Key Technologies

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Patient Derived Xenografts-A PDX platform will be established with more than 70 metastasectomies obtained from mCRC patients.

 

Exome Sequencing Analysis - Exome-enriched genomic tumour DNA from PDXs will be used by VIB to generate raw sequence data using the Illumina® Genome Analyzer II. This approach will provide over 30-fold coverage for at least 90% of all exons.

 

RNA Analysis - RNA from PDX samples (responders and non-responders) will be isolated, amplified, labeled and hybridized to Affymetrix® Human Gene arrays to correlate gene expression with outcome.

 

Contrast Enhanced Ultrasound (CEUS)- will be used as a cost effective pre-clinical modality to longitudinally monitor effects of treatment on tumour vasculature using a Visualsonics Vevo® 770 ultrasound scanner with 704 (40MHz) probe

A Visualsonics Vevo® 770 ultrasound scanner

 

Immunohistochemistry studies- will be employed to assess pharmacodynamic & phenotypic treatment effects for REG response phenotypes: eg tumour cell proliferation (Ki67); microvessel density (CD 31); vessel maturation (Smooth Muscle Actin); angiogenesis (VEGFR2, TIE2); cell death (TUNEL); inhibition of cell signaling pathways (pERK1/2); quantification of macrophages (F4/80).

The TUNEL assay for detection of apoptosis in formalin fixed tissue. In brief, 10micron sections were prepared and TUNEL staining was performed on (B) positive control heart tissue (treated with 1 unit Dnase in 100ul) and (A) non-Dnase treated heart tissue. 20X Magnification.

 

Reverse phase protein array (RPPA) analysis of PDX tumours- The RPPA approach will examine total and phosphorylated protein for a panel of human protein tyrosine kinases & pathway effectors/related proteins of interest for each REG Response Phenotype using a targeted library of 100 validated antibodies

Description of RPPA workflow process. Protein lysates are prepared and plated into 384-well plates. Samples are printed onto nitrocellulose-coated slides and stained with the relevant antibodies. Staining intensity is measured using the relevant software and statistically analysed (diagram courtesy of Professor Lance Liotta).

 

Pre-clinical Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI)-

Functional imaging will be performed at the European Institute for Molecular Imaging (EIMI). REG effects on vessel parameters, tumour microvessel density and tumour vessel size, will be determined by acquiring ΔR2 and ΔR2* maps following administration of classical (Gd) or novel contrast agents.

A small animal MRI scanner Bio-Spec 94/20 (Bruker BioSpin MRI GmbH, Germany). 

 

Multivariate statistics - Multivariate statistics methods such as Principal Component or Cluster Analyses allow us to study the effect of several emergent biomarker signatures in combination. 

 

Systems Biology approaches: Systems Biology approaches use computational models to study genomic regulation and proteomic pathways on different biological scales. This includes the study of protein signaling in a cell, or the proliferation of tumour and non-tumour cells within a tissue.