Abstract 475: Characterization of miR-21 expression in budding colon cancer cells using digital images of multiplex fluorescence stained slides obtained by confocal scanning microscopy

Abstract

Abstract This study was undertaken to analyze the presence and prevalence of microRNA-21 (miR-21) positive budding cancer cells in colon cancer using confocal scanning microscopy. Budding cancer cells are locally invasive cancer cells with increased metastatic properties and characteristics of epithelial to mesenchymal transition (EMT). Expression of miR-21 in stromal fibroblastic cells in colorectal cancers is well documented (1,2), whereas miR-21 expression in budding cells is poorly described. In order to characterize miR-21 positive budding cells, we first developed a multiplex fluorescence staining method by combining miR-21 in situ hybridization with immunohistochemical staining for cytokeratin and budding cell marker laminin-γ2, and stained 16 colon cancer cases (stage II, n=5, stage III, n=11). We then employed a confocal scanning microscope to obtain 15-55 GB digital images covering areas of 10-40 mm2 of the invasive front. The high resolution of the confocal digital images allowed detailed examination of the 4-fluorophore-stained slides using extended focus and z-stack images, e.g. in the discrimination of epithelial cells from adjacent stromal cells. All cytokeratin-positive budding cells were evaluated for the presence of miR-21 and laminin-γ2. Five out of the 16 cases had more than 10% miR-21 positive budding cells, a few of which were also laminin-γ2 positive. All five cases were stage III cancers. The presence of miR-21 in the budding cancer cells was not associated with the level of tumor budding. These observations suggest that the miR-21 expression in tumor budding cells increases with the progression of the cancer and is independent of laminin-γ2.