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

It is estimated that metastasis is the main reason in about 90% of cancer deaths. Therefore, a better understanding of the key molecular players in determining and regulating this process is urgently needed. Previous studies in fixed cells in culture have shown, that certain microRNA (miRNA) molecules are in significantly higher numbers in exosomes of highly metastatic cells in comparison to low metastatic cells [1]. However, in real patient tissues, issues such as the cell heterogeneity in the primary tumor and the definition of metastatic clones are a major challenge in the context of metastasis.

In this project, we used human colon tissue sections, which are relevant samples for routine diagnosis in pathology, to study the chromatin nanostructure of different cells types and the presence and distribution of five metastatically relevant miRNAs (miR-21, miR-31, mi-R210, miR-135b and miR-218).

DNA-sequences complementary to the above-mentioned metastatically relevant miRNA were synthesized and modified so that a fluorescent dye was incorporated in the middle of the sequence. Human formalin-fixed paraffin-embedded (FFPE) colon tissues were sliced into 10mm thick sections and fluorescent in situ hybridization (FISH) with the different miRNA probes was performed at normal and colon cancer tissue sections followed by a subsequent nuclear counterstain with Sytox Orange (SO).

Confocal images allowed the quantification of the different miRNAs in human tissue samples. Pro-metastatic miRNAs were found in significantly higher numbers of molecules in epithelial cells of tumor samples in comparison with their normal counterparts. More variability and a strong patient-associated quantification was observed for miR-218, an anti-metastatic miRNA.

We successfully performed, for the first time, single molecule localization microscopy (SMLM) for the chromatin nanostructure and human microRNAs at the single molecule level in routine patient tissues. Preliminary results showed the distribution of miR-21 in the periphery and the inside of a tumor cell nucleus. Vesicle-like structures outside the nuclear region of sizes ranging from 110 to 240 nm appear to be loaded with this miRNA.

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