Cancer associated fibroblasts predict for poor outcome and promote periostin-dependent invasion in oesophageal adenocarcinoma

Interactions between cancer cells and cancer-associated fibroblasts (CAF) play an important role in tumour development and progression. In this study we investigated the functional role of CAF in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of EAC patients (183 patients) for CAF markers related to disease mortality. We characterized CAF and normal oesophageal fibroblasts (NOF) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3-D organotypic culture and xenograft models were used to examine effects on EAC cell function, and dissect molecular mechanisms regulating invasion. Most EAC (93%) contained CAF with a myofibroblastic (?-SMA-positive) phenotype, which correlated significantly with poor survival (p?=?0.016; HR 7. 1 (1.7-29.4). Primary CAF, isolated from EAC, have a contractile, myofibroblastic phenotype, and promote EAC cell invasion in vitro (Transwell assays, p?=?<0.05; organotypic culture, p?<?0.001) and in vivo (p?=?<0.05). In vitro, this pro-invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAF, and acts as a ligand for EAC cell integrins ?v?3 and ?v?5, promoting activation of the PI3kinase/Akt pathway. In patient samples, periostin expression at the tumour cell/stromal interface correlates with poor overall and disease-free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF-secreted periostin and EAC-expressed integrins results in PI3 kinase/Akt activation and increased tumour cell invasion. Most EAC contain a myofibroblastic CAF-rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patient

Role of Mcl-1 turnover in the control of neutrophil apoptosis

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

scSNV: accurate dscRNA-seq SNV co-expression analysis using duplicate tag collapsing

Abstract Identifying single nucleotide variants has become common practice for droplet-based single-cell RNA-seq experiments; however, presently, a pipeline does not exist to maximize variant calling accuracy. Furthermore, molecular duplicates generated in these experiments have not been utilized to optimally detect variant co-expression. Herein, we introduce scSNV designed from the ground up to “collapse” molecular duplicates and accurately identify variants and their co-expression. We demonstrate that scSNV is fast, with a reduced false-positive variant call rate, and enables the co-detection of genetic variants and A>G RNA edits across twenty-two samples

MiR-145 Expression Accelerates Esophageal Adenocarcinoma Progression by Enhancing Cell Invasion and Anoikis Resistance

<div><p>Background</p><p>Carcinoma of the esophagus has a high case fatality ratio and is now the 6th most common cause of cancer deaths in the world. We previously conducted a study to profile the expression of miRNAs in esophageal adenocarcinoma (EAC) pre and post induction therapy. Of the miRNAs differentially expressed post induction chemoradiation, miR-145, a known tumor suppressor miRNA, was upregulated 8-fold following induction therapy, however, its expression was associated with shorter disease-free survival. This unexpected result was explored in this current study.</p><p>Methods</p><p>In order to study the role of miR-145 in EAC, miRNA-145 was overexpressed in 3 EAC cell lines (OE33, FLO-1, SK-GT-4) and one ESCC cell line (KYSE-410). After validation of the expression of miR-145, hallmarks of cancer such as cell proliferation, resistance to chemotherapy drugs or anoikis, and cell invasion were analyzed.</p><p>Results</p><p>There were no differences in cell proliferation and 5 FU resistance between miR145 cell lines and the control cell lines. miR-145 expression also had no effect on cisplatin resistance in two of three cell lines (OE33 and FLO-1), but miR-145 appeared to protect SK-GT-4 cells against cisplatin treatment. However, there was a significant difference in cell invasion, cell adhesion and resistance to anoikis. All three EAC miR-145 cell lines invaded more than their respective controls. Similarly, OE33 and SK-GT-4 miR-145 cell lines were able to survive longer in a suspension state.</p><p>Discussion</p><p>While expression of miR-145 in ESCC stopped proliferation and invasion, expression of miR-145 in EAC cells enhanced invasion and anoikis resistance. Although more work is required to understand how miR-145 conveys these effects, expression of miR-145 appears to promote EAC progression by enhancing invasion and protection against anoikis, which could in turn facilitate distant metastasis.</p></div

MiR-145 inhibited cell proliferation, delayed wound closure and enhanced anoikis in ESCC cells.

<p>(A) Cell proliferation and (B) wound healing assay of KYSE-410 pcmv and KYSE-410 miR-145 cells. miR-145 expression in KYSE-410 led to decreased numbers of colonies after cell suspension culture (C) and enhanced PARP and caspase 3 cleavage (D).*: p<0.05, n = 3.</p

MiR-145 accelerated wound closure and enhanced cell invasion.

<p>Wound healing assay (A) with pcmv and miR-145 cell lines. The wound length was assessed after 8 h culture. *: p<0.05, n = 3. Cell invasion assay with pcmv and miR-145 cell lines (B). *: p<0.05, n = 3.</p

MiR-145 did not generally affect the EAC resistance to chemotherapy drugs.

<p>OE33, FLO-1 and SK-GT-4 (pcmv and miR-145) cells were cultured for 72 h with either (A) cisplatin (5 µM) or (B) 5-fluorouracil (35 µM) and their respective control. Live cell number was then assessed. Results show the percentage of live cells compared to the control treatment. *: p<0.05, n = 3.</p

MiR-145 enhanced the clonogenic potential of OE33 and SK-GT-4 after suspension culture.

<p>Photo images of clonogenic assay after 72 h suspension culture with OE33 and SK-GT-4 (A). Results showed the average percentage of colonies formed after suspension culture compare to the number of colonies formed in monolayer (B). *: p<0.05, n = 2.</p

MiR-145 expression did not affect EAC cells proliferation.

<p>Cell proliferation assay of the pcmv and miR-145 EAC cell lines, n = 3</p

MiR-145 protected EAC cells against anoikis.

<p>pcmv and miR-145 EAC cell lines were cultured in suspension for 72 h. The levels of cleaved PARP and cleaved caspase 3 were assessed by Western Blotting.</p