation of HER2 and FGFR2 pathways. The same effect was found in vivo, were co-injection with HER2+ tumour associated fibroblasts promoted tumour aggressiveness and resistance through HER2 and FGFR2 activation CONCLUSION These results highlight the importance of microenvironment in supporting tumor progression after chemotherapy. Citation Format: Patricia Fernandez Nogueira, Mario Mancino, Gemma Fuster, Estel Enreig, Elisabeth Ametller, Paloma Bragado, Vanessa Almendro, Pedro Gascón, Pedro Gascón. Fibroblast growth factor receptor 2 - HER2 transactivation: Role of fbroblasts in the acquisition and maintenance of anti-Her2 target therapies resistance in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 752.

Abstract 227: Leptin induces a fibroblast and endothelial cell crosstalk in tumor stroma

Abstract Background: The tissue microenvironment plays a major role in tumor development and recurrence. It is known that leptin secreted by cancer and adipose cells can modify the tumor stroma, which is composed of immune, fibroblast, and endothelial cells (EC). Leptin affects both normal and cancer associated fibroblasts. We have previously shown that leptin can modify EC directly via an increase of angiogenic features; which was linked to VEGFR signaling and Notch expression independent of VEGF. It is hypothesized that leptin induces a communication between fibroblast and endothelial cells via induction of Notch signaling, which may be related to exosome production. Methods: Non-expressing Ob-R fibroblasts, and fibroblasts transfected with Ob-R plasmid, were used to investigate leptin's actions on Notch activation. Fibroblasts were challenged with leptin (0.6, 1.2, 6.2 nM) and an inhibitor of Notch signaling activation (DAPT). Fibroblast culture supernatants were added to and human umbilical vein endothelial cells (HUVEC) cultures. Notch proteins and RNA expression and molecular markers of activated fibroblasts and derived exosomes were determined via Western blot, RT-PCR, and multiplex analyses, respectively. EC angiogenic features were also determined. Results: Our findings suggest that leptin induces fibroblast activation, which triggers the expression of Notch and the development of angiogenic features by EC. Conclusions: A novel mechanism is proposed by which leptin promotes tumor stroma development via Notch activation and a crosstalk between fibroblasts and EC. Cancer therapies targeting leptin signaling could be a new strategy for interfering with cellular processes that promotes changes of tumor stroma towards a more aggressive tumor environment. Citation Format: Viola Lanier. Leptin induces a fibroblast and endothelial cell crosstalk in tumor stroma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 227.

Barbatiman and chitosan creams as adjuvants in rabbit skin wound healing

In this study, 5% barbatiman and 5% chitosan creams were evaluated as adjuvants in the tissue repair process by secondary intention of rabbit’s skin wounds. Four equidistant wounds were induced in the dorsal skin of 20 adult male rabbits, which were submitted to healing by secondary intention and treated with 5% chitosan cream (QC, n=5), 5% barbatiman cream (BC, n=5), 2% allantoin cream (n=5), and base cream (n=5). The creams were applied with the aid of disposable spatulas after washing the wounds. The wounds were daily analyzed by clinical examination for 21 days and histological analyses were performed on the 3rd, 14th, and 21st day after induction. The microscopic evaluation of the wounds of all groups showed macroscopic features of the healing process at different time intervals. The QC and BC treatments helped in the skin repair process in rabbits when compared to the other two treatments. They induced fibroblast activation and early collagen deposition, and modulated re-epithelialization and neovascularization. Thus, it was concluded that BC and QC are efficient and economically feasible as adjuvants in the healing process of skin wounds in rabbits.

LXA4 actions direct fibroblast function and wound closure

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A4 (LXA4), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function.

Necrotic myocardial cells release damage-associated molecular patterns that provoke fibroblast activation in vitro and trigger myocardial inflammation and fibrosis in vivo.

BackgroundTissue injury triggers inflammatory responses that promote tissue fibrosis; however, the mechanisms that couple tissue injury, inflammation, and fibroblast activation are not known. Given that dying cells release proinflammatory “damage-associated molecular patterns” (DAMPs), we asked whether proteins released by necrotic myocardial cells (NMCs) were sufficient to activate fibroblasts in vitro by examining fibroblast activation after stimulation with proteins released by necrotic myocardial tissue, as well as in vivo by injecting proteins released by necrotic myocardial tissue into the hearts of mice and determining the extent of myocardial inflammation and fibrosis at 72 hours.Methods and ResultsThe freeze–thaw technique was used to induce myocardial necrosis in freshly excised mouse hearts. Supernatants from NMCs contained multiple DAMPs, including high mobility group box-1 (HMGB1), galectin-3, S100β, S100A8, S100A9, and interleukin-1α. NMCs provoked a significant increase in fibroblast proliferation, α–smooth muscle actin activation, and collagen 1A1 and 3A1 mRNA expression and significantly increased fibroblast motility in a cell-wounding assay in a Toll-like receptor 4 (TLR4)- and receptor for advanced glycation end products–dependent manner. NMC stimulation resulted in a significant 3- to 4-fold activation of Akt and Erk, whereas pretreatment with Akt (A6730) and Erk (U0126) inhibitors decreased NMC-induced fibroblast proliferation dose-dependently. The effects of NMCs on cell proliferation and collagen gene expression were mimicked by several recombinant DAMPs, including HMGB1 and galectin-3. Moreover, immunodepletion of HMGB1 in NMC supernatants abrogated NMC-induced cell proliferation. Finally, injection of NMC supernatants or recombinant HMGB1 into the heart provoked increased myocardial inflammation and fibrosis in wild-type mice but not in TLR4-deficient mice.ConclusionsThese studies constitute the initial demonstration that DAMPs released by NMCs induce fibroblast activation in vitro, as well as myocardial inflammation and fibrosis in vivo, at least in part, through TLR4-dependent signaling.

Abstract 15392: Inflammation and Ischemic Heart Failure: Monocyte-Mediated Cardiac Fibroblast Activation and Matrix Remodeling Through a Direct Cell-Cell Contact Mechanism

BACKGROUND: Following myocardial infarction (MI), activated cardiac myofibroblasts facilitate extracellular matrix (ECM) remodeling to prevent mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity, however, the mechanisms are unclear. In this study, we explored the effects of peripheral blood monocytes on human cardiac fibroblast activation in a 3D ECM microenvironment. METHODS/RESULTS: Human cardiac fibroblasts isolated from surgical human heart biopsies were seeded into 3D collagen matrices. Peripheral blood monocytes isolated from healthy human donors were co-cultured with fibroblasts. Monocytes increased fibroblast activation measured by collagen ECM contraction (17.9±11.1% increase; p<0.01) and resulted in local ECM remodeling observed by confocal microscopy. Under co-culture conditions that prevent cell-cell contact but allow interaction via paracrine factors, monocytes had minimal effects on fibroblast activation (6.4±7.0 vs.17.9±11.1% increase, respectively; p<0.01). Multiplex analysis of the co-culture media revealed an increase in the paracrine factors Transforming Growth Factor-beta 1 (TGF-β1) and Matrix Metalloproteinase 9 when monocytes and fibroblasts were cultured under cell-cell contact conditions (162.2±11.7pg/mL and 17.5±0.5ng/mL, respectively, vs. 21.8±5.7pg/mL and 4.9 ±0.4ng/mL; p<0.001). TGF-β1 blockade abolished monocyte induced cardiac fibroblast activation, as did β1-integrin. These data suggest direct cell-cell interaction between monocytes and cardiac fibroblasts through β1-integrin results in TGF-β1 release facilitating fibroblast activation and matrix remodeling. CONCLUSION: For the first time, we demonstrate that peripheral blood monocytes stimulate human cardiac fibroblast activation through a mechanism involving TGF-β1 release as a consequence of direct cell-cell interaction through β1-integrin. These data implicate inflammation as a driver of cardiac fibrosis post-MI, highlighting potential novel therapeutic targets for the treatment of ischemic HF.

AB0203 S100A4 Serum Levels Correlate with Disease Activity, Skin Fibrosis and Lung Involvement in Systemic Sclerosis

BackgroundIn our previous study we demonstrated that S100A4 is overexpressed in scleroderma (SSc) skin, SSc fibroblasts and preclinical models of SSc in a TGF-β dependent manner. We showed that S100A4...

OP0240 Signal Transducer and Activator of Transcription 3 Regulates Fibroblast Activation as A Downstream Mediator of Transforming Growth Factor-Beta

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by uncontrolled activation of fibroblasts with increased deposition of extracellular matrix. Persistent activation of TGFβ signalling is thought to play a key-role...

A3.16 Serum S100A4 correlates with skin fibrosis, lung involvement and disease activity in systemic sclerosis

Background In our previous study we demonstrated that S100A4 is overexpressed in scleroderma (SSc) skin, fibroblasts and preclinical models of SSc in a TGF-β dependent manner. We showed that S100A4...

Inhibition of casein kinase II reduces TGFβ induced fibroblast activation and ameliorates experimental fibrosis

ObjectivesCasein kinase II (CK2) is a constitutively active serine/threonine protein kinase that plays a key role in cellular transformation and tumorigenesis. The purpose of the study was to characterise whether...

Canonical Wnt signalling as a key regulator of fibrogenesis – implications for targeted therapies?

Canonical Wnt signalling belongs to the so-called morphogen pathways and plays essential roles in development and tissue homeostasis. Being such a crucial regulatory pathway, Wnt signalling is tightly controlled at different levels. However, uncontrolled activation of canonical Wnt signalling has been implicated into the pathogenesis of various human disorders. In the last years, aberrant Wnt signalling has been demonstrated in fibrotic diseases including systemic sclerosis (SSc). In this review, we will discuss the current state of research on canonical Wnt signalling in SSc. Activation of canonical Wnt signalling induces fibroblast activation with subsequent myofibroblast differentiation and excessive collagen release resulting in tissue fibrosis. Genetic or pharmacological blockade of Wnt activation ameliorates experimental fibrosis in different preclinical models. These findings have direct translational implications because several small molecule inhibitors of Wnt signalling are currently evaluated in clinical trials and some already showed first promising results.

P190B RhoGAP Overexpression in the Developing Mammary Epithelium Induces TGFβ-dependent Fibroblast Activation

Rho GTPases mediate stromal-epithelial interactions that are important for mammary epithelial cell (MEC) morphogenesis. Increased extracellular matrix (ECM) deposition and reorganization affect MEC morphogenesis in a Rho GTPase-dependent manner. Although the effects of altered ECM on MEC morphogenesis have been described, how MECs regulate stromal deposition is not well understood. Previously, we showed that p190B RhoGAP overexpression disrupts mammary gland morphogenesis by inducing hyperbranching in association with stromal alterations. We therefore hypothesized that MEC overexpression of p190B regulates paracrine interactions to impact fibroblast activation. Using a combination of in vivo morphometric and immunohistochemical analyses and primary cell culture assays, we found that p190B overexpression in MECs activates fibroblasts leading to increased collagen, fibronectin, and laminin production and elevated expression of the collagen crosslinking enzyme lysyl oxidase. Phosphorylation of the TGF-β effector SMAD2 and expression of the TGF-β target gene αSma were increased in p190B-associated fibroblasts, suggesting that elevated TGF-β signaling promoted fibroblast activation. Mechanical tension and TGF-β cooperate to activate fibroblasts. Interestingly, active TGF-β was elevated in conditioned medium from p190B overexpressing MECs compared to control MECs, and p190B overexpressing MECs exhibited increased contractility in a collagen gel contraction assay. These data suggest that paracrine signaling from the p190B overexpressing MECs may activate TGF-β signaling in adjacent fibroblasts. In support of this, transfer of conditioned medium from p190B overexpressing MECs onto wildtype fibroblasts or co-culture of p190B overexpressing MECs with wildtype fibroblasts increased SMAD2 phosphorylation and mRNA expression of ECM genes in the fibroblasts when compared to fibroblasts treated with control CM or co-cultured with control MECs. The increased ECM gene expression and SMAD2 phosphorylation were blocked by treatment with a TGF-β receptor inhibitor. Taken together, these data suggest that p190B overexpression in the mammary epithelium induces fibroblast activation via elevated TGF-β paracrine signaling.

Increased KGF Expression Promotes Fibroblast Activation in a Double Paracrine Manner Resulting in Cutaneous Fibrosis

Fibrotic disorders of the skin share the characteristic features of increased production and deposition of extracellular matrix components by activated fibroblasts. Their clinical course ranges from benign with localized cutaneous involvement to a systemic, life-threatening disease. The molecular cause for fibroblast activation remains unknown, yet epithelial-mesenchymal interactions draw mounting attention in the research field of fibrogenesis. We examined keratinocyte growth factor (KGF), a crucial molecule in fibroblast-keratinocyte cross talk, exemplarily in keloid and scleroderma, and found its expression to be increased in disease-derived fibroblasts and tissues compared with healthy controls. This overexpression induces fibroblast activation through a double paracrine mode of action. Upon KGF stimulation, the keratinocytes produced and secreted OSM (oncostatin M). Fibroblasts were in turn activated by OSM reacting with the increased expression of collagen type I-α1, fibroblast activation protein, and enhanced migration. The observed increase in collagen expression and fibroblast migration can be traced back to OSM-regulated STAT3 phosphorylation, leading to enhanced urokinase plasminogen activator expression. Hence, we propose a causative loop in the pathogenesis of fibrosing disorders of the skin mediated by the overexpression of KGF in mesenchymal cells.

Progression of genotype-specific oral cancer leads to senescence of cancer-associated fibroblasts and is mediated by oxidative stress and TGF-β

Keratinocyte senescence acts as a barrier to tumor progression but appears to be lost in late pre-malignancy to yield genetically unstable oral squamous cell carcinomas (GU-OSCC); a subset of OSCC possessing wild-type p53 and are genetically stable (GS-OSCC). In this study, fibroblasts from GU-OSCC were senescent relative to fibroblasts from GS-OSCC, epithelial dysplastic tissues or normal oral mucosa, as demonstrated by increased senescence-associated β-galactosidase (SA β-Gal) activity and overexpression of p16(INK4A). Keratinocytes from GU-OSCC produced high levels of reactive oxygen species (ROS) and this was associated with an increase in the production of transforming growth factor-β1 (TGF-β1) and TGF-β2 in stromal fibroblasts. Treatment of normal fibroblasts with keratinocyte conditioned media (CM) from GU-OSCC, but not GS-OSCC or dysplastic keratinocytes with dysfunctional p53, induced fibroblast senescence. This phenomenon was inhibited by antioxidants and anti-TGF-β antibodies. Fibroblast activation by TGF-β1 preceded cellular senescence and was associated with increased ROS levels; antioxidants inhibited this reaction. Senescent fibroblasts derived from GU-OSCC or normal fibroblasts treated with CM from GU-OSCC or hydrogen peroxide, but not non-senescent fibroblasts derived from GS-OSCC, promoted invasion of keratinocytes in vitro. Epithelial invasion was stimulated by fibroblast activation and amplified further by fibroblast senescence. The data demonstrate that malignant keratinocytes from GU-OSCC, but not their pre-malignant counterparts, produce high levels of ROS, which, in turn, increase TGF-β1 expression and induce fibroblast activation and senescence in a p5-independent manner. Fibroblasts from GU-OSCC were particularly susceptible to oxidative DNA damage because of high levels of ROS production, downregulation of antioxidant genes and upregulation of pro-oxidant genes. The results demonstrate the functional diversity of cancer-associated fibroblasts and show that malignant keratinocytes from GU-OSCC reinforce their malignant behavior by inducing fibroblast activation and senescence through ROS and TGF-β-dependent mechanisms.

Celecoxib induces proliferation and Amphiregulin production in colon subepithelial myofibroblasts, activating erk1–2 signaling in synergy with EGFR

The COX-2 inhibitor Celecoxib, tested in phase III trials for the prevention of sporadic colon adenomas, reduced the appearance of new adenomas, but was unable to affect the incidence of colon cancer. Moreover the 5years follow-up showed that patients discontinuing Celecoxib treatment had an increased incidence of adenomas as compared to the placebo arm. In the APC(min/+) mouse model short term treatment with Celecoxib reduced gut adenomas, but a prolonged administration of the drug induced fibroblast activation and intestinal fibrosis with a final tumor burden. The way Celecoxib could directly activate human colon myofibroblasts (MF) has not yet been investigated.We found that MF are activated by non toxic doses of Celecoxib. Celecoxib induces erk1–2 and Akt phosphorylation within 5′. This short term activation is apparently insufficient to cause phenotypic changes, but the contemporary triggering of EGFR causes an impressive synergic effect inducing MF proliferation and the neo-expression and release of Amphiregulin (AREG), a well known EGFR agonist involved in colon cancer progression. As a confirm to these observations, the erk inhibitor U0126 and the EGFR inhibitors Tyrphostin and Cetuximab were able to contrast AREG induction.Our data provide evidence that Celecoxib directly activates MF empowering EGFR signaling. According to these results the association with EGFR (or erk1–2) inhibitors could abolish the off-target activity of Celecoxib, possibly extending the potential of this drug for colon cancer prevention.

Gene expression in extratumoral microenvironment predicts clinical outcome in breast cancer patients

IntroductionA gene expression signature indicative of activated wound responses is common to more than 90% of non-neoplastic tissues adjacent to breast cancer, but these tissues also exhibit substantial heterogeneity. We hypothesized that gene expression subtypes of breast cancer microenvironment can be defined and that these microenvironment subtypes have clinical relevance.MethodsGene expression was evaluated in 72 patient-derived breast tissue samples adjacent to invasive breast cancer or ductal carcinoma in situ. Unsupervised clustering identified two distinct gene expression subgroups that differed in expression of genes involved in activation of fibrosis, cellular movement, cell adhesion and cell-cell contact. We evaluated the prognostic relevance of extratumoral subtype (comparing the Active group, defined by high expression of fibrosis and cellular movement genes, to the Inactive group, defined by high expression of claudins and other cellular adhesion and cell-cell contact genes) using clinical data. To establish the biological characteristics of these subtypes, gene expression profiles were compared against published and novel tumor and tumor stroma-derived signatures (Twist-related protein 1 (TWIST1) overexpression, transforming growth factor beta (TGF-β)-induced fibroblast activation, breast fibrosis, claudin-low tumor subtype and estrogen response). Histological and immunohistochemical analyses of tissues representing each microenvironment subtype were performed to evaluate protein expression and compositional differences between microenvironment subtypes.ResultsExtratumoral Active versus Inactive subtypes were not significantly associated with overall survival among all patients (hazard ratio (HR) = 1.4, 95% CI 0.6 to 2.8, P = 0.337), but there was a strong association with overall survival among estrogen receptor (ER) positive patients (HR = 2.5, 95% CI 0.9 to 6.7, P = 0.062) and hormone-treated patients (HR = 2.6, 95% CI 1.0 to 7.0, P = 0.045). The Active subtype of breast microenvironment is correlated with TWIST-overexpression signatures and shares features of claudin-low breast cancers. The Active subtype was also associated with expression of TGF-β induced fibroblast activation signatures, but there was no significant association between Active/Inactive microenvironment and desmoid type fibrosis or estrogen response gene expression signatures. Consistent with the RNA expression profiles, Active cancer-adjacent tissues exhibited higher density of TWIST nuclear staining, predominantly in epithelium, and no evidence of increased fibrosis.ConclusionsThese results document the presence of two distinct subtypes of microenvironment, with Active versus Inactive cancer-adjacent extratumoral microenvironment influencing the aggressiveness and outcome of ER-positive human breast cancers.

The Role of the Ethylacetate Fraction from Hydnocarpi Semen in Acute InflammationIn VitroModel

We previously reported that Hydnocarpi Semen (HS) has a wound healing effect on diabetic foot ulcer lesion in mice. In this study, ethylacetate (EtOAc) fraction from HS extract were evaluated for their wound healing activity by using in vitro acute inflammation model. GC and GC/MS analysis shows that the main constituents in EtOAc fraction are chaulmoogric acid, hydnocarpic acid, and gorlic acid. EtOAc fraction activated macrophages to increase the production of TNF-α. The fraction also increased the production of TGF-β and VEGF, which induced fibroblast activation and angiogenesis. These results suggest that the mechanism that the fraction helps to enhance healing of skin wound is possibly associated with the production of TNF-α, as well as secretion of VEGF, TGF-β and HS may have a new bioactive material for the treatment of skin wound.

Ultraviolet exposure of melanoma cells induces fibroblast activation protein-α in fibroblasts: Implications for melanoma invasion

Fibroblast activation protein-α (FAP-α) promotes tumor growth and cell invasiveness through extracellular matrix degradation. How ultraviolet radiation (UVR), the major risk factor for malignant melanoma, influences the expression of FAP-α is unknown. We examined the effect of UVR on FAP-α expression in melanocytes, keratinocytes and fibroblasts from the skin and in melanoma cells. UVR induces upregulation of FAP-α in fibroblasts, melanocytes and primary melanoma cells (PM) whereas keratinocytes and metastatic melanoma cells remained FAP-α negative. UVA and UVB stimulated FAP-α-driven migration and invasion in fibroblasts, melanocytes and PM. In co-culture systems UVR of melanocytes, PM and cells from regional metastases upregulated FAP-α in fibroblasts but only supernatants from non-irradiated PM were able to induce FAP-α in fibroblasts. Further, UV-radiated melanocytes and PM significantly increased FAP-α expression in fibroblasts through secretory crosstalk via Wnt5a, PDGF-BB and TGF-β1. Moreover, UV radiated melanocytes and PM increased collagen I invasion and migration of fibroblasts. The FAP-α/DPPIV inhibitor Gly-ProP(OPh)2 significantly decreased this response implicating FAP-α/DPPIV as an important protein complex in cell migration and invasion. These experiments suggest a functional association between UVR and FAP-α expression in fibroblasts, melanocytes and melanoma cells implicating that UVR of malignant melanoma converts fibroblasts into FAP-α expressing and ECM degrading fibroblasts thus facilitating invasion and migration. The secretory crosstalk between melanoma and tumor surrounding fibroblasts is mediated via PDGF-BB, TGF-β1 and Wnt5a and these factors should be evaluated as targets to reduce FAP-α activity and prevent early melanoma dissemination.

Bacillus Calmette Guerin induces fibroblast activation both directly and through macrophages in a mouse bladder cancer model.

BackgroundBacillus Calmette-Guerin (BCG) is the most effective treatment for non-muscle invasive bladder cancer. However, a failure in the initial response or relapse within the first five years of treatment has been observed in 20% of patients. We have previously observed that in vivo administration of an inhibitor of nitric oxide improved the response to BCG of bladder tumor bearing mice. It was described that this effect was due to a replacement of tumor tissue by collagen depots. The aim of the present work was to clarify the mechanism involved in this process.Methodology/Principal FindingsWe demonstrated that BCG induces NIH-3T3 fibroblast proliferation by activating the MAPK and PI3K signaling pathways and also differentiation determined by alpha-smooth muscle actin (alpha-SMA) expression. In vivo, intratumoral inoculation of BCG also increased alpha-SMA and collagen expression. Oral administration of L-NAME enhanced the pro-fibrotic effect of BCG. Peritoneal macrophages obtained from MB49 tumor-bearing mice treated in vivo with combined treatment of BCG with L-NAME also enhanced fibroblast proliferation. We observed that FGF-2 is one of the factors released by BCG-activated macrophages that is able to induce fibroblast proliferation. The involvement of FGF-2 was evidenced using an anti-FGF2 antibody. At the same time, this macrophage population improved wound healing rate in normal mice and FGF-2 expression was also increased in these wounds.Conclusions/SignificanceOur findings suggest that fibroblasts are targeted by BCG both directly and through activated macrophages in an immunotherapy context of a bladder murine model. We also described, for the first time, that FGF-2 is involved in a dialog between fibroblasts and macrophages induced after BCG treatment. The fact that L-NAME administration improves the BCG effect on fibroblasts, NO inhibition, might represent a new approach to add to the conventional BCG therapy.