Applying toponome imaging system for studying colon cancer

Introduction In a proof of principle study, we have applied an automated fluorescence toponome imaging system (TIS) in order to examine, whether TIS can find protein network structures distinguishing cancerous from normal colon tissue from same patient. Methods/Materials Cancer specimen and corresponding normal tissue were harvested at colectomy from a single patient. 5μm sections were then prepared for TIS using a battery of different antibodies, including a number of putative CSC markers. Expression of multiple protein clusters was determined and Combinatorial Molecular Phenotypes (CMPs) were analysed, using specific image-analysis tools. Results By using a three symbol code and a power of combinatorial molecular discrimination (PCMD) of 221 per sub-cellular data point in one single tissue section, we demonstrate an in situ protein network structure, visualized as a mosaic of 6,813 protein clusters (Combinatorial molecular phenotype or CMPs) in the cancerous part of the colon. By contrast, in the histologically normal colon, TIS identifies nearly 5 times the number of protein clusters as compared to the cancerous part (32,009). Discussion and Conclusion By sub-cellular visualization procedures we found, that many cell surface membrane molecules were closely associated with the cell cytoskeleton as unique CMPs in the normal part of the colon, while the same molecules were disassembled in the cancerous part, suggesting presence of dysfunctional cytoskeleton-membrane complexes. As expected, glandular and stromal cell signatures were found, but interestingly also potentially TIS signatures identifying a very restricted subset of cells expressing several putative stem cell markers, all restricted to the cancerous tissue. The detection of these signatures is based on the extreme searching depth, high degree of dimensionality, and sub-cellular resolution capacity of TIS. These findings provide the technological rationale for the feasibility of a complete colon cancer toponome to be established by massive parallel high throughput/high content TIS mapping.