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Enhanced Ligase based histochemical techniques

Final Report Summary - ENLIGHT (Enhanced Ligase based Histochemcial Techniques)

In situ analysis of cells and tissues has for many years been an essential part of pathological research and diagnosis primarily within cancer, and a number of specific biomarkers of predictive and prognostic value for various cancers have been identified. In situ analysis of proteins is dominated by immunohistochemistry where sections of tissues are tested for the presence of proteins by specific antibodies, while in situ analysis of nucleic acid sequences is dominated by in situ hybridisation. Due to limitations in these technologies there is a significant need for new efficient techniques and procedures for more advanced analyses. A major challenge is to develop improved means for more detailed studies of biomolecules in situ, in order to determine their abundance, sub-cellular localisation and secondary modifications, as well as how they interact with other molecules and participate in signalling and control of cellular function.

The main purpose of the ENLIGHT project was to develop new analytical procedures with the sensitivity and specificity required to study individual nucleic acid and protein molecules and their functional status (interactions and posttranslational modifications) in their normal context in single cells and tissues ('in situ') and on tissue lysate microarrays. The project was based on two fundamental recent technological inventions, the 'proximity ligation assay' technology (also called PLA) for protein analysis and 'padlock probing' for DNA analysis. These are the first technologies to offer the sensitivity and specificity required for studies of single bio-molecules in single cells and tissue in situ.

The detection by microscopy of characteristic biomarkers in cancer cells is based on specific staining of tissue sections or cells isolated from patients and mounted on glass slides. The staining results in coloured or fluorescent signals at those locations where the biomarkers are expressed, and the number and / or intensity of the signals often reflects the presence (diagnosis), severity (prognosis) and preferred treatment (prediction) of the disease. The quantification of high numbers and varying intensities of signals is very cumbersome (if possible) without assistance from computers and dedicated software programs.

The ENLIGHT project provided novel molecular and software tools to the scientific community in order to study protein and DNA biomarkers in cultured cells and clinical specimens with a higher resolution than possible before the project. More specifically, we developed a generic PLA method for detailed studies of protein biomolecules in situ. We explored and optimised the method in order to analyse individual proteins, interactions between proteins, post-translational protein modifications and the sub-cellular localisation of these molecules. We furthermore used the padlock probe technology to distinguish single-nucleotide differences between different mitochondrial genomes, and their localisation in situ. Relative to state-of-the-art procedures, we have shown that these methods can provide significantly improved in situ analyses. We also developed automated image analysis procedures to complement the molecular methods, in order to achieve quantitative information about what molecules or molecule complexes are present in a sample and their tissue or sub-cellular localisation (i.e. in which cellular substructures). Finally, we generated important new biological knowledge on the role of several biomarkers in tumourigenesis and mitochondrial diseases thanks to the novel molecular methods and image analysis procedures developed in the project.

Uppsala University invented the PLA principle before the ENLIGHT project started. During the project, the Uppsala lab explored new ways to use PLA. To improve the usefulness of the technology and to facilitate technology transfer to other laboratories, the possibility to use secondary species-specific proximity probes were explored by Uppsala University in the ENLIGHT project, omitting the need of covalent conjugation of oligonucleotides to each new pair of antibodies. Modifications of the methods enabled readout by flow-cytometers, bright-field microscopy and the method was shown to have advantages in high-content drug screening. Further improvements of the method was made to allow multiplexed detection.

ENLIGHT provided the scientific community with two new and unique molecular tools to study individual proteins or nucleic acids and their functional status in single cells and clinical specimens. This project consisted of three European SMEs (Olink AB, Visiopharm A/S, ImmunSystem AB) and one large established company (Dako Denmark A/S), all with high future potential. These companies were successfully brought together with academic scientists (Uppsala University, VTT Technical Research Centre in Finland, Edinburgh University, Leiden University Medical Centre). We expect that the in situ techniques developed in this project, and the scientific knowledge created, in the longer run will lead to improved disease prevention, more rapid and accurate cancer diagnosis, and better treatment opportunities.

Relative to state-of-the-art procedures, these methods are expected to provide significantly improved in situ analyses in terms of specificity, sensitivity (single molecule detection), possibility to study biomarker localisation, analysis of protein interactions and protein modifications, and an opportunity for simultaneous analysis of multiple markers (multiplex analysis). Automated image analysis procedures were developed, i.e. software-based classification of molecules and their localisation in tissues or cells. The software provides a rapid way to analyse many samples as well as user-independent, unbiased data classification. The results from this project have provided important new commercial opportunities for products addressing significant market needs, thereby allowing the participating European companies to build sustainable businesses at the forefront of biotechnology.