Human Esophageal Cell Research Articles

Sialylated keratan sulfates on MUC5B are Siglec-8 ligands in the human esophagus.

Human sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed on subsets of immune cells. Siglec-8 is an immune inhibitory Siglec on eosinophils and mast cells, which are effectors in allergic disorders including eosinophilic esophagitis. Inhibition occurs when Siglec-8 is crosslinked by multivalent Siglec ligands in target tissues. Previously we discovered a high-affinity Siglec-8 sialoglycan ligand on human airways composed of terminally sialylated keratan sulfate chains carried on a single protein, DMBT1. Here we extend that approach to another allergic inflammatory target tissue, human esophagus. Lectin overlay histochemistry revealed that Siglec-8 ligands are expressed predominantly by esophageal submucosal glands, and are densely packed in submucosal ducts leading to the lumen. Expression is tissue-specific; esophageal glands express Siglec-8 ligand whereas nearby gastric glands do not. Extraction and resolution by gel electrophoresis revealed a single predominant human esophageal Siglec-8 ligand migrating at >2MDa. Purification by size exclusion and affinity chromatography, followed by proteomic mass spectrometry, revealed the protein carrier to be MUC5B. Whereas all human esophageal submucosal cells express MUC5B, only a portion convert it to Siglec-8 ligand by adding terminally sialylated keratan sulfate chains. We refer to this as MUC5B S8L. Material from the esophageal lumen of live subjects revealed MUC5B S8L species ranging from ~1-4MDa. We conclude that MUC5B in the human esophagus is a protein canvas on which Siglec-8 binding sialylated keratan sulfate chains are post-translationally added. These data expand understanding of Siglec-8 ligands and may help us understand their roles in allergic immune regulation.

Human esophageal cancer stem-like cells escape the cytotoxicity of natural killer cells via down-regulation of ULBP-1

BackgroundCancer stem-like cells (CSCs) play an important role in initiation and progression of aggressive cancers, including esophageal cancer. Natural killer (NK) cells are key effector lymphocytes of innate immunity that directly attack a wide variety of cancer cells. NK cell-based therapy may provide a new treatment option for targeting CSCs. In this study, we aimed to investigate the sensitivity of human esophageal CSCs to NK cell-mediated cytotoxicity.MethodsCSCs were enriched from human esophageal squamous cell carcinoma cell lines via sphere formation culture. Human NK cells were selectively expanded from the peripheral blood of healthy donors. qRT-PCR, flow cytometry and ELISA assays were performed to examine RNA expression and protein levels, respectively. CFSE-labeled target cells were co-cultured with human activated NK cells to detect the cytotoxicity of NK cells by flow cytometry.ResultsWe observed that esophageal CSCs were more resistant to NK cell-mediated cytotoxicity compared with adherent counterparts. Consistently, esophageal CSCs showed down-regulated expression of ULBP-1, a ligand for NK cells stimulatory receptor NKG2D. Knockdown of ULBP-1 resulted in significant inhibition of NK cell cytotoxicity against esophageal CSCs, whereas ULBP-1 overexpression led to the opposite effect. Finally, the pro-differentiation agent all-trans retinoic acid was found to enhance the sensitivity of esophageal CSCs to NK cell cytotoxicity.ConclusionsThis study reveals that esophageal CSCs are more resistant to NK cells through down-regulation of ULBP-1 and provides a promising approach to promote the activity of NK cells targeting esophageal CSCs.

Anlotinib inhibits growth of human esophageal cancer TE-1 cells by negative regulating PI3K/Akt signaling pathway

Anlotinib is effective in treatment of many kinds of malignant cancer, but its antineoplastic effects on esophageal cancer remains unclear. This study aims to investigate its impact on esophageal cancer and the underlying mechanisms. Anlotiniband 5-fluorouracil + cisplatin (5-FU + DDP) was administered separately to human esophageal cancer TE- 1 cells tumor xenograft mouse models every 3 days. Tumor size and body weight were measured before each treatment and at the end of the experiment. In vitro studies were conducted using TE- 1 cells to examine the effects of Anlotinib. Cell viability, migration, proliferation, apoptosis, cell cycle, their regulatory proteins and the transcriptomic changes were analyzed. Anlotinib reduced tumor size, tumor weight, and the ratio of tumor weight to body weight in vivo. It decreased the viability of TE- 1 cells, with a 50% growth-inhibitory concentration of 9.454 μM for 24 h, induced apoptosis, and arrested TE- 1 cell cycle in the S phase. It inhibited migration and proliferation while negatively regulating the PI3K/Akt signaling pathway. Enhanced expressions of P21, Bax, and lowered expressions of cyclin A1, cyclin B1, CDK1, PI3K, Akt, p-Akt, and Bcl-2 were observed after Anlotinib treatment. Anlotinib exhibits antineoplastic activity against human esophageal cancer TE- 1 cells by negatively regulating the PI3K/Akt signaling pathway, consequently altering the expressions of proteins related to proliferation, apoptosis, and the cell cycle.

Shikonin Induces Autophagy and Apoptosis in Esophageal Cancer EC9706 Cells by Regulating the AMPK/mTOR/ULK Axis.

Shikonin is a plant medicine extracted from Lithospermum, which dominate influential antioxidant and antitumor effect. Here, we report that shikonin was capable of inducing human esophageal cancer EC9706 cell apoptosis and autophagy, in a time- and dose-dependent manner. Shikonin exposure repressed cell viability and migration and invasion capabilities and caused EC9706 cell autophagy and apoptosis by activating the AMPK/mTOR/ULK axis. Autophagy inhibition secured EC9706 cells against shikonin-induced autophagy and apoptosis and reversed the upregulation of AMPK and ULK phosphorylation and downregulation of mTOR phosphorylation provoked by shikonin. In summary, shikonin instigates EC9706 cell apoptosis and autophagy using the target AMPK/mTOR/ULK signal pathway axis, which provides a potential new target to treat human esophageal cancer.

ZPR1 is an immunodiagnostic biomarker and promotes tumor progression in esophageal squamous cell carcinoma.

To evaluate the potential of zinc finger protein 1 (ZPR1) as a diagnostic biomarker and explore the underlying role for esophageal squamous cell carcinoma (ESCC). A human proteome microarray was customized to identify anti-ZPR1 autoantibody, and enzyme-linked immunosorbent assay (ELISA) was adopted to assess the diagnostic performance of anti-ZPR1 autoantibody in 294 patients with ESCC and 294 normal controls. The expression of ZPR1 protein was measured by immunohistochemistry. The effect of ZPR1 on the proliferation, migration, and invasion of ESCC cells was investigated through CCK-8, wound healing, and Transwell assays. The expression level of anti-ZPR1 autoantibody (fold change = 2.77) in ESCC patients was higher than that in normal controls. The receiver operating characteristic (ROC) analysis manifested anti-ZPR1 autoantibody achieved area under the ROC curve (AUC) of 0.726 and 0.734 to distinguish ESCC from normal controls with sensitivity of 50.0% and 42.3%, and specificity of 91.0% and 92.0% in the test group and validation group, respectively. The positive rate of ZPR1 protein was significantly higher in ESCC tissues (75.5%, 80/106) than paracancerous tissues (9.4%, 5/53). Compared with the human normal esophageal cell line, the expression level of ZPR1 mRNA and protein in ESCC lines (KYSE150, Eca109, and TE1) had an increased trend. The knockdown or overexpression of ZPR1 reduced and enhanced the proliferation, migration, and invasion of ESCC cell, respectively. ZPR1 was a potential immunodiagnostic biomarker for noninvasive detection and could be a promotional factor in tumor progression of ESCC.

An alternative technique for cyclization synthesis, in vitro anti-esophageal cancer evaluation, and molecular docking of novel thiazolidin-4-one derivatives

In this work, two novel series of thiazolidin-4-one derivatives (9a–e and 10a–e) constituting ten compounds were synthesized under batch and ultrasound irradiation. It is found that the use of sonochemical methodology in the thiazolidin-4-ones synthesis offers several ecological advantages such as an effective, inexpensive, easy applicable, eco-friendly, and waste-free chemicals than the batch method. Different techniques including NMR (1H and 13C), mass spectra, and infrared radiation (FTIR) were employed to characterize the chemical structures of all the synthesized thiazolidin-4-one derivatives. The purified derivatives were tested for their anti-esophageal cancer activity against a panel of human esophageal cancer cell line (SKGT-4). The in vitro cytotoxic and apoptotic activities of these derivatives were evaluated by MTT and AO/EB assays, respectively. Among these derivatives, thiazolidin-4-one derivatives 9e and 10b were found to have the most potency toward SKGT-4 human esophageal cells. When compared to the reference standard (cisplatin, IC50 5.21±0.41 µg/mL), the values (IC50) of the derivatives 9e and 10b were found as 17.6 ± 0.06 and 18.5 ± 0.01 μg/mL, respectively. The collected data from AO/EB assay revealed these derivatives are able to stimulate the apoptotic effect in the SKGT-4 cells with a selective pathway compared to their behaviors in the normal Vero cells. Moreover, DFT-assisted calculations with theoretical level: B3LYP/6–31G(d,p) were adopted to optimize the geometrical structures of the derivatives 9e and 10b. To elucidate the possible binding mechanisms of these derivatives 9e and 10b with the active sites of human esophageal proteins (PDB ID: 6DUK, 2LEO, and 5HZN), the docking study was accomplished. According to the docking analysis, the derivatives 9e and 10b showed efficient interactions with the target receptors with low values of binding affinity. Thus, the biological tests and docking scores suggested that the synthesized thiazolidin-4-one derivatives 9e and 10b may be considered as a potential candidate for esophageal cancers treatments.

Pulsed Electric Field Ablation of Esophageal Malignancies and Mitigating Damage to Smooth Muscle: An In Vitro Study.

Cancer ablation therapies aim to be efficient while minimizing damage to healthy tissues. Nanosecond pulsed electric field (nsPEF) is a promising ablation modality because of its selectivity against certain cell types and reduced neuromuscular effects. We compared cell killing efficiency by PEF (100 pulses, 200 ns-10 µs duration, 10 Hz) in a panel of human esophageal cells (normal and pre-malignant epithelial and smooth muscle). Normal epithelial cells were less sensitive than the pre-malignant ones to unipolar PEF (15-20% higher LD50, p < 0.05). Smooth muscle cells (SMC) oriented randomly in the electric field were more sensitive, with 30-40% lower LD50 (p < 0.01). Trains of ten, 300-ns pulses at 10 kV/cm caused twofold weaker electroporative uptake of YO-PRO-1 dye in normal epithelial cells than in either pre-malignant cells or in SMC oriented perpendicularly to the field. Aligning SMC with the field reduced the dye uptake fourfold, along with a twofold reduction in Ca2+ transients. A 300-ns pulse induced a twofold smaller transmembrane potential in cells aligned with the field, making them less vulnerable to electroporation. We infer that damage to SMC from nsPEF ablation of esophageal malignancies can be minimized by applying the electric field parallel to the predominant SMC orientation.

Baicalein Inhibits the Growth of Transplanted Esophageal Cancer in Mice and the Effect on the Expression of PAK4

We studied the mechanism underlying the effect of baicalein on the growth of transplanted esophageal cancer in NOG mice and its effect on the expression of PAK4. For that purpose, we developed a new model of transplanted esophageal cancer (human esophageal cancer OE19 cells (107 cells/ml) were inoculated to NOG mice). Three experimental groups with transplanted esophageal cancer cells received baicalein in different doses (1, 1.5, and 2 mg/kg). In 32 days, the tumors were resected, and the expression of PAK4 and the level of activated PAK4 were assayed by reverse transcription PCR and Western blotting, respectively. The results showed a dose-depending anti-tumor effect of baicalein on the transplanted esophageal cancer in NOG mice: this effect of baicalein (determined by the size and weight of the tumor) increased with increasing the dose of the substance. Furthermore, the anti-tumor effect of baicalein was also confirmed by reduction of PAK4 expression. Thus, baicalein can inhibit tumor growth by inhibiting activation of PAK4. Therefore, our results showed that baicalein could inhibit the growth of esophageal cancer cells by inhibiting the activity of PAK4, which can be an important mechanism of its antitumor effect.

Abstract B014: Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma

Abstract Introduction: Mutations of the tumor suppressor p53 are detected up to 80% of esophageal squamous cell carcinoma (ESCC) cases, which in turn correlate with high metastatic rates and poor prognosis. To understand the mutant p53-mediated mechanisms in promoting ESCC metastasis, we conducted RNA-Seq and cytokine array on isogenic primary and metastatic tumor cells harvested from our mouse model of esophageal cancer harboring Trp53R172H/-, in which we have identified Colony stimulating factor 1 (Csf1) to be upregulated. Our goal is to investigate the role and mediators of CSF1 signaling through its cognate receptor CSF1R by which missense p53 mutations can promote tumor invasion and lung metastasis in ESCC. Methods: We have utilized novel L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP mice and isolated tumor cells to model metastatic ESCC. We conducted ChIP-Seq analysis for p53 on tumor cells derived from lung metastases in our mouse models. Furthermore, we have genetically and pharmacologically targeted CSF1/CSF1R signaling axis to assess its role in ESCC tumor invasion and lung metastasis. In addition to studying tumor intrinsic mechanisms, we have established quantitative multiplex immunofluorescence (qmIF) and flow cytometry approaches to characterize the changes in tumor microenvironment (TME). Results And Discussion: We demonstrate that metastatic ESCC has increased Csf1 expression compared to primary tumors, and this is dependent upon p53 mutation status, which is reinforced by the TCGA data and patient-derived tissue microarrays (TMAs). The overlay of the RNA-Seq with the ChIP-Seq analysis indicates that Csf1 is a direct gene target of p53-R172H with enriched binding motifs. Furthermore, based on the proximity ligation assay (PLA) with murine ESCC tumor cells, bromodomain and extra-terminal motif (BET) protein BRD4 interacts with p53-R172H to induce Csf1 expression. We show that the BRD4-CSF1 axis fosters tumor invasion, subcutaneous tumor growth and metastatic burden in a mutant p53 background. In accordance, upon inhibiting this signaling pathway in the tail-vein injection lung metastasis models, we have identified reduced expression of CD31+ cells and decreased infiltration of F4/80+CD163+ and F4/80+CD206+ M2-polarized macrophages at the metastatic tumor sites, indicating that the CSF1/CSF1R pathway plays a critical role in shaping the pro-metastatic and immunosuppressive TME. Finally, analysis of the esophageal cancer datasets reveals that specific p53 mutations are associated with markedly differential overall survival rates and Csf1 expression. Based upon these results, we are generating human and murine esophageal cells with distinct DNA contact and conformational p53 mutations via base editing. Conclusion: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1/CSF1R signaling pathway in fostering ESCC tumor invasion and lung metastasis that may be applicable to other squamous cell cancers. We believe this can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Qiaosi Tang, Katherine M. Cunningham, Kensuke Sugiura, Karen Dunbar, Kausik Regunath, Kensuke Suzuki, Andres J. Klein-Szanto, Lois Resnick-Silverman, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B014.

MiR-4443 promotes radiation resistance of esophageal squamous cell carcinoma via targeting PTPRJ

BackgroundRadiotherapy is one of the main treatments for esophageal squamous cell carcinoma (ESCC), but its efficacy is limited by radioresistance. MicroRNAs play a crucial role in posttranscriptional regulation, which is linked to the cancer response to radiation.MethodsWe successfully established a radioresistant cell line model by using fractionated irradiation. qRT-PCR was adopted to detect the expression of miR-4443 in human normal esophageal cell lines, tumor cells, and radioresistant cells. Next, CCK-8, colony formation, apoptosis, and cell cycle assays were used to assess the biological effect of miR-4443. Weighted gene coexpression network analysis (WGCNA) was performed to identify potential radiosensitivity-related genes. Additionally, we predicted the probable targets of the miRNA using bioinformatic methods and confirmed them using Western blot.ResultsmiR-4443 was significantly upregulated in radioresistant ESCC cells. Enhancement of miR-4443 further decreased the radiosensitivity of ESCC cells, while inhibition of miR-4443 increased the radiosensitivity of ESCC cells. Notably, miR-4443 modulated radiosensitivity by influencing DNA damage repair, apoptosis, and G2 cycle arrest. By using WGCNA and experimental validation, we identified PTPRJ as a key target for miRNA-4443 to regulate radiosensitivity. The effects of miR-4443 overexpression or inhibition could be reversed by increasing or decreasing PTPRJ expression.ConclusionIn this study, miR-4443 is found to promote radiotherapy resistance in ESCC cells by regulating PTPRJ expression, which provides a new perspective and clue to alleviate radioresistance.

Abstract 13129: Human in vitro Study on Pulsed Electric Field Cardiac Ablation Cell Sensitivity

Introduction: Catheter cardiac ablation is a standard treatment for atrial fibrillation for patients that do not respond to drugs. Pulsed electric field (PEF) cardiac ablation has been proposed as a nonthermal technology to cause cell death by irreversible electroporation (IRE). IRE has shown to minimize off target damage. The lack of systematic studies to inform optimal PEF ablation parameters has significantly slowed down device development and regulatory process. Hypothesis: As part of a larger effort to develop a standard laboratory assay for PEF parameter optimization, in this study we investigate tissue specificity of IRE cardiac ablation treatments. Methods: Human induced pluripotent stem cell cardiomyocytes derived (hiPSC-CMs, Fujifilm Cellular Dynamic, Inc.), human esophageal muscle cells (HESMC, ScienCell Research Laboratories), and human bronchial epithelial cells (HBEC3-KT, ATCC) were cultured in monolayer formats on 96-well NanoFiber plates. PEF were delivered by an FID pulse generator connected to two needle electrodes (1.7 mm center-to-center distance). PEF parameters investigated in this study are: phase duration tp=1, 5, 10 μs; phase intensity Vp=284, 372, 568V; pulse repetition frequency PRF=10, 100, 1000 Hz, pulse number p#=100. We quantified IRE areas 4 hours post-treatment by propidium iodide (PI). Results: Preliminary results show that increasing the tp, Vp and decreasing the PRF resulted in a larger IRE area in all the cell types studied. For the range of PEF parameters investigated, a treatment with same tp, p# and PRF required higher Vp to induce IRE in HESMC and HBEC3-KT than hiPSC-CMs, i.e., 100 pulses with tp=5 μs and PRF=1000 Hz, to achieve the same IRE area, hiPSC-CMs needed half Vp than HESMC and HBEC3-KT. Conclusions: Our data suggests that same PEF treatments induced larger IRE areas in hiPSC-CM than in HESMC and HBEC3-KT. Future work will focus on validating these results in tissues to assess safety of IRE cardiac ablation devices.

Sodium selenite supplementation does not reduce the effectiveness of X-ray irradiation treatment on human cancerous esophageal TE-8 cells

Sodium selenite supplementation at a concentration 50 nM before X-ray irradiation was suggested to protect non-cancerous human esophageal CHEK-1 cells from irradiation-induced damage. This present study investigated those effects on cancerous human esophageal cell line. The human cancer esophageal cell line, TE-8, was cultured and supplemented for the cytotoxicity assay, the GPx-1 activity, the cell viability assay, clonogenic assay and western blot analysis. An apoptosis biomarker, Cleaved PARP, was used. The results show that cell survival post-irradiation of supplemented-cells had the same effect as the cells treated by irradiation only, tended to decrease the cell viability (p=0.27), and decrease the survival rate of cancerous cells (p=1.00). The cleaved PARP level was higher in supplemented-and irradiated- cells than cells with irradiation alone. These results suggest that 50 nM sodium selenite supplementation prior to irradiation does not reduce the effectiveness of irradiation treatment on cancerous cells.

Human cardiomyocytes are more susceptible to irreversible electroporation by pulsed electric field than human esophageal cells.

Pulse electric field‐based (PEF) ablation is a technique whereby short high‐intensity electric fields inducing irreversible electroporation (IRE) are applied to various tissues. Here, we implemented a standardized in vitro model to compare the effects of biphasic symmetrical pulses (100 pulses, 1–10 μs phase duration (d), 10–1000 Hz pulse repetition rate (f)) using two different human cellular models: human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) and human esophageal smooth muscle cells (hESMCs) cultured in monolayer format. We report the PEF‐induced irreversibly electroporated cell monolayer areas and the corresponding electric field thresholds (EFTs) for both cardiac and esophageal cultures. Our results suggest marked cell type specificity with EFT estimated to be 2–2.5 times lower in hiPSC‐CMs than in hESMCs when subjected to identical PEF treatments (e.g., 0.90 vs 1.85 kV/cm for the treatment of 100 pulses with d = 5 μs, f = 10 Hz, and 0.65 vs 1.67 kV/cm for the treatment of 100 pulses with d = 10 μs, f = 10 Hz). PEF treatment can result in increased temperature around the stimulating electrodes and lead to unanticipated thermal tissue damage that is proportional to the peak temperature rise and to the duration of the PEF‐induced elevated temperatures. In our study, temperature increases ranged from less than 1°C to as high as 30°C, however, all temperature changes were transient and quickly returned to baseline and the highest observed ∆T returned to 50% of its maximum recorded temperature in tens of seconds.

Lentinan Enhances the Function of Oxaliplatin on the Esophageal Tumors by Persuading Immunogenic Cell Death.

Objective The focus of this research was to look at the effects of the combination of the lentinan (LNT) and oxaliplatin (Oxa) on the apoptosis of human esophageal cancer cells, as well as the underlying mechanism. Methods LNT and Oxa were used to treat EC-109 human esophageal cancerous cells at various doses, and the cell survival rate was measured using the Cell Counting Kit-8 (CCK-8) assay. In addition, 24 h after treatment of EC-109 cells with a combination of LNT and Oxa, flow cytometry was used to analyze their apoptotic effect on these cells. Additionally, LNT on EC-109 cell apoptotic upshot was assessed via measuring the consequence of LNT on the mRNA and protein expression levels pertaining to immunogenic cell death factors CALR, HSP90, and HSP70 by qPCR (quantitative real-time polymerase chain reaction) and western blot analysis, correspondingly. Results Cell proliferation was inhibited only when EC-109 cells were added with LNT at 1,200 μg/mL to the maximum concentrations, but the combination of LNT and Oxa at a low dose (800 μg/mL and 20 μM, respectively) significantly increased their sensitivity to Oxa and reduced their proliferation (P < 0.05), and their apoptosis was significantly increased by LNT (P < 0.05). The immunogenic cell death-related genes CALR, HSP90, and HSP70 had dramatically enhanced mRNA and protein expression levels after therapy with a combination of LNT and Oxa (P < 0.05). Conclusion These data imply that LNT increases the susceptibility of esophageal cancerous cells to Oxa by driving EC-109 cells to display immunogenic death. Therefore, LNT combined with Oxa may be an effective method in esophageal cancer management.

Risk Stratification and Validation of Eleven Autophagy-Related lncRNAs for Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC), the most prevalent subtype of esophageal cancer, ranks sixth in cancer-related mortality, making it one of the deadliest cancers worldwide. The identification of potential risk factors for ESCC might help in implementing precision therapies. Autophagy-related lncRNAs are a group of non-coding RNAs that perform critical functions in the tumor immune microenvironment and therapeutic response. Therefore, we aimed to establish a risk model composed of autophagy-related lncRNAs that can serve as a potential biomarker for ESCC risk stratification. Using the RNA expression profile from 179 patients in the GSE53622 and GSE53624 datasets, we found 11 lncRNAs (AC004690.2, AC092159.3, AC093627.4, AL078604.2, BDNF-AS, HAND2-AS1, LINC00410, LINC00588, PSMD6-AS2, ZEB1-AS1, and LINC02586) that were co-expressed with autophagy genes and were independent prognostic factors in multivariate Cox regression analysis. The risk model was constructed using these autophagy-related lncRNAs, and the area under the receiver operating characteristic curve (AUC) of the risk model was 0.728. To confirm that the model is reliable, the data of 174 patients from The Cancer Genome Atlas (TCGA) esophageal cancer dataset were analyzed as the testing set. A nomogram for ESCC prognosis was developed using the risk model and clinic-pathological characteristics. Immune function annotation and tumor mutational burden of the two risk groups were analyzed and the high-risk group displayed higher sensitivity in chemotherapy and immunotherapy. Expression of differentially expressed lncRNAs were further validated in human normal esophageal cells and esophageal cancer cells. The constructed lncRNA risk model provides a useful tool for stratifying risk and predicting the prognosis of patients with ESCC, and might provide novel targets for ESCC treatment.

Abstract 6124: Interleukin-1β and exosomal M6PR secreted by serglycin-overexpressing esophageal cancer cells instigate fibroblasts and endothelial cells to promote esophageal cancer progression

Abstract Non-tumor cells can be recruited and educated by cancer cells to facilitate cancer progression. Previously, we found that serum serglycin (SRGN), a secretory proteoglycan, was an independent prognostic marker for patients with esophageal squamous cell carcinoma (ESCC), and that the autocrine pro-invasive effect of SRGN on ESCC cells was mediated by midkine (MDK). Here, we investigated the effects of cancer cell-derived SRGN on human esophageal fibroblasts (HEF) and human umbilical vein endothelial cells (HUVECs). We found that conditioned medium from SRGN-overexpressing ESCC cells (SRGN-CM) promoted the migration and proliferation of HEF. After SRGN-CM treatment, HEF showed increased expression of fibroblast activation protein alpha (FAP), hepatocyte growth factor (HGF) and amphiregulin, and could enhance tumor growth in vivo. In addition, exosomes derived from SRGN-overexpressing ESCC cells (SRGN-Exo) enhanced the tube formation ability of HUVECs. We found that the effects of SRGN-CM on activation, migration and proliferation of HEF were mediated by MDK. To elucidate the mechanisms by which SRGN upregulates HGF and amphiregulin, and promotes endothelial tube formation, cytokine array and mass spectrometry were performed to analyze differentially expressed proteins in SRGN-CM and SRGN-Exo respectively. The results showed upregulated secretion of interleukin (IL)-1β, IL-18 and tumor necrosis factor-α in SRGN-CM, as well as enriched cation-dependent mannose-6-phosphate receptor (M6PR), integrin alpha-5, teneurin-2 and neurogenic locus notch homolog protein 2 in SRGN-Exo, which were validated by Western blot. These effects were dependent on the glycosaminoglycan chains on SRGN. Our data also showed that the enhanced secretion of IL-1β promoted the expression of HGF in HEF by activating extracellular signal-regulated kinase/activating protein-1. Treatment with SU11274, a c-Met (the receptor of HGF) inhibitor, attenuated the proliferation of ESCC cells co-cultured with HEF, which further indicates that IL-1β-induced HGF from HEF plays a significant role in the tumor microenvironment. In addition, the upregulated exosomal M6PR was found to mediate the enhancing effect of SRGN-Exo on endothelial tube formation ability. Notably, the expression level of M6PR in serum samples of patients with ESCC was positively correlated with that of SRGN and with poor survival. Taken together, SRGN overexpression in ESCC cells created a tumor-promoting microenvironment by altering the ESCC cell secretome including exosomes to exert influence on HEF and HUVECs. [This study was supported by Research Grants Council of the Hong Kong SAR, China, GRF Project No. 17100819] Citation Format: Dongdong Yan, Di Cui, Yun Zhu, Cecilia Ka Wing Chan, Chung Hang Jonathan Choi, Tengfei Liu, Sai Wah Tsao, Stephanie Ma, Annie Lai-Man Cheung. Interleukin-1β and exosomal M6PR secreted by serglycin-overexpressing esophageal cancer cells instigate fibroblasts and endothelial cells to promote esophageal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6124.

Multiparametric High-Content Cell Painting Identifies Copper Ionophores as Selective Modulators of Esophageal Cancer Phenotypes.

Esophageal adenocarcinoma is of increasing global concern due to increasing incidence, a lack of effective treatments, and poor prognosis. Therapeutic target discovery and clinical trials have been hindered by the heterogeneity of the disease, the lack of “druggable” driver mutations, and the dominance of large-scale genomic rearrangements. We have previously undertaken a comprehensive small-molecule phenotypic screen using the high-content Cell Painting assay to quantify the morphological response to a total of 19,555 small molecules across a panel of genetically distinct human esophageal cell lines to identify new therapeutic targets and small molecules for the treatment of esophageal adenocarcinoma. In this current study, we report for the first time the dose–response validation studies for the 72 screening hits from the target-annotated LOPAC and Prestwick FDA-approved compound libraries and the full list of 51 validated esophageal adenocarcinoma-selective small molecules (71% validation rate). We then focus on the most potent and selective hit molecules, elesclomol, disulfiram, and ammonium pyrrolidinedithiocarbamate. Using a multipronged, multitechnology approach, we uncover a unified mechanism of action and a vulnerability in esophageal adenocarcinoma toward copper-dependent cell death that could be targeted in the future.

COX-2 strengthens the effects of acid and bile salts on human esophageal cells and Barrett esophageal cells

AimsInvestigate the effect and mechanism of COX-2 on viability, intestinal metaplasia, and atypia in human esophageal squamous and Barrett esophageal cell lines.MethodsHuman esophageal squamous and Barrett esophageal cell lines were transfected with a COX-2 expression vector and a COX-2 siRNA, and then were treated with acid, bile salts, and a mixture of both. Cell viability, the expression of COX-2, NF-κB(p65), CDX-2, MUC2, c-myb, and BMP-4, and the morphology and microstructure of cells were then observed.ResultsThe viability of COX-2 overexpressed cells was significantly higher than that of control cells, while the viability of COX-2 siRNA-treated cells was significantly lower than that of control cells. Intestinal metaplasia and atypia were observed in cells overexpressing COX-2. Acid, bile salts, and their mixture inhibited the viability of these two cell lines, but the inhibitory effect of the mixture was stronger than a single treatment in either. SiRNA mediated knockdown of COX-2 strengthened the antiproliferative effects of the mixture on HET-1A and BAR-T cells. The expression of p-p65, CDX-2, and BMP-4 was positively correlated with COX-2 expression, while the expression levels of p65, MUC2, and c-myb remained unchanged.ConclusionCOX-2 may influence the viability, atypia, and intestinal metaplasia of human esophageal cells and Barrett esophageal cells. Activation of the p-p65, CDX-2, and BMP-4 signaling pathways by COX-2 may be part of this mechanism.

Overexpression of Adenovirus E1A Reverses Transforming Growth Factor-β-induced Epithelial-mesenchymal Transition in Human Esophageal Cancer Cells.

The epithelial-mesenchymal transition (EMT), a normal biological process by which epithelial cells acquire a mesenchymal phenotype, is associated with migration, metastasis, and chemoresistance in cancer cells, and with poor prognosis in patients with esophageal cancer. However, therapeutic strategies to inhibit EMT in tumor environments remain elusive. Here, we show the therapeutic potential of telomerase-specific replication- competent oncolytic adenovirus OBP-301 in human esophageal cancer TE4 and TE6 cells with an EMT phenotype. Transforming growth factor-β (TGF-β) administration induced the EMT phenotype with spindleshaped morphology, upregulation of mesenchymal markers and EMT transcription factors, migration, and chemoresistance in TE4 and TE6 cells. OBP-301 significantly inhibited the EMT phenotype via E1 accumulation. EMT cancer cells were susceptible to OBP-301 via massive autophagy induction. OBP-301 suppressed tumor growth and lymph node metastasis of TE4 cells co-inoculated with TGF-β-secreting fibroblasts. Our results suggest that OBP-301 inhibits the TGF-β-induced EMT phenotype in human esophageal cancer cells. OBP-301-mediated E1A overexpression is a promising antitumor strategy to inhibit EMT-mediated esophageal cancer progression.

Metabolomics and proteomics study reveals the effects of benzo[a]pyrene on the viability and migration of KYSE-150 esophageal cells

A representative polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P), has been widely detected in environmental compartments and is highly carcinogenic to humans. Oral ingestion of B[a]P is the dominant exposure pathway. The esophagus acts as the first contact point when B[a]P enters the human body. However, its role in the development of human esophageal cancer is rarely discussed. Herein, we employed untargeted metabolomics in combination with proteomics to explore B[a]P-related intracellular responses in human esophageal cell lines. Our results demonstrated that B[a]P exposure induced significant metabolic disorders, further leading to overproduction of reactive oxygen species (ROS) and disturbance of the cellular viability process and migration ability of esophageal cells. In response, glutathione (GSH) was consumed to meet the demand for cellular detoxification, and thioredoxin (TXN) was upregulated to balance the cellular redox. These alterations caused the reregulation of some specific protein families, including S100A proteins, ribosomal proteins, and histone H1 proteins. Such changes impeded the viability and migration of esophageal cells, which could adversely affect wound healing of the epithelium. These cellular responses indicate that B[a]P will cause serious cellular damage to esophageal cells and increase the carcinogenic risk even as a result of short-term exposure. SynopsisOur omics study demonstrated how benzo[a]pyrene hampered the migration of esophageal cells and proposed a plausible mechanism underlying its carcinogenicity, which may contribute to our understanding of environmental pollutants.