Synthesis of single cell, single molecule RNA probes for the detection of enogenous microRNAs in human cancer to dentify metastatically relevant cance cell fractions in heterogenous tumors

Cancer metastasis is the major cause of death of cancer patients, and novel diagnostic methods are urgently needed to prevent metastasis. Especially, methods are needed to identify metastatically critical cells within the heterogeneity of diverse cells in patients’ primary tumors, to predict which patient, despite an initial curative treatment, would need additional (targeted) therapy to prevent metastasis. Our recent work has characterized the most critical microRNAs for metastasis in a systematic approach at resected metastasis tissues, and we have already succeeded in introducing the first single cell, single molecule laser superresolution microscopy to visualize specific microRNA molecules marked with photostable cyanine-styryl dyes in subcellular compartments (including exosomes) of fixed single cells in vitro in tumour cell lines. Own work and publications of others have confirmed the importance of the action of particular pro-metastatic miRs in exosomes, which can communicate a cross-talk between cancer and surrounding cells, and also helps cancer cells to prime the metastatic niche in distant organ compartments. Also, the differential interaction of miRs with different 3’UTR targets, depending on a specific cellular context, might be able to support, or inhibit, a pro-metastatic phenotype in a given cell.
The aim of our project therefore is to attempt to synthesize, develop and establish, for the first time, a single cell, single molecule superresolution protocol able to detect, and visualize, 1) naturally occurring miRs in single cells by sequence-specific fluorescence nucleic acid “light up” probes 2) Naturally occurring miRs in single living cells and 3) miRs within single cells of tissue sections of primary tumors of patients. We also will try to attempt multicolour protocols by using differently labelled nucleic acid probes to visualize microRNA intracellular patterns critical for metastasis. 

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