Hamoudi, R

Khan, S

Levi Seah, R

Nomikou, N

UCL Discovery

Molecular characterisation of FFPE pancreatic tumours treated with 5-Fluorouracil (5-FU) and Sonodynamic Therapy (SDT) using whole transcriptome analysis Introduction  •Current standards of care in pancreatic cancer (PC), such as surgical resection and chemoradiotherapy, remain ineffective in improving overall survival rates in PC •Sonodynamic therapy (SDT) is a novel treatment modality that utilises ultrasound in conjunction with sonosensitisers to destroy tumors in a site-specific manner1 •This study aimed to investigate the effect of 5-Fluorouracil (5-FU) and SDT on expression levels of genes involved in aberrant signaling in PC using Next Generation Sequencing technology such as the Ion Proton™ System    •Bioinformatics analysis was performed using R/bioconductor and Database for Annotation, Visualization and Integrated Discovery (DAVID)   Discussion  •BCL3 expression was lower in both SDT and 5-FU treated samples as compared to untreated control sample, suggesting both treatment modalities cause lower BCL3 expression levels •Functional clustering revealed the involvement of G-Protein coupled receptors (GPCR) and signal transduction pathways in PC •Bioinformatics analysis also revealed two genes that showed the highest levels of differential expression between treated and untreated samples:  •ATP1B1 had 8.94 times lower expression levels in 5-FU sample compared to control, and this plays an integral role in the membrane protein Na+/K+-ATPase involved in energy production3 •RUNDC1 had 6.99 times higher expression levels in SDT sample compared to control, and this is associated with a transcription factor that is involved with ubiquitination4  •Further work will validate the presence of ATP1B1 and RUNDC1 using qRT-PCR, by performing in vitro studies on untreated and treated cell lines Saif-U-Rehman Khan, Ryan Levi Seah, Rifat Hamoudi, Nikolitsa Nomikou UCL Division of Surgery & Interventional Science Materials & Methods    •RNA extraction was performed on 3 FFPE specimens of BxPC-3 human pancreatic adenocarcinoma cells in a mouse model that were subjected to the following treatments2:  1.Untreated (Control) 2.440uM 5-FU  3.O2MB-RB* and 440uM 5-FU  treated with ultrasound (SDT)  •Sample validation was performed using qRT-PCR, qPCR and a bioanalyser •Whole transcriptome amplification was performed using an Ion AmpliSeq™ RNA Library Kit Whole transcriptome sequencing was performed using the Ion Proton™ System on amplified transcriptomes  * Oxygen-carrying microbubbles with covalently attached rose bengal on their surface   Results Figure 1. Mean E-ΔCT of three samples. E-ΔCT was calculated by taking 2-(CT gene of interest – CT reference gene) for each sample. Error bars represent standard error of  the mean where n = 3 Figure 2. Unsupervised hierarchical clustering was applied to normalised  RNA-seq values using R/Bioconductor.  A heatmap representation of  differentially expressed genes among  all samples was generated  •Statistically significant differences in BCL3 expression levels between SDT and control (p = 0.001) and 5-FU and control (p = 0.001) were observed (Figure 1.) •Clustering's heatmap shows different transcriptomic signatures between three transcripts suggesting that each treatment targets different transcriptomic signature (Figure 2.) References  1.Trendowski. Chemotherapy Research and Practice. 2015, 2015. 2.McEwan et. al. Biotmaterials. 2016, 80. 3.National Centre for Biotechnology Information. 2016.  4.STRING. 2016. 

Molecular characterisation of FFPE pancreatic tumours treated with 5-Fluorouracil (5-FU) and sonodynamic therapy (SDT) using whole transcriptome analysis

http://discovery.ucl.ac.uk/1530525/1/Khan_Molecular%20characterisation%20of%20FFPE%20pancreatic%20tumours.pdf

Molecular characterisation of FFPE pancreatic tumours treated with 5-Fluorouracil (5-FU) and sonodynamic therapy (SDT) using whole transcriptome analysis

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