Esophageal Fibroblasts Research Articles

429 Maturational Changes in Esophageal Fibroblasts: Implications Upon the Natural History and Treatment of Eosinophilic Esophagitis

429 Maturational Changes in Esophageal Fibroblasts: Implications Upon the Natural History and Treatment of Eosinophilic Esophagitis

Correlation of cancer-associated fibroblasts with tumour cell invasion and chemoresistance in oesophageal adenocarcinoma

BackgroundThe incidence of oesophageal adenocarcinoma is rising rapidly; treatment options are associated with high morbidity and poor prognosis. The initiating events are believed to be related to chronic reflux in the lower oesophagus leading to mucosal and stromal inflammation. The transforming growth factor β (TGFβ) superfamily mediates the inflammatory response in a range of tissues, and TGFβ signalling is seen in the stroma of gastrointestinal cancers. Furthermore, TGFβ is the dominant driver in the activation of resident stromal fibroblasts into the secretory and contractile myofibroblast phenotype, characterised by expression of α smooth muscle actin (αSMA) and observed in most cancer-associated fibroblasts (CAF). We hypothesised that CAF are present in oesophageal adenocarcinoma and that they have a direct effect on tumour biology leading to increased invasion and chemoresistance. MethodsPrimary normal oesophageal fibroblasts (NOF), and CAF were cultured directly from oesophageal resection specimens, and their phenotype confirmed by western blotting and immunofluorescence for αSMA. We assessed functional ability of NOF and CAF to promote oesophageal adenocarcinoma cell invasion and chemoresistance using organotypic culture, transwell invasion assays, collagen-1 gel contraction assays, siRNA gene silencing, and colony forming assays. The multifunctional extracellular matrix protein, periostin, which has tumour promoting effects in other cancers, was examined as a potential CAF-secreted molecular target in oesophageal adenocarcinoma. t tests were done for all statistical analyses. Survival data were analysed by Kaplan-Meier plots (SPSS, version 19). FindingsCAF showed high αSMA expression and increased collagen contractility compared with NOF, suggesting a myofibroblast like phenotype (p<0·01). Compared with NOF, oesophageal adenocarcinoma cell invasion doubled in response to CAF-conditioned medium in transwell invasion assays and in organotypic models containing co-cultured fibroblasts and oesophageal adenocarcinoma cells (OE33 and FLO-1 cell lines, p<0·05). Reduction of CAF-secreted periostin by siRNA and immunodepletion resulted in a significant (p=0·02) reduction in cancer cell invasion in transwell invasion assays that could be rescued with recombinant periostin, and a complete loss of invasion in organotypic culture. Moreover, collagen gel contraction was abolished by periostin downregulation in CAF. NOF exposed to TGFβ for 72 h demonstrated myofibroblast transdifferentiation including high αSMA expression, secretion of periostin, and the ability to significantly promote oesophageal adenocarcinoma cell invasion to a similar degree as CAF. There was a significant correlation between periostin expression assessed in 183 oesophageal resections and disease-specific survival (high periostin=worse survival, p<0·05). CAF-conditioned medium supported more cancer cell colony formation in the presence of cisplatin and fluorouracil than did NOF-conditioned medium (p<0·05). When CAF-conditioned medium was depleted of periostin, this protective effect was lost, but could be rescued with recombinant periostin. InterpretationThis study highlights the role of the tumour microenvironment in cancer development and progression in the oesophagus. CAF are present in the oesophagus and promote oesophageal adenocarcinoma cell invasion and chemoresistance via periostin secretion in vitro. The CAF phenotype can be induced by exposing oesophageal NOF to TGFβ in vitro and this is likely to represent the molecular driving force for fibroblast transdifferentiation in vivo. Manipulation of CAF transdifferentiation and interruption of CAF-secreted periostin signalling in cancer cells might offer novel therapeutic targets in oesophageal adenocarcinoma. FundingNational Institute of Health Research, UK Medical Research Council.

T-Helper 2 Cytokines, Transforming Growth Factor β1, and Eosinophil Products Induce Fibrogenesis and Alter Muscle Motility in Patients With Eosinophilic Esophagitis

Patients with eosinophilic esophagitis (EoE) often become dysphagic from the combination of organ fibrosis and motor abnormalities. We investigated mechanisms of dysphagia, assessing the response of human esophageal fibroblasts (HEFs), human esophageal muscle cells (HEMCs), and esophageal muscle strips to eosinophil-derived products. Biopsy specimens were collected via endoscopy from the upper, middle, and lower thirds of the esophagus of 18 patients with EoE and 21 individuals undergoing endoscopy for other reasons (controls). Primary cultures of esophageal fibroblasts and muscle cells were derived from 12 freshly resected human esophagectomy specimens. Eosinophil distribution was investigated by histologic analyses of full-thickness esophageal tissue. Active secretion of EoE-related mediators was assessed from medium underlying mucosal biopsy cultures. We quantified production of fibronectin and collagen I by HEF and HEMC in response to eosinophil products. We also measured the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by, and adhesion of human eosinophils to, HEFs and HEMCs. Eosinophil products were tested in an esophageal muscle contraction assay. Activated eosinophils were present in all esophageal layers. Significantly higher concentrations of eosinophil-related mediators were secreted spontaneously in mucosal biopsy specimens from patients with EoE than controls. Exposure of HEFs and HEMCs to increasing concentrations of eosinophil products or co-culture with eosinophils caused HEFs and HEMCs to increase secretion of fibronectin and collagen I; this was inhibited by blocking transforming growth factor β1 and p38 mitogen-activated protein kinase signaling. Eosinophil binding to HEFs and HEMCs increased after incubation of mesenchymal cells with eosinophil-derived products, and decreased after blockade of transforming growth factor β1 and p38 mitogen-activated protein kinase blockade. Eosinophil products reduced electrical field-induced contraction of esophageal muscle strips, but not acetylcholine-induced contraction. In an analysis of tissues samples from patients with EoE, we linked the presence and activation state of eosinophils in EoE with altered fibrogenesis and motility of esophageal fibroblasts and muscle cells. This process might contribute to the development of dysphagia.

408 TH2 Cytokines Stimulate Eotaxin-3 Secretion by Esophageal Fibroblasts: An Effect That Is Not Blocked by Omeprazole.

408 TH2 Cytokines Stimulate Eotaxin-3 Secretion by Esophageal Fibroblasts: An Effect That Is Not Blocked by Omeprazole.

Abstract 4946: Novel photoimmunotherapy (PIT) targeting cancer-associated fibroblasts (CAFs) for esophageal cancer.

Abstract Introduction: CAFs are thought to play an essential role in cancer invasion and metastasis. We have developed the novel therapy targeting CAFs. Originally, CAFs have been defined as fibroblasts which express α-SMA in cancer tissue. As α-SMA express in cytoplasm, it can't become the target by itself. However recently it was reported that fibroblast activation protein (FAP) is functional and specific surface protein for CAFs. The aim of this study is to analyze CAFs and FAP expression of esophageal cancer and to develop the new therapy that targeted CAFs by PIT for esophageal cancer. Here we suggest a novel method of an antitumor effect by killing CAF itself. Matherials and Methods: TE-4 and TE-8 (esophageal squamous cell carcinoma), FEF3 (fetal esophageal fibroblast 3),GEP-FEF3 cell lines were used in this study. 1) The specimens of esophageal cancer were double immunostained with α-SMA and FSP1 antibodies to count CAFs. 2) In vivo study, TE cells and TE cells+activated FEF3 in the two groups were compared with tumor progression. 3) The interaction of activated FEF3 and TE cells was examined about the expression of α-SMA and FAP by western blotting and flowcytometry. 4) We examined the surface expression of FAP in activated FEF3 by immunohistochemistory(IHC). 5) We used a target-specific photosensitizer based on a near-infrared(NIR) phthalocyanine dye, IR700, conjugated to monoclonal antibodies targeting FAP. In vitro and in vivo, we observed whether cell proliferation and tumorigenicity are controlled after irradiation with NIR light in FEF3 expressing FAP conjugated FAP-IR700. Results: 1) The expression of CAFs was recognized as a deeper invasion depth, moreover the CAFs has tended to express more in the periphery and the leading parts. 2) In vivo, the tumors co-injected TE cells and CAFs tended to grow more than cancer cell alone. 3) In western blotting, FAP expression of FEF3 activated by TGF-β and co-cultured with TE cells was detected more than that of normal status.In flowcytometry, FAP expression of FEF3 activated by TGF-β, co-cultured with TE cells and cultured in TE cells conditioned medium was detected more than that of normal status. 4) In IHC, FAP expression of FEF3 activated by TGF-β and co-cultured with TE cells was increased more than that of normal status.5.We could suppress the proliferation of activated FEF3 cells in vitro with FAP-IR700 mediated PIT. And in vivo, the tumor with FAP-IR700 was also suppressed. Conclusion: In conclusion, we demonstrate that CAF contributed to tumor invasion and progression in surgical specimens and it was represented in vivo, which indicated that CAF is essential for cancer development. Furthermore, here we suggest novel therapeutic concept of “Targeting CAF by PIT” for cancer progression. The PIT for CAF is a unique strategy and can bring us a new type theory as combination targeting cancer cells and their fundamental microenvironment. Citation Format: Shinichiro Watanabe, Kazuhiro Noma, Shinichi Urano, Toshiaki Ohara, Yuuri Hashimoto, Hiroshi Tazawa, Toshiyoshi Fujiwara. Novel photoimmunotherapy (PIT) targeting cancer-associated fibroblasts (CAFs) for esophageal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4946. doi:10.1158/1538-7445.AM2013-4946

Esophageal epithelial and mesenchymal cross-talk leads to features of epithelial to mesenchymal transition in vitro

BackgroundEsophageal fibrosis is a complication of eosinophilic esophagitis (EoE) which has been attributed to both subepithelial fibrosis and to epithelial to mesenchymal transition (EMT), a process by which epithelial cells acquire mesenchymal features. Common to both causes of EoE-fibrosis is the notion that granulocyte-derived TGF-β, induces myofibroblast differentiation of the target cell. To date, the role of esophageal epithelial cells as effector cells in esophageal fibrosis has never been explored. Herein, we investigated consequences of cross-talk between esophageal epithelial cells and fibroblasts, and identified profibrotic cytokines which influence the development of EMT in vitro. Methods and resultsStimulation of primary fetal esophageal fibroblasts (FEF3) with conditioned media (CEM) from esophageal epithelial cells (EPC2-hTERT), primed FEF3 cells to secrete IL-1β and TNFα, but not TGFβ. To determine whether these cytokines signaled in a paracrine fashion to esophageal epithelial cells, FEF3 cells were stimulated with CEM, followed by transfer of this fibroblast conditioned media (FCM) to EPC2-hTERT cells. Epithelial FCM stimulation increased expression of mesenchymal markers and reduced E-cadherin expression, features of EMT which were TNFα and IL-1β-dependent. Using organotypic culture models, primary EoE epithelial cells exhibited features of EMT compared to non-EoE cells, corresponding to patterns of EMT in native biopsies. ConclusionsEsophageal epithelial cell and fibroblast cross-talk contributes to esophageal fibrosis. Our results suggest that features of EMT can develop independent of TGF-β and granulocytes, which may have important implications in treatment of EoE.

600 Esophageal Epithelial and Mesenchymal Cross-Talk Leads to Features of Epithelial to Mesenchymal Transition (EMT) In Vitro

Background and Aims: Subepithelial fibrosis has recently been recognized as a histologic feature of eosinophilic esophagitis (EoE), a chronic inflammatory disease which leads to esophageal stricture, dysphagia, and recurrent food impaction. It is widely believed that activation by pro-fibrogenic cytokines including TGF-β, TNF-α, and IL-1β, stimulates fibroblasts to acquire the activated phenotype of myofibroblasts, morphologic intermediates between fibroblasts and smooth muscle cells which synthesize extracellular matrix (ECM) components. In the context of EoE, it has been suggested that these pro-fibrotic cytokines are released by circulating innate immune effector cells including mast cells and eosinophils, leading to fibroblast differentiation to the myofibroblast phenotype. To date, the potential role of esophageal epithelial cells as effector cells in EoE-associated tissue remodeling has never been investigated. In the current study, we investigated the consequences of crosstalk between esophageal epithelial cells and esophageal fibroblasts, and explored the ability of esophageal epithelial cells to develop features characteristic of epithelial to mesenchymal transition (EMT) In Vitro. Methods and Results: To determine whether factors secreted by esophageal epithelial cells can influence fibroblast expression of pro-fibrotic genes, we stimulated primary human fetal esophageal fibroblasts (FEF3 cells) with conditioned epithelial media (CEM) harvested from confluent non-transformed immortalized esophageal epithelial cells (EPC2-hTERT). Using qRT-PCR, we quantified FEF3 expression of profibrotic cytokines TGF-β, TNF-α, and IL-1β at various time points after CEM stimulation. Surprisingly, we observed a robust induction in mRNA expression of IL-1β and TNF-α, with a modest induction of TGF-β after stimulation. Based upon this unexpected result, we hypothesized that fibroblast derived factors might induce epithelial to mesenchymal transition (EMT) In Vitro. To test this hypothesis, we cultured FEF3 cells in CEM for 2 days, and transferred this fibroblast-conditioned CEM onto EPC2-hTERT cells for 5 days. Surprisingly, this led to enhanced esophageal epithelial expression of α-smooth muscle actin (SMA) and decreased expression of epithelial-specific e-cadherin, features suggestive of EMT. Similar changes in α-SMA and e-cadherin expression were observed after EPC2-hTERT cells were cultured with recombinant TGF-β, TNF-α, and IL-1β. Conclusions: There is a complex interplay between the esophageal epithelium and its underlying matrix. Our results suggest for the first time that paracrine signals from epithelium prime adjacent fibroblasts to express profibrotic cytokines and that these cytokines may, in turn, play a role in epithelial to mesenchymal transition by human esophageal epithelial cells In Vitro.

Abstract 1495: Role of stromal fibroblasts in the progression of esophageal squamous cell carcinoma

Abstract Objectives: The prevalence of esophageal squamous-cell carcinoma (ESCC) is high in Asia, including Japan. Cancer results from the accumulated effects of many genetic alterations. However, the microenvironment of cancer cells has recently been shown to strongly influence the biologic properties of cancer. In this study, we focused on interrelations between cancer and fibroblasts, the main component of cancer stroma. Methods: We purchased 22 cell lines of esophageal squamous-cell carcinoma from RIKEN Cell Bank and the Japanese Collection of Research Bioresources. For fibroblasts we used human esophageal fibroblasts isolated from surgical specimens. To examine interactions between esophageal squamous-cell carcinoma and fibroblasts, the following in vitro experiments were performed: 1) cell proliferation assays; 2) cell migration assays; 3) 3-dimensional organotypic culture; and 4) time-lapse cinematography of 3-dimentional culture. For in vivo experiments, cancer cells and fibroblasts were co-transplanted subcutaneously in NOD/SCID mice to assess the effects of fibroblasts on tumorigenicity. We also retrospectively analyzed 97 ESCC patients to study the relationship between tumor stroma and pathological findings. Results: Fibroblast culture supernatant increased the cell count in 14 of 22 cell lines. The stimulating effect of fibroblast supernatant could be explained by various growth factors and cytokines; HGF and IL-6 stimulated the proliferation of 6 and 5 cancer cell lines, respectively, while FGF1, FGF7, and FGF10 increased cell proliferation of 7, 6, and 6 cancer cell lines, respectively. When the MET inhibitor PHA-665752 was added to the fibroblast culture supernatant, the culture supernatant-induced proliferation of was abrogated in 2 cell lines. Among 10 cell lines whose proliferation was induced by FGFs, addition of FGFR inhibitor PD-173074 to the fibroblast culture supernatant abrogated the effect of the culture supernatant in 4 cell lines. In migration assays, the presence of fibroblasts significantly increased the migration of cancer cells in 3 of 6 representative cell lines. In organotypic culture, stromal invasion was seen only in presence of fibroblasts. Time-lapse cinematography confirmed that clusters of cancer cells progressively migrated towards the fibroblast meshwork. In vivo tumor growth was promoted by co-injection of fibroblasts in 13 of 22 cancer cell lines. In clinicopathological study, the extent of stromal fibrosis was significantly related to depth of invasion and venous invasion of tumor cells. Conclusion: In many esophageal cancer cell lines, cell proliferation was promoted by the presence of fibroblasts both in vivo and in vitro. The stimulatory effect of fibroblast could be accounted for by various growth factors and cytokines, such as HGF, interleukin-6 and/or FGFs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1495. doi:1538-7445.AM2012-1495

Development and characterization of an organotypic model of Barrett's esophagus

Understanding the molecular and cellular processes underlying the development, maintenance, and progression of Barrett's esophagus (BE) presents an empirical challenge because there are no simple animal models and standard 2D cell culture can distort cellular processes. Here we describe a three-dimensional (3D) cell culture system to study BE. BE cell lines (CP-A, CP-B, CP-C, and CP-D) and esophageal squamous keratinocytes (EPC2) were cultured on a matrix consisting of esophageal fibroblasts and collagen. Comparison of growth and cytokeratin expression in the presence of all-trans retinoic acid or hydrochloric acid was made by immunohistochemistry and Alcian Blue staining to determine which treatments produced a BE phenotype of columnar cytokeratin expression in 3D culture. All-trans retinoic acid differentially affected the growth of BE cell lines in 3D culture. Notably, the non-dyplastic metaplasia-derived cell line (CP-A) expressed reduced squamous cytokeratins and enhanced columnar cytokeratins upon ATRA treatment. ATRA altered the EPC2 squamous cytokeratin profile towards a more columnar expression pattern. Cell lines derived from patients with high-grade dysplasia already expressed columnar cytokeratins and therefore did not show a systematic shift toward a more columnar phenotype with ATRA treatment. ATRA treatment, however, did reduce the squamoid-like multilayer stratification observed in all cell lines. As the first study to demonstrate long-term 3D growth of BE cell lines, we have determined that BE cells can be cultured for at least 3 weeks on a fibroblast/collagen matrix and that the use of ATRA causes a general reduction in squamous-like multilayered growth and an increase in columnar phenotype with the specific effects cell-line dependent.

Suppression of transforming growth factor-beta type II receptor expression by RNA interference inhibits postoperative anastomotic stricture in esophageal cancer patients

Objective To study the effects of inhibition of transforming growth factor-beta type Ⅱ receptor (TGF-βRⅡ ) in esophageal fibroblasts on scar formation.Methods Primary esophageal fibroblasts were cultured from endoscopic biopsy specimens of esophageal anastomotic stricture after operation in 5 patients with esophageal cancer and identified by using immunocytochemistry.TGF-βR Ⅱ specific small interfering RNA (siRNAs) designed from the human gene sequence were transfected into cultured fibroblasts.The tansfection efficiency was observed by fluorescence microscope,the expression of TGF-βRⅡand fibronectin were detected by Western blotting and reverse transcription-polymerase chain reaction ( RTPCR),and wound scratch assay was used to evaluate migration of esophageal fibroblasts.Results Primary esophageal fibroblasts were successfully cultured and positive for Vimentin.The transfection efficiency of siRNAs was above 85％ at 48 h after transfection.As compared with control groups,in TGF-βRⅡ specific siRNA targeted fibroblasts,the expression of TGF-βRⅡ and fibronectin was inhibited (P ＜ 0.05) and cell migration was decreased by (70.6 ± 9.5) ％.Conclusion TGF-βRⅡ specific siRNA targeted esophageal fibroblasts can inhibit the scar formation. Key words: Esophageal carcinoma; Anastomotic stricture; RNA interference; Transforming growth factor-beta type Ⅱ receptor

Ionizing Radiation Enhances Esophageal Epithelial Cell Migration and Invasion Through a Paracrine Mechanism Involving Stromal-Derived Hepatocyte Growth Factor

Esophageal cancer is the sixth leading cause of cancer death worldwide and the seventh leading cause of cancer death in the U.S. male population. Ionizing radiation exposure is a risk factor for development of esophageal squamous cell carcinoma, a histological subtype of esophageal cancer that is highly aggressive and is associated with poor patient prognosis. This study investigated the effects of ionizing radiation on the microenvironment and intercellular communication as it relates to esophageal carcinogenesis. We demonstrate that normal esophageal epithelial cells exhibited increased migration and invasion when cultured in the presence of irradiated stromal fibroblasts or with conditioned medium derived from irradiated stromal fibroblasts. Cytokine antibody arrays and ELISAs were used to identify hepatocyte growth factor (HGF) as an abundant protein that is secreted by esophageal fibroblasts at twofold increased levels in culture medium after γ irradiation. Reverse transcription qPCR analysis confirmed an approximately 50% increase in mRNA levels for HGF at 1 h in irradiated fibroblasts compared to unirradiated controls. Recombinant HGF stimulated increased wound healing, migration and invasion of esophageal epithelial cells, while blocking antibodies against HGF significantly decreased migration and invasion of epithelial cells in coculture with irradiated fibroblasts. Since HGF is known to direct cell migration, invasion and metastasis in a variety of tissues, including the esophagus, its modulation by ionizing radiation may have important implications for nontargeted pathways that influence radiation carcinogenesis in the esophagus.

NF‐κB is activated in oesophageal fibroblasts in response to a paracrine signal generated by acid‐exposed primary oesophageal squamous cells

Oesophageal exposure to duodenogastro-oesophageal refluxate leads to reflux oesophagitis and is implicated in the development of Barrett's metaplasia (BM). NF-κB signalling in epithelial cells is associated with the activation of transcription factors believed to be central to BM development, whilst NF-κB activation in fibroblasts plays a critical role in matrix remodelling. Our aim was to study the effects of acid exposure on NF-κB activation in primary human oesophageal fibroblasts (HOFs) and primary and immortalized oesophageal squames and to investigate any epithelial/stromal interactions in the response of these cells to acid. Primary HOFs and primary and immortalized oesophageal epithelial cells were exposed to acid (pH 7 - pH 4 ≤ 120 min) in single or pulsed treatments. Conditioned medium from epithelial cells following acid exposure was also applied to fibroblasts. Cell viability was determined by MTT-ESTA. NF-κB activation was determined by cellular localization of NF-κB/p65 visualized by immunofluorescence. Conditioned medium from oesophageal epithelial cells, subjected to pH 5 pulsatile exposure, activated NF-κB in fibroblasts, with some inter-patient variability, but these conditions did not directly activate NF-κB in the epithelial cells themselves. Significant NF-κB activation was seen in the epithelial cells but only with greater acidity and exposure times (pH 4, 60-120 min). Our findings show that acid exposure can cause indirect activation of stromal cells by epithelial-stromal interactions. This may contribute to the pathogenesis of oesophageal diseases, and the inter-patient variability may go some way to explain why some patients with reflux oesophagitis develop BM and others do not.

Abstract 3104: Stromal fibroblasts promote progression of esophageal squamous cell carcinoma cells both in vitro and in vivo

Abstract Objectives: The purpose of the study was to clarify the role of fibroblasts in the proliferation and growth pattern of esophageal squamous cell carcinoma and to experimentally study interactions between cancer cells and fibroblasts. Methods: We used 22 esophageal squamous cell carcinoma cell lines (mainly, TE series and KYSE series obtained from the RIKEN Cell Bank and the Japanese Collection of Research Bioresources, respectively) and human esophageal fibroblasts that had been established in our laboratory from surgical specimens. Cancer cells were cultured on Matrigel in the presence or absence of fibroblasts in the Matrigels, and their growth patterns were examined. Cell count assays were performed to examine the effects of the fibroblast culture supernatant, growth factors, cytokines, and various inhibitors on cancer cell growth. We also examined the influence of co-injection of fibroblasts on tumor growth in immunodeficient mice. Results: When plated on Matrigel, nearly all cell lines spread and grew in an invasive manner in the presence of fibroblasts, while in the absence of fibroblasts, most cell lines formed round or spheroidal colonies. Cell-count assay showed that in most cancer cells, cell proliferation was stimulated by addition of fibroblast supernatant. In particular, cell proliferation increased by more than 50% in 11 cell lines. Addition of hepatocyte growth factor (HGF) or interleukin-6 elicited a definitive proliferative response in only 2 and 3 cell lines, respectively. Both KYSE-220 and KYSE-50 proliferated in the presence of HGF, but the addition of MET inhibitor (PHA665752) suppressed the proliferation of only the former. In vivo tumor growth was stimulated by co-injection of fibroblasts in 9 of 22 cancer cell lines. Conclusions: Fibroblast had a profound influence on cell growth pattern on Matrigel. In many esophageal cancer cell lines, cell proliferation was promoted by the presence of fibroblasts both in vivo and in vitro. The stimulatory effect of fibroblast could be accounted for by HGF and/or interleukin-6 in some, but not in all, esophageal cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3104. doi:10.1158/1538-7445.AM2011-3104

Modulating Cell Adhesion Dynamics on Carbon Nanotube Monolayer Engineered with Extracellular Matrix Proteins

Although it has been demonstrated that carbon nanotubes (CNTs) may have potentials for tissue engineering applications because of their unparalleled physical properties, little has been known on the cell adhesion mechanisms on model CNT monolayer pertaining to the design of novel cell therapeutics device. In this study, the adhesion dynamics of primary porcine esophageal fibroblasts (PEFs) on CNT monolayer were elucidated with confocal reflectance interference contrast microscopy (C-RICM) integrating with phase contrast microscopy. Moreover, CNT monolayer (CNT-ML) was functionalized with two typical extracellular matrix (ECM) proteins including collagen type I (COL) and fibronectin (FN) in order to promote its biocompatibility. First, it is shown by atomic force microscopy that the topographical features of CNT-ML were dependent on the types of immobilized ECM protein. Second, significant time lag in adhesion contact evolution (around 10 min) for PEFs was found on both CNT-ML and CNT-COL compared to the negligible time lag on CNT-FN. It was found that adhesion energy of PEFs on the CNT-COL and CNT-FN surfaces reached steady state at 60 and 30 min after cell seeding compared to 70 min on CNT-ML surface. At steady state, the adhesion energy of PEFs on the CNT-COL and CNT-FN surfaces was about twice as much than that on the CNT-ML surface. Moreover, immobilization of collagen or fibronectin on CNT monolayer led to an increase in seeding efficiency and proliferation rate of PEFs. Scanning electron microscopy and immunostaining together demonstrated that PEFs displayed an elongated morphology and highly polarized actin network on both CNT-COL and CNT-FN surfaces, whereas PEFs displayed nonuniform cell morphology and actin organization on the CNT-ML surface. Overall, our results demonstrated that the biophysical responses and biological behavior of PEFs on unmodified or functionalized CNT monolayer were different. Functionalization of CNT through extracellular matrix protein immobilization effectively promotes cell adhesion and proliferation, which may provide information for designing CNT-based biomaterials or novel cell therapeutics devices in biomedical engineering.

A Biodegradable Esophageal Stent in the Treatment of a Corrosive Esophageal Stenosis in a Child

A Biodegradable Esophageal Stent in the Treatment of a Corrosive Esophageal Stenosis in a Child

Temporal Effect of Functional Blocking of β1 Integrin on Cell Adhesion Strength under Serum Depletion

Cell adhesion is generally concomitant to the formation of focal adhesion. Although it is well-known that focal adhesion plays an important role in the functional regulations of anchorage dependent cells, previous experimental studies have not provided quantitative description of the relation between focal adhesion and biophysical responses of cells. Furthermore, there is lack of knowledge on the importance of the beta1 integrin subunit to the dynamic responses of cells during initial cell seeding. In this study, we attempt to bridge the quantitative connection between focal adhesion density and cell-substrate interactions and evaluate the influence on functional blocking of beta1 integrin on adhesion strength. Total internal reflection fluorescence microscopy (TIRFM), fluorescence microscopy, and phase contrast microscopy was employed to study the time-dependent evolvement of vinculin pattern, distribution of actin filament, and morphological change, respectively, during 4 h of culture for porcine esophageal fibroblasts (non-blocked and beta1-blocked) on a fibronectin-coated surface. Micropipet aspiration technique was used to study the change of mechanotransduction through the determination of adhesion force and strength. It is shown in our experimental results that spread area, adhesion force, and adhesion strength increases over time on the two types of cells. Throughout the culture period, the two key mechanotransduction parameters of non-blocked cells is higher than those of beta1-blocked cells. Interestingly, adhesion strength initially ascends, then begins to diminish at a critical time point, and finally resumes increasing linearly against the increase of focal adhesion density. This variation as mentioned above can be explained by peeling and fracture models based on the dissimilar vinculin pattern of cells after being cultured for different time periods. Moreover, the averaged focal adhesion strength and non-focal adhesion strength of beta1-blocked cells are significantly less than those of non-blocked of cells. The weaker adhesion strength on beta(1)-blocked cells is directly caused by lower focal and non-focal adhesion strength, as well as by smaller focal adhesion density.

Periostin facilitates eosinophil tissue infiltration in allergic lung and esophageal responses

Periostin is an extracellular matrix protein that has been primarily studied in the context of the heart, where it has been shown to promote cardiac repair and remodeling. In this study, we focused on the role of periostin in an allergic eosinophilic inflammatory disease (eosinophilic esophagitis (EE)) known to involve extensive tissue remodeling. Periostin was indeed markedly overexpressed (35-fold) in the esophagus of EE patients, particularly in the papillae, compared with control individuals. Periostin expression was downstream from transforming growth factor-β and interleukin-13, as these cytokines were elevated in EE esophageal samples and markedly induced periostin production by primary esophageal fibroblasts (107- and 295-fold, respectively, at 10 ng ml−1). A functional role for periostin in eliciting esophageal eosinophilia was demonstrated, as periostin-null mice had a specific defect in allergen-induced eosinophil recruitment to the lungs and esophagus (66 and 72% decrease, respectively). Mechanistic analyses revealed that periostin increased (5.8-fold) eosinophil adhesion to fibronectin. As such, these findings extend the involvement of periostin to esophagitis and uncover a novel role for periostin in directly regulating leukocyte (eosinophil) accumulation in T helper type 2-associated mucosal inflammation in both mice and humans.

The Essential Role of Fibroblasts in Esophageal Squamous Cell Carcinoma–Induced Angiogenesis

Esophageal squamous cell carcinoma (ESCC) is known to be a highly angiogenic tumor. Here, we investigated the role of the stromal fibroblasts in the ESCC-induced angiogenic response using a novel 3-dimensional model. A novel assay was developed where cocultures of ESCC and esophageal fibroblasts induced human microvascular endothelial cell (HMVEC) vascular network formation in a 3-dimensional collagen gel. Biochemical studies showed that the ESCC-induced activation of the fibroblasts was required to induce vascular network formation via a transforming growth factor (TGF)-beta and vascular endothelial growth factor (VEGF)-dependent pathway. Conditioned media from a panel of 4 ESCC lines transdifferentiated normal esophageal fibroblasts into myofibroblasts via TGF-beta signaling. The presence of fibroblasts was essential for efficient HMVEC network formation, and the addition of ESCC cells to these cultures greatly enhanced the angiogenic process. The role of TGF-beta in this process was shown by the complete inhibition of network formation following TGF-beta inhibitor treatment. Finally, we showed that ESCC-derived TGF-beta regulates angiogenesis through the release of VEGF from the fibroblasts and that the VEGF release was blocked following TGF-beta inhibition. This study shows the essential role of fibroblasts in the ESCC angiogenic-induced response and suggests that the pharmacologic targeting of the TGF-beta signaling axis could be of therapeutic benefit in this deadly disease.

Adhesion dynamics of porcine esophageal fibroblasts on extracellular matrix protein-functionalized poly(lactic acid)

Effective attachment of esophageal cells on biomaterials is one important requirement in designing engineered esophagus substitute for esophageal cancer treatment. In this study, poly(lactic acid) (PLA) was subjected to surface modification by coupling extracellular matrix (ECM) proteins on its surface to promote cell adhesion. Two typical ECM proteins, collagen type I (COL) and fibronectin (FN), were immobilized on the PLA surface with the aid of glutaraldehyde as a cross linker between aminolyzed PLA and ECM proteins. By using confocal reflectance interference contrast microscopy (C-RICM) integrating with phase contrast microscopy, the long-term adhesion dynamics of porcine esophageal fibroblasts (PEFs) on four types of surfaces (unmodified PLA, PLA-COOH, PLA-COL and PLA-FN) was investigated during 24 h of culture. It is demonstrated by C-RICM results that PEFs form strong adhesion contact on all four types of surfaces at different stages of cell seeding. Among the four surfaces, PEFs on the PLA-FN surface reach the maximum adhesion energy (9.5 × 10−7 J m−2) in the shortest time (20 min) during the initial stage of cell seeding. After adhesion energy reaches the maximum value, PEFs maintain their highly deformed geometries till they reached a steady state after 20 h of culture. F-actin immunostaining results show that the evolvement of spatial organization of F-actin is tightly correlated with the formation of adhesion contact and cell spreading. Furthermore, the cell attachment ratio of PEFs on PLA in 2 h is only 26% compared with 88% on PLA-FN, 73% on PLA-COL and 36% on PLA-COOH. All the results demonstrate the effect of surface functionalization on the biophysical responses of PEFs in cell adhesion. Fibronectin-immobilized PLA demonstrates promising potential for application as an engineered esophagus substitute.

The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation

Esophageal cancer is a prototypic squamous cell cancer that carries a poor prognosis, primarily due to presentation at advanced stages. We used human esophageal epithelial cells as a platform to recapitulate esophageal squamous cell cancer, thereby providing insights into the molecular pathogenesis of squamous cell cancers in general. This was achieved through the retroviral-mediated transduction into normal, primary human esophageal epithelial cells of epidermal growth factor receptor (EGFR), the catalytic subunit of human telomerase (hTERT), and p53(R175H), genes that are frequently altered in human esophageal squamous cell cancer. These cells demonstrated increased migration and invasion when compared with control cells. When these genetically altered cells were placed within the in vivo-like context of an organotypic three-dimensional (3D) culture system, the cells formed a high-grade dysplastic epithelium with malignant cells invading into the stromal extracellular matrix (ECM). The invasive phenotype was in part modulated by the activation of matrix metalloproteinase-9 (MMP-9). Using pharmacological and genetic approaches to decrease MMP-9, invasion into the underlying ECM could be suppressed partially. In addition, tumor differentiation was influenced by the type of fibroblasts within the stromal ECM. To that end, fetal esophageal fibroblasts fostered a microenvironment conducive to poorly differentiated invading tumor cells, whereas fetal skin fibroblasts supported a well-differentiated tumor as illustrated by keratin "pearl" formation, a hallmark feature of well-differentiated squamous cell cancers. When inducible AKT was introduced into fetal skin esophageal fibroblasts, a more invasive, less-differentiated esophageal cancer phenotype was achieved. Invasion into the stromal ECM was attenuated by genetic knockdown of AKT1 as well as AKT2. Taken together, alterations in key oncogenes and tumor suppressor genes in esophageal epithelial cells, the composition and activation of fibroblasts, and the components of the ECM conspire to regulate the physical and biological properties of the stroma.