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ENLIGHT<br />
New molecular methods and image<br />
analysis tools for analysis of cancer<br />
biomarkers in situ<br />
Summary<br />
The ENLIGHT project has the purpose to develop analytical<br />
procedures with the sensitivity and specifi city required<br />
to study individual nucleic acid and protein molecules,<br />
and also interacting molecules, in their normal context in<br />
cells and tissues (in situ) and in tissue lysate microarrays.<br />
A spectrum of reagents will be developed for the analysis<br />
of specifi c markers of particular interest in oncology. Furthermore,<br />
we will establish software and algorithms for<br />
automatic user-independent in situ image analysis of single<br />
molecule-events. These methods and algorithms will<br />
be applied to evaluate candidate biomarkers of special<br />
relevance for tumour biology and cancer pathology.<br />
Problem<br />
In situ analysis of cells and tissues has been an essential part<br />
of pathological research and diagnosis primarily within cancer<br />
for many years, and a number of specifi c biomarkers of<br />
predictive and prognostic value for various cancers have<br />
been identifi ed. In situ analysis of nucleic acid sequences is<br />
dominated by in situ hybridization, while in situ analysis of<br />
proteins is dominated by immunohistochemistry where sections<br />
of tissues are tested for the presence of proteins by<br />
specifi c antibodies. Due to limitations in these technologies<br />
there is a signifi cant need in the scientifi c community for<br />
new effi cient techniques and procedures for more advanced<br />
analyses. A major challenge is to develop improved means<br />
for more detailed studies of biomolecules in situ, in order to<br />
determine their abundance, sub-cellular localization, and<br />
secondary modifi cations, as well as how they interact with<br />
other molecules and participate in signalling and control of<br />
cellular function.<br />
Aims<br />
The fi rst aim is to develop new molecular methods and<br />
assays for the analysis of individual DNA and protein molecules<br />
in situ. The project is based on two fundamental<br />
technological inventions, padlock probing and proximity<br />
ligation. These are the fi rst technologies to off er the sensitivity<br />
and specifi city required for studies of single bio-molecules<br />
in situ. The padlock probe technology is used to interrogate<br />
30<br />
Keywords | <strong>Cancer</strong> | biomarker | in situ analysis | technology development | proximity ligation | padlock probing |<br />
nucleic acids and to distinguish closely similar sequence variants,<br />
while the proximity ligation assay (PLA) is applied to<br />
analyze individual proteins, interactions between proteins,<br />
and post-translational protein modifi cations.<br />
As a second aim we will develop automated image analysis<br />
procedures to complement the molecular methods. The<br />
objective is to achieve quantitative information about what<br />
molecules or molecule complexes are present in the sample<br />
and their tissue or sub-cellular localization (i.e. in which<br />
cellular substructures).<br />
The third aim is to use the new methods and image analysis<br />
procedures to clarify the role of molecular biomarkers in<br />
tumourigenesis, primarily concerning the AP-1 and HER<br />
protein families and for mitochondrial DNA. The methods<br />
will also be tested in a high-throughput microarray analysis<br />
system. The cancer biomarker will primarily be investigated<br />
in a research setting, but we will also explore the utility of<br />
the markers for diagnostic analyses.<br />
Expected results<br />
This project is expected to provide new means to study<br />
biomarkers for oncogenesis, and to generate novel insights<br />
in cancer biology. We expect that the in situ techniques<br />
developed in this project, and the scientifi c knowledge<br />
created, in the longer run will lead to improved disease<br />
prevention, more rapid and accurate cancer diagnosis, and<br />
better treatment opportunities.<br />
New analytical means will be developed to analyse cancer<br />
biomarkers in situ. Relative to state-of-the-art procedures,<br />
these methods are expected to provide signifi cantly<br />
improved in situ analyses in terms of specifi city, sensitivity<br />
(single molecule detection), possibility to study biomarker<br />
localisation, analysis of protein interactions and protein<br />
modifi cations, and an opportunity for simultaneous analysis<br />
of multiple markers (multiplex analysis). Automated image<br />
analysis procedures will be developed, i.e. software-based<br />
classifi cation of molecules and their localisation in tissues<br />
or cells. The software will provide a rapid way to analyse<br />
many samples as well as user-independent, unbiased data<br />
classifi cation.<br />
The results from this project are furthermore expected<br />
to provide new commercial opportunities for products<br />
addressing signifi cant market needs, thereby allowing the<br />
participating European SMEs to build sustainable businesses<br />
at the forefront of biotechnology.<br />
Potential applications<br />
<strong>Cancer</strong> biology and diagnosis.<br />
CANCER RESEARCH PROJECTS FUNDED UNDER THE SIXTH FRAMEWORK PROGRAMME
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