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MicroRNA-21 (miR-21) expression in stromal fibroblastic cells in colorectal cancer is well-documented, whereas miR-21 expression in tumor budding cells (TBCs) is poorly described. TBCs are locally invasive carcinoma cells with increased metastatic properties and characteristics of epithelial to mesenchymal transition. This study was conducted to better characterize the expression of miR-21 in TBCs. First, chromogenic miR-21 in situ hybridization (ISH) staining was performed in 58 colon adenocarcinomas with evident TBCs. Then, to obtain unambiguous identification of miR-21 in the TBCs, twenty cases were selected for an additional multiplex fluorescence analysis combining miR-21 ISH with cytokeratin and laminin-5γ2 immunofluorescence. Employing confocal slide scanning microscopy, comprehensive digital images of the invasive front (10-40 mm 2 ) were obtained from 16 of the 20 cases, and miR-21 expression was evaluated in cytokeratin-positive TBCs. The high resolution of the confocal digital slide images allowed a detailed examination of the confocal stacks of the multiplex-stained tissue sections. The cases with the highest fraction of miR-21 positive TBCs were all stage III cancers defined by the presence of regional lymph node metastasis. Some of the miR-21 positive TBCs were also laminin-5γ2 positive. The confocal image stacks also revealed that some TBCs were actually directly connected to malignant glands. In conclusion, miR-21 expression was unambiguously identified in TBCs by evaluation of digital slides obtained by confocal slide scanning microscopy. In addition, the digital confocal slides provided a more detailed understanding of local cancer cell invasion by allowing evaluation of the cell structures in three dimensions.

miR-21 expression analysis in budding colon cancer cells by confocal slide scanning microscopy