Human Esophageal Adenocarcinoma Cells Research Articles

Abstract 4559: SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma

Abstract Background: Resistance to conventional treatments is a significant barrier to esophageal adenocarcinoma (EAC) therapy. TGF-β signaling appears to be important for EAC tumorigenesis; however, its role in EAC chemoresistance remains understudied. This study investigates the role of SMAD3 in EAC resistance to the frontline chemotherapeutic drug oxaliplatin. Methods: Publicly available datasets were analyzed to determine SMAD3 expression in EAC versus normal samples. Western blot, quantitative real-time polymerase chain reaction, immunofluorescence staining, and flow cytometric analyses were utilized to validate bioinformatic results. Human EAC cell lines (OE33 and OE19) were treated with pharmacologic inhibitors or transfected with small interfering RNA and assessed for DNA damage and apoptotic cell death. Patient-derived organoids and patient-derived xenograft models of EAC were used to confirm our findings in vivo. Results: SMAD3 was significantly upregulated in EAC versus normal tissue samples. Total and phosphorylated SMAD3 protein levels were upregulated in EAC tissues accompanied by increased expression of several genes regulated by SMAD3. Notably, oxaliplatin-resistant EAC cells exhibited overexpression of SMAD3. Chemotherapy non-responding patients showed enrichment of SMAD3 gene expression when compared to responders in the clinical dataset GSE165252. Knockdown or inhibition of SMAD3 hindered DNA repair and sensitized EAC cells to oxaliplatin treatment in vitro and in vivo. Mechanistically, SMAD3 promoted ATM phosphorylation by interacting with protein phosphatase 2A (PP2A) and sequentially inhibiting the binding of PP2A to ATM. Conclusion: Our results reveal a pivotal role for SMAD3 in mediating oxaliplatin resistance in EAC and thus hold a significant potential in guiding future drug development and combination strategies. Citation Format: Farah Ballout, Heng Lu, Nadeem Bhat, Lei Chen, Dunfa Peng, Zheng Chen, Alexander Zaika, Oliver McDonald, Wael El-Rifai. SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4559.

Generation and multiomic profiling of a TP53/CDKN2A double-knockout gastroesophageal junction organoid model.

Inactivation of the tumor suppressor genes tumor protein p53 (TP53) and cyclin-dependent kinase inhibitor 2A (CDKN2A) occurs early during gastroesophageal junction (GEJ) tumorigenesis. However, because of a paucity of GEJ-specific disease models, cancer-promoting consequences of TP53 and CDKN2A inactivation at the GEJ have not been characterized. Here, we report the development of a wild-type primary human GEJ organoid model and a CRISPR-edited transformed GEJ organoid model. CRISPR-Cas9-mediated TP53 and CDKN2A knockout (TP53/CDKN2AKO) in GEJ organoids induced morphologic dysplasia and proneoplastic features in vitro and tumor formation in vivo. Lipidomic profiling identified several platelet-activating factors (PTAFs) among the most up-regulated lipids in CRISPR-edited organoids. PTAF/PTAF receptor (PTAFR) abrogation by siRNA knockdown or a pharmacologic inhibitor (WEB2086) reduced proliferation and other proneoplastic features of TP53/CDKN2AKO GEJ organoids in vitro and tumor formation in vivo. In addition, murine xenografts of Eso26, an established human esophageal adenocarcinoma cell line, were suppressed by WEB2086. Mechanistically, TP53/CDKN2A dual inactivation disrupted both the transcriptome and the DNA methylome, likely mediated by key transcription factors, particularly forkhead box M1 (FOXM1). FOXM1 activated PTAFR transcription by binding to the PTAFR promoter, further amplifying the PTAF-PTAFR pathway. Together, these studies established a robust model system for investigating early GEJ neoplastic events, identified crucial metabolic and epigenomic changes occurring during GEJ model tumorigenesis, and revealed a potential cancer therapeutic strategy. This work provides insights into proneoplastic mechanisms associated with TP53/CDKN2A inactivation in early GEJ neoplasia, which may facilitate early diagnosis and prevention of GEJ neoplasms.

Simvastatin Inhibits Histologic Changes Associated with Gastroduodenal Reflux in a Murine Model.

Observational studies demonstrate a protective effect of statins on the development and progression of esophageal adenocarcinoma. The role of statins in the prevention of reflux-induced esophageal changes remains unknown. Using a mixed gastroduodenal reflux mouse model, we hypothesized that oral administration of simvastatin would attenuate reflux-induced mucosal changes of the distal esophagus. Human Barrett's (CPB) and esophageal adenocarcinoma (FLO1 and OE19) cells were treated with simvastatin. Cell proliferation and apoptosis were evaluated using the MTS proliferation and annexin V apoptosis assays, respectively. A reflux mouse model was generated by performing a side-to-side anastomosis between the gastroesophageal junction and first portion of the duodenum (duodeno-gastroesophageal anastomosis, DGEA). DGEA mice were fed a standard or simvastatin-containing diet following surgery. Mice were euthanized 6weeks post-operatively. Simvastatin significantly decreased proliferation and increased apoptosis in all cell lines. Compared to control animals, mice undergoing DGEA who were fed a standard diet demonstrated a fourfold increase in mucosal thickness and significant increase in proliferating cells (p < 0.0001). DGEA mice fed a simvastatin-containing diet had an attenuated response to reflux, with a significant reduction in mucosal hyperplasia and proliferation (p < 0.0001). DGEA mice fed a simvastatin-containing diet demonstrated significant upregulation of procaspase-3 (p = 0.009) and cleaved caspase-3 (p = 0.034) in the distal esophagus. We demonstrate for the first time a reduction in reflux-induced histologic changes of the distal esophagus following oral administration of simvastatin in vivo. These findings identify simvastatin as a potential preventative agent to inhibit the development and progression of reflux-induced esophageal injury.

The Cytotoxic Effect of Iraqi Rumex Acetosella against Breast and Esophagus Cancer Cells

Background: cell lines derived from cancer cells are frequently used in research, including use as a model to understand cancer and to identify potential new treatments. The aim of this article has been prepared to evaluate the anticancer effect of Rumex Acetosella that has been studied for their anticancer activity on esophagus and breast cancer cell line. Method: AMJ13 (new breast cancer cell line (AMJ13) has been established from an Iraqi breast cancer patient and SK-GT-4 cells (Human esophageal adenocarcinoma cell line) were treated with whole methanolic extract of the leaves of Rumex acetosella. the MTT assay to determine the anticancer activity was done using 96-well plates where cell lines were seeded at 1 × 104cells/well after 24 hrs.m the cells were treated with tested compounds at different concentrations. Cell viability was measured after 72 hrs of treatment by removing the medium, adding 28 μL of 2 mg/mL solution of MTT, and incubating the cells for 2.5 h at 37 °C. After removing the MTT solution, the crystals remaining in the wells were solubilized by the addition of 130 μL of DMSO (Dimethyl Sulphoxide) followed by 37 °C incubation for 15 min with shaking. The absorbency was determined on a microplate reader at 492 nm. Conclusion: Plant extract from Rumex Acetosella showed particularly strong anticancer capabilities since it inhibited actual tumor progression in a breast adenocarcinoma mouse model. Our results suggest that whole plant extracts are promising anticancer reagents.

Expression of Adhesion Molecules in a Gastroduodenal Reflux Murine Model

BackgroundVarious adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), have been shown to play a role in inflammation as well as contribute to tumor progression and prognosis. We hypothesized that gastroduodenal reflux upregulates ICAM-1 and VCAM-1 expression in the distal esophagus, serving as possible early markers of pathologic esophageal disease. MethodsNormal human esophageal epithelial cells (HET1A), Barrett cells (CPB), and esophageal adenocarcinoma cells (FLO1 and OE33) were treated with deoxycholic acid at increasing concentrations for 24 hours. Adhesion molecule expression was assessed using immunoblotting. A surgical mouse reflux model was generated by performing a side-to-side anastomosis between the gastroesophageal junction and the first portion of the duodenum (duodenum-gastroesophageal anastomosis). Esophageal sections were evaluated using hematoxylin and eosin staining, immunohistochemistry, and immunofluorescence. ResultsDeoxycholic acid induced a significant increase in ICAM-1 and VCAM-1 expression in HET1A, CPB, FLO1, and OE33 cells. Animals undergoing duodenum-gastroesophageal anastomosis demonstrated a significant increase in mucosal hyperplasia (P < .0001) and cellular proliferation (P < .0001) compared with control animals. Immunofluorescence and Western blot analysis of the lower esophagus demonstrated significant upregulation of ICAM-1 (P = .005), with no change in VCAM-1 expression (P = .82). ConclusionsOur results reveal that ICAM-1 and VCAM-1 are upregulated in response to in vitro reflux treatment of normal esophageal epithelial cells. However, our investigation using a mouse reflux model found ICAM-1 is noticeably upregulated without a concomitant increase in VCAM-1. These findings identify ICAM-1, but not VCAM-1, as a potential player in early esophageal disease developing from chronic reflux exposure.

Cellular molecular and proteomic profiling deciphers the SIRT1 controlled cell death pathways in esophageal adenocarcinoma cells

Worldwide prevalence of esophageal adenocarcinomas with high rates of mortality coupled with increased mutations in esophageal cells warrants investigation to understand deregulation of cell signaling pathways leading to cancer. To this end, the current study was undertaken to unravel the cell death signatures using the model human esophageal adenocarcinoma cell line-OE33. The strategy involved targeting the key epigenetic modulator SIRT1, a histone deacetylase by a small molecule inhibitor - sirtinol. Sirtinol induced a dose-dependent inhibition of cell viability under both normoxic and hypoxic conditions with long term impact on proliferation as shown by clonogenic assays. Signature apoptotic signaling pathways including caspase activation and decreased Bcl-2 were observed. Proteomic analysis highlighted an array of entities affected including molecules involved in replication, transcription, protein synthesis, cell division control, stress-related proteins, spliceosome components, protein processing and cell detoxification/degradation systems. Importantly, the stoichiometry of the fold changes of the affected proteins per se could govern the cell death phenotype by sirtinol. Sirtinol could also potentially curb resistant and recurrent tumors that reside in hypoxic environments. Overall, in addition to unraveling the cellular, molecular and proteomics basis of SIRT1 inhibition, the findings open up avenues for designing novel strategies against esophageal adenocarcinoma.

Statins Inhibit Toll-Like Receptor 4–Mediated Growth of Human Esophageal Adenocarcinoma Cells

BackgroundEsophageal adenocarcinoma (EAC) is a lethal malignancy with poor prognosis. Pharmacologic inhibitors of inflammation, such as statins, have been shown to decrease the risk of development and progression of esophageal cancer, but the mechanism of this protection is unclear. The objective of this study was to elucidate the effect of statins on toll-like receptor 4-mediated–proliferation of human EAC cells and identify the mechanism responsible for these observed effects. MethodsHuman EAC cells (OE33 and FLO1) were treated with simvastatin or atorvastatin for increasing doses and time periods. Toll-like receptor 4 (TLR4) expression was assessed. Cells were pretreated with statin followed by lipopolysaccharide (LPS). Cell proliferation and expression of signaling proteins were evaluated. FLO1 cells were injected into the flank of nude mice. Mice received intraperitoneal injections of simvastatin, atorvastatin, or control solution and tumor volume was measured. ResultsOE33 and FLO1 cells demonstrated decreased TLR4 expression after treatment with simvastatin or atorvastatin for 8 h (P < 0.05). LPS increased proliferation, whereas pretreatment with statin abolished this response (P < 0.05). Statins decreased expression and activation of LPS-induced signaling proteins, including MyD88, TRAF6, Akt, and NF-κB (P < 0.05). Mice receiving daily statin injections demonstrated smaller tumors than control mice (P < 0.001 at day 33). ConclusionsTreatment of EAC cells with simvastatin or atorvastatin decreases TLR4-mediated proliferation and in vivo tumor growth. Decreased TLR4 expression and subsequent reduction in MyD88-dependent signaling could be a mechanism by which statins act to reduce tumor growth rates.

Combination effect of lapatinib with foretinib in HER2 and MET co-activated experimental esophageal adenocarcinoma

Recent studies have demonstrated that HER2 and MET receptor tyrosine kinases are co-overexpressed in a subset esophageal adenocarcinoma (EAC). We therefore studied the usefulness of combining HER2 and MET targeting by small-molecule inhibitors lapatinib and foretinib, respectively, both in in-vitro and in-vivo models of experimental EAC. We characterized MET and HER2 activation in a panel of human EAC cell lines, and the differential susceptibility of these EAC cell lines to single agent or combination of foretinib and lapatinib. We then explored the antitumor efficacy with survival advantage following foretinib and lapatinib monotherapy and in combination in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC. The OE33 EAC cell line with strong expression of phosphorylated both MET and HER2, demonstrated reduced sensitivity to foretinib and lapatinib when used as a single agent. The co-administration of foretinib and lapatinib effectively inhibited both MET and HER2 phosphorylation, enhanced inhibition of cell proliferation and xenograft tumor growth by inducing apoptosis, and significantly enhanced mouse overall survival, overcoming single agent resistance. In the OE19 EAC cell line with mainly HER2 phosphorylation, and the ESO51 EAC cell line with mainly MET phosphorylation, profound cell growth inhibition with induction of apoptosis was observed in response to single agent with lack of enhanced growth inhibition when the two agents were combined. These data suggest that combination therapy with foretinib and lapatinib should be tested as a treatment option for HER2 positive patients with MET-overexpressing EAC, and could be a novel treatment strategy for specific EAC patients.

Abstract 289: MET activation mediates lapatinib resistance in experimental esophageal adenocarcinoma

Abstract Introduction: A major target for esophageal adenocarcinoma (EAC) therapies is the human epidermal growth factor receptor 2 (HER2). Unfortunately, patients treated with lapatinib, a dual EGFR and HER2 inhibitor, frequently develop resistance. Lapatinib fails to improve patient survival in HER2-postive EAC, and the mechanisms contributing to resistance remain largely unknown. Recent studies have demonstrated that HER2 and MET receptor tyrosine kinases are co-overexpressed in a subset EAC. In this study, we therefore studied the role of MET activation in lapatinib resistance mechanisms in experimental EAC. Methods: We first characterized MET and HER2 activation in a panel of human EAC cell lines, and the differential susceptibility of these EAC cell lines to single agent or combination of MET and HER2 inhibitions by foretinib and lapatinib, respectively. We then explored the antitumor efficacy with survival advantage following foretinib and lapatinib monotherapy and in combination in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC. We also tested MET and HER activation status with sensitivity to their inhibitors in newly generated lapatinib resistant OE19 EAC cells (OE19-LPR). Results: The OE33 EAC cell line with phosphorylation of both MET and HER2, demonstrated reduced sensitivity to foretinib and lapatinib when used as single agents. The co-administration of foretinib and lapatinib effectively inhibited both MET and HER2 phosphorylation, synergistically inhibited cell proliferation and xenograft tumor growth, induced apoptosis, and significantly enhanced overall mouse survival, thus overcoming single agent resistance. In the OE19 EAC cell line with only HER2 phosphorylation and the ESO51 EAC cell line with only MET phosphorylation, profound cell growth inhibition with induction of apoptosis was observed in response to single agent with lack of enhanced growth inhibition when the two agents were combined. Interestingly, OE19-LPR cells showed significant upregulated expression of phosphorylated MET compared to parent OE19 cells, detected by both Activesignal assay and Western blot analysis. In addition, OE19-LPR cells showed significantly reduced sensitivity to lapatinib compared to parent OE19 cells, and the co-administration of foretinib and lapatinib synergistically inhibited cell proliferation in OE19-LPR cells. Conclusion: These data suggest that resistance to HER2 targeted therapies in HER2 and MET overexpressed EAC cells can be due to MET activation. Thus MET and HER2 targeted therapy appears to be a sensible treatment strategy for HER2 positive MET-overexpressing EAC. Citation Format: Md Sazzad Hassan, Fiona Williams, Lucia Petrova, Niranjan Awasthi, Margaret Schwarz, Roderich Schwarz, Urs von Holzen. MET activation mediates lapatinib resistance in experimental esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 289.

Submicron infrared imaging of an oesophageal cancer cell with chemical specificity using an IR-FEL

This work reports the use of an infrared spectroscopic version of scanning near-field optical microscopy (IR-SNOM) and shows that it is possible to reveal subcellular entities via their chemical constituents in a label-free human oesophageal adenocarcinoma cell (OE33). This work presents the first high spatial resolution (∼0.15 μm) study of features imaged at various wavelengths within a cancer cell. The clear illumination of sub-micron sized moieties within a cell is a major step forward and is a key requirement for understanding cancer and for the study of other diseases and healthy tissue. The stable and tuneable light source was provided by the infrared free electron laser on the ALICE accelerator at Daresbury. The images reveal a structure with a size and wavelength absorption that are consistent with a chromosome and open the possibility of observing other localized structures, such as microvesicles, that play an important role in the development and spread of cancers.

Toll-Like Receptor-4 Is a Mediator of Proliferation in Esophageal Adenocarcinoma

Chronic inflammation from reflux disease has been implicated as part of the development of esophageal adenocarcinoma. Toll-like receptors (TLRs), a component of the innate immune system, have been implicated in mediating hyperplasia and metaplasia in response to inflammatory stimuli. Increased TLR4 in human esophageal cancer has been correlated with its carcinogenesis. We hypothesized that TLR4 mediates proliferation of human esophageal adenocarcinoma cells. Normal human esophageal (HET1A) and adenocarcinoma (OE33, FLO-1) cell lines were cultured using standard techniques. TLR4 was measured at baseline and in response to reflux stimuli. All cell lines were treated with the TLR4 agonist lipopolysaccharide for 48 hours, and growth response was measured. Changes in myeloid differentiation primary response 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6), and nuclear factor-κB (NF-κB) activity were measured during lipopolysaccharide treatment. All cell lines had NF-κB inhibited, and growth rate response was measured. TLR4 was expressed in all cell lines, with increased baseline expression in adenocarcinoma cell lines (p < 0.05). Reflux stimuli increased TLR4 expression (p < 0.01) in normal esophageal cells. After treatment with lipopolysaccharide, all cell lines showed significant increases in proliferation (p < 0.05) due to the NF-κB pathway, and their growth rate was reduced with NF-κB inhibition (p < 0.05). TLR4 is consistently detectable in esophageal cell lines and most highly expressed in adenocarcinoma. TLR4 expression increases in an inflammatory model of reflux disease. TLR4 activation results in increased proliferation due to the TLR4-MyD88-TRAF6-NF-κB signaling pathway, and inhibition of NF-κB leads to decreased esophageal cell growth. These findings suggest TLR4 may be a target to suppress esophageal cancer growth.

Abstract 4826: Synergistic effects of foretinib with lapatinib in MET and HER2 co-activated experimental esophageal adenocarcinoma

Abstract Introduction: Recent studies have demonstrated that HER2 and MET receptor tyrosine kinases are co-overexpressed in a subset esophageal adenocarcinoma (EAC). We therefore studied the usefulness of combining HER2 and MET targeting by small-molecule inhibitors foretinib and lapatinib both in-vitro and in-vivo models of experimental EAC. Methods: In this study we first characterized MET and HER2 activation in a panel of human EAC cell lines and the differential susceptibility of these EAC cell lines to single agents or combinations of foretinib, a multi-kinase MET inhibitor, with HER2 targeted agent lapatinib. We evaluated the levels of phosphorylation status of MET and HER2 proteins using western blot in EAC cell lines. Foretinib and lapatinib, as single agent or in combination were tested for effect on cell growth as detected by WST-1 assay and on cell apoptosis as detected by western blot of cleavage of caspase 3 and poly ADP ribose polymerase (PARP). In addition, we explored the antitumor efficacy with survival advantage following foretinib and lapatinib mono and combination therapies for two weeks in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC. Results: The OE33 EAC cell line with phosphorylation of both MET and HER2, demonstrated reduced sensitivity to foretinib and lapatinib when used as a single agent. The co-administration of foretinib and lapatinib effectively inhibited both MET and HER2 phosphorylation, synergistically inhibited cell growth and induced apoptosis, overcoming single agent resistance. In the OE19 EAC cell line with only HER2 phosphorylation and the ESO51 EAC cell line with only MET phosphorylation, profound cell growth inhibition with induction of apoptosis was observed in response to single agent foretinib and lapatinib, respectively, with lack of enhanced growth inhibition when the two drugs were combined. Foretinib in combination with lapatinib treatment resulted in significantly higher antitumor efficacy and survival benefit compared with foretinib or lapatinib treatment alone. In subcutaneous xenografts using OE33 cells, average net tumor growth after two weeks in different therapy groups was 247.83 mm3 in control, 216.71 mm3 after foretinib (p=0.49), 239.68 mm3 after lapatinib (p=0.74), and 108.06 mm3 after foretinib plus lapatinib (p=0.0011). In the OE33 survival model there was a significant increase in median animal survival after two weeks foretinib plus lapatinib treatment (71 days) compared to control (60 days, p=0.0021), to foretinib therapy (63 days, p=0.0019) or to lapatinib (61 days, p=0.0019) therapy. Conclusion: These data suggest that combination therapy with foretinib and lapatinib should be tested as a treatment option for HER2 positive patients with MET-overexpressing EAC. Therefore, this combination therapy could be a novel treatment strategy for EAC with MET and HER co-activation. Citation Format: Md Sazzad Hassan, Fiona Williams, Niranjan Awasthi, Margaret A. Schwarz, Roderich E. Schwarz, Urs von Holzen. Synergistic effects of foretinib with lapatinib in MET and HER2 co-activated experimental esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4826.

MiR-21 Suppresses Anoikis through Targeting PDCD4 and PTEN in Human Esophageal Adenocarcinoma.

Anoikis is a form of apoptosis induced upon cell detachment from extracellular matrix. It has been determined that acquisition of resistance to anoikis is a critical step for tumor cell metastasis. MiR-21, the most prominent oncomiR, plays an important role in tumor progression. In this study, we revealed that up-regulation of miR-21 in human esophageal adenocarcinoma (EA) is associated with lymph node metastasis and poor survival rate. Because of the established anti-apoptosis effect of miR-21, it is tempting to speculate that miR-21 might contribute to tumor metastasis by regulating anoikis. qRT-PCR analysis demonstrated that miR-21 expression in OE33/AR cells (subpopulation of human EA OE33 cells that acquired resistance to anoikis) was significantly increased. Also, transfection of miR-21 mimics provided OE33 cells resisting to anoikis. By luciferase assays, we verified that PDCD4 and PTEN were the functional targets of miR-21. In mouse model, via tail vein injection experiment, we showed that the metastasis formation of OE33 cells in vivo could be mediated by changing the miR-21 expression pattern. Taken together, our findings suggested that miR-21 was involved in the regulation of anoikis in human EA cells. Targeting miR-21 may provide a novel strategy to prevent metastasis.

Hypoxia imaging with 18F-FAZA PET/CT predicts radiotherapy response in esophageal adenocarcinoma xenografts

BackgroundEsophageal cancer is an aggressive disease with poor survival rates. A more patient-tailored approach based on predictive biomarkers could improve outcome. We aimed to predict radiotherapy (RT) response by imaging tumor hypoxia with 18F-FAZA PET/CT in an esophageal adenocarcinoma (EAC) mouse model. Additionally, we investigated the radiosensitizing effect of the hypoxia modifier nimorazole in vitro and in vivo.MethodsIn vitro MTS cell proliferation assays (OACM5 1.C SC1, human EAC cell line) were performed under normoxic and hypoxic (< 1%) conditions: control (100 μL PBS), nimorazole, irradiation (5, 10 or 20 Gy) with or without nimorazole. In vivo, subcutaneous xenografts were induced in nude mice (OACM5 1.C SC1). Treatment was given daily for 5 consecutive days: (A) control (600 μl NaCl 0.9% intraperitoneally (IP)) (N = 5, n = 7), (B) RT (5 Gy/d) (N = 11, n = 20), (C) combination (nimorazole (200 mg/kg/d IP) 30 min before RT) (N = 13, n = 21). N = number of mice, n = number of tumors. 18F-FAZA PET/CT was performed before treatment and tumor to background (T/B) ratios were calculated. Relative tumor growth was calculated and tumor sections were examined histologically (hypoxia, proliferation).ResultsA T/B ≥ 3.59 on pre-treatment 18F-FAZA PET/CT was predictive for worse RT response (sensitivity 92.3%, specificity 71.4%). Radiation was less effective in hypoxic tumors (T/B ≥ 3.59) compared to normoxic tumors (T/B < 3.59) (P = 0.0025). In vitro, pre-treatment with nimorazole significantly decreased hypoxic radioresistance (P < 0.01) while in vivo, nimorazole enhanced the efficacy of RT to suppress cancer cell proliferation in hypoxic tumor areas (Ki67, P = 0.064), but did not affect macroscopic tumor growth.ConclusionsTumor tissue hypoxia as measured with 18F-FAZA PET/CT is predictive for RT response in an EAC xenograft model. The radiosensitizing effect of nimorazole was questionable and requires further investigation.

An evaluation of the application of the aperture infrared SNOM technique to biomedical imaging

A single human oesophageal adenocarcinoma cell (OE33) has been imaged using aperture infrared scanning near-field optical microscopy (IR-SNOM) in transmission and reflection and also by Fourier-transform infrared (FTIR) microspectroscopy in transmission only. This work presents the first images obtained in both transmission and reflection of the same specimen using the aperture IR-SNOM technique. The results have been used to compare the two SNOM modes and also the two techniques, which have complementary capabilities. The SNOM technique necessitates a very stable source and a careful choice of wavelengths, since it is too slow to yield images at the thousands of wavelengths obtained with FTIR. However the SNOM technique is not diffraction limited and with careful fabrication of tips can yield images with high spatial resolution. There is no significant correlation between the SNOM images obtained in transmission and reflection and the correlations between images obtained at different wavelengths vary with the different imaging modes. These results are attributed to the strong dependence of the evanescent wave on both the wavelength and the distance between the tip and the source of the signal within the sample. While both transmission and reflection SNOM images show some correlation with topography this is not a dominant effect. These results indicate that with suitable calibration a combination of reflection and transmission aperture IR-SNOM measurements has the potential to reveal information on the depth distribution of the chemical structure of a specimen.

Animal Model: Xenograft Mouse Models in Esophageal Adenocarcinoma.

Researchers often use murine models of esophageal cancer to evaluate novel therapies prior to clinical protocol treatment. Subcutaneous xenograft models are often used for testing the efficacy of anticancer agents in many cancers including esophageal adenocarcinoma. However, mice subcutaneous esophageal adenocarcinoma models only represent local tumor growth and do not provide any information about a survival benefit for a particular anticancer regimen, which is very crucial for experimental treatment efficacy. In addition, anticancer agents may well inhibit subcutaneous tumor growth without effecting overall animal survival. Herein, we describe a peritoneal dissemination mouse xenograft model for survival outcome analysis with intraperitoneal injection of human esophageal adenocarcinoma cell lines.

Antitumor and antimetastatic effects of walnut oil in esophageal adenocarcinoma cells

Walnuts contain many components including specific fatty acids, which could be active against cancer. Even though the anticarcinogenic effect of some of the individual fatty acids in walnut oil has been described, the effect of walnut oil itself on esophageal cancer cells hasn't yet been investigated. We aimed to investigate whether walnut oil affects tumor growth and metastatic potential in esophageal cancer cells. The human esophageal adenocarcinoma cell line, OE19, was treated with different doses of walnut oil and cell viability, apoptosis/necrosis and cell cycle analyses were performed using WST-1 assay and flow cytometry respectively. Adhesion, colony formation and wound healing assays were performed to assess the antimetastatic effects of walnut oil. NFkB expression was evaluated with western blot analysis. Walnut oil decreased the cell viability of esophageal cancer cells in a dose-dependent manner. 20mg/mL walnut oil reduced cell viability by ∼50% when compared with control. The analysis revealed that necrosis and accumulation of cells in G0/G1 phase was induced in the cells treated with high doses of walnut oil. It also down-regulated the protein levels of NFkB. Walnut oil suppressed the adhesion, migration and colony formation of the cells. High-dose short-term administration of walnut oil reduces the cell viability and metastatic ability of esophageal cancer cells, while exhibiting anticarcinogenic effect by inducing necrosis and cell cycle arrest at the G0/G1 phase, probably through suppression of the NFkB pathway. These data indicate that walnut oil, and by extension walnut consumption, may have beneficial effects in esophageal cancer in humans. This should be tested by clinical trials in the future.

Abstract LB-094: Treatment with the G9a antagonist, BRD4770, inhibits esophageal adenocarcinoma cell growth

Abstract Cancer has been considered a heterogeneous disease, which mainly arises from the accumulation of genetic mutations, leading to a loss of tumor suppressor genes, as well as to an activation of oncogenes. The epigenetic mechanisms, such as DNA and histone modifications, are processes that regulate gene expression, and an alteration of these can contribute to cancer progression. Methylation on histone 3 lysine 9 (H3K9), in particular di-methyl (H3K9me2), is mainly mediated by the nuclear histone lysine methyltransferase G9a. Preliminary data showed that G9a was overexpressed in aggressive and highly metastatic forms of cancer. However, to date, this pathway has remained unstudied in esophageal adenocarcinoma. Interestingly, epigenetic drugs, such as those that target G9a, have the potential to reverse the adverse effects acquired downstream from genetic mutations, which currently cannot be directly repaired or targeted. In the current study, we utilize BRD4770, an S-adenosylmethionine (SAM) mimetic inhibitor of G9a, to determine the benefits of inhibiting this pathway in the context of esophageal adenocarcinoma. We hypothesize that pharmacological targeting of G9a by BRD4770 inhibits esophageal adenocarcinoma cell growth. To examine G9a-mediated dimethyl histone H3K9 levels in esophageal adenocarcinoma, we performed immunohistochemistry on paraffin-embedded esophageal adenocarcinoma tissues with an H3K9me2 antibody. In order to confirm specificity of BRD4770 in regulating the dimethyl histone H3K9 levels, and thus trimethyl H3K9 levels, in esophageal adenocarcinoma, we treated human esophageal adenocarcinoma cells (SKGT-4) with different doses of BRD4770 at various time points for western blot analysis using antibodies against histone H3K9me2 and H3K9me3. Moreover, we found that treatment of SKGT-4 cells with BRD4770 significantly inhibits cell proliferation by MTS assay, as well as clonogenic survival. Therefore, to determine the mechanism(s) by which BRD4770 inhibits the growth of esophageal adenocarcinoma cells, we utilized Caspase-3, Senescence, and Autophagy Assays. Our data shows that inhibition of G9a by BRD4770 does not induce Caspase 3 activity, while it significantly increases senescence and autophagy. Combined, our results suggest that BRD4770 has an important pharmacological role in regulating methyl histone H3K9 levels that may lead to inhibition of esophageal adenocarcinoma cell growth via an increase in senescence and autophagy. Further studies are focused on in vivo models to understand these effects in more detail, however the fact that an anti-G9a drug antagonizes esophageal adenocarcinoma cell growth raises optimism for its potential in the treatment of this malignancy. Note: This abstract was not presented at the meeting. Citation Format: Mabel G. Perez-Oquendo, Gwen A. Lomberk, Raul A. Urrutia, Sarah Kossak, Navtej Buttar, Sounak Gupta. Treatment with the G9a antagonist, BRD4770, inhibits esophageal adenocarcinoma cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-094. doi:10.1158/1538-7445.AM2017-LB-094

Abstract 2826: A novel intraperitoneal metastatic xenograft mouse model for survival outcome assessment of esophageal adenocarcinoma

Abstract Introduction: Esophageal adenocarcinoma (EAC) has become the dominant type of esophageal cancer in United States. EAC is the fastest growing cancer in the western world and the overall 5 year survival rate of EAC is below 20 percent. Most patients present with locally advanced or widespread metastatic disease, where current treatment is largely ineffective. Prognosis for EAC patients remains poor even with combination therapies due to high resistance to chemotherapy. Therefore, new therapeutic approaches are urgently needed. Improvement of esophageal adenocarcinoma outcome requires well-characterized animal models in which to evaluate novel therapeutics. In this study we aimed to establish a peritoneal dissemination xenograft mouse model of esophageal adenocarcinoma that would support survival outcome analyses. Methods: To find the best candidate cell line from 7 esophageal adenocarcinoma cell lines of various origin, we injected them intraperitoneally and subcutaneously into severe combined immunodeficiency (SCID) mice and examined the tumor progression and survival outcomes. Human esophageal adenocarcinoma cell lines of Caucasian/Hispanic origin ESO26, OE33, ESO51, SK-GT-2, OE19, OACM5.1C and Flo-1 originating from gastroesophageal junction, distal esophagus and gastric cardia/fundus were injected intraperitoneally/subcutaneously into SCID mice. The peritoneal/xenograft tumor formation and mouse survival were compared among different groups. Results: All cell lines injected subcutaneously formed tumors within 3 months at variable rates. All cell lines except OACM5.1C formed intraperitoneal tumors within 3 months at variable rates. Median animal survival with peritoneal dissemination was 108 days for ESO26 cells (5X106), 65 days for OE33 cells (5X106), 88 days for ESO51 cells (5X106), 76 days for SK-GT-2 cells (5X106), 55 days for OE19 cells (5X106), 45 days for OE19 cells (10X106) and 82 days for Flo-1 cells (5X106). Interestingly, only in the OE19 model all mice (7/7 for 5X106 and 5/5 for10X106) developed bloody ascites with liver metastasis after intraperitoneal injection. The median survival time of these animals was the shortest (45 days for 10X106 cells). In addition, median survival was significantly increased after paclitaxel treatment compared with the control group (57 days versus 45 days, p=0.0034) along with a significant decrease of the relative subcutaneous tumor volume (p=0.023). Conclusion: Peritoneal esophageal adenocarcinoma xenografts were successfully established after intraperitoneal injection of OE19 cells. This animal model of peritoneal dissemination for survival outcome will provide a useful survival outcome assessment model for the preclinical evaluation of cancer therapeutics in esophageal adenocarcinoma. Citation Format: Md Sazzad Hassan, Niranjan Awasthi, Roderich E. Schwarz, Margaret A. Schwarz, Urs von Holzen. A novel intraperitoneal metastatic xenograft mouse model for survival outcome assessment of esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2826. doi:10.1158/1538-7445.AM2017-2826

A novel intraperitoneal metastatic xenograft mouse model for survival outcome assessment of esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) has become the dominant type of esophageal cancer in United States. The 5-year survival rate of EAC is below 20% and most patients present with locally advanced or widespread metastatic disease, where current treatment is largely ineffective. Therefore, new therapeutic approaches are urgently needed. Improvement of EAC patient outcome requires well-characterized animal models in which to evaluate novel therapeutics. In this study we aimed to establish a peritoneal dissemination xenograft mouse model of EAC that would support survival outcome analyses. To find the best candidate cell line from 7 human EAC cell lines of different origin named ESO26, OE33, ESO51, SK-GT-2, OE19, OACM5.1C and Flo-1 were injected intraperitoneally/subcutaneously into SCID mice. The peritoneal/xenograft tumor formation and mouse survival were compared among different groups. All cell lines injected subcutaneously formed tumors within 3 months at variable rates. All cell lines except OACM5.1C formed intraperitoneal tumors within 3 months at variable rates. Median animal survival with peritoneal dissemination was 108 days for ESO26 cells (5X106), 65 days for OE33 cells (5X106), 88 days for ESO51 cells (5X106), 76 days for SK-GT-2 cells (5X106), 55 days for OE19 cells (5X106), 45 days for OE19 cells (10X106) and 82 days for Flo-1 cells (5X106). Interestingly, only in the OE19 model all mice (7/7 for 5X106 and 5/5 for10X106) developed bloody ascites with liver metastasis after intraperitoneal injection. The median survival time of these animals was the shortest (45 days for 10X106 cells). In addition, median survival was significantly increased after paclitaxel treatment compared with the control group (57 days versus 45 days, p = 0.0034) along with a significant decrease of the relative subcutaneous tumor volume (p = 0.00011). Thus peritoneal dissemination mouse xenograft model for survival outcome assessment after intraperitoneal injection of OE19 cells will be very useful for the evaluation of cancer therapeutics.