Introduction/Aim. Thymidine analogues are DNA-incorporating synthetic nucleosides that are widely used for detecting proliferating cells in tissues. Unique among them is 5-ethynyl-2’-deoxyuridine (EdU), primarily due to the chemical nature of its detection after its incorporation into replicating DNA. Detection is fast, efficient, and more importantly preserves cellular structure and integrity making it ideal for use in (1) pull-down assays to extract well-conserved RNA for genomic profiling studies; and (2) evaluation of the well-preserved protein expression for histological analysis. In this study, we aim to develop a simple yet efficient method utilising EdU as a marker for a combined histological and molecular profiling of the clinically relevant dividing cells and the relatively quiescent counterpart for their comparative analysis.
Methods. Tumour model was established by intracranial injection of glioma cells in mice. Multifluorescence imaging using the combined techniques of immunofluorescence, fluorescence in situ hybridization, and “click chemistry” for EdU detection were used to analyze cellular phenotypes in brain sections harbouring a tumour. EdU-positive and EdU-negative tumour cells were fractionated for RNA extraction using flow cytometry and subsequent gene profiling was determined by quantitative real time PCR.
Results. EdU positive cells were discretely detected in the established tumour model. Multifluorescence imaging identified and quantified proliferating and non-proliferating tumour-infiltrating host cells and human-derived tumour cells. GFAP+ mouse astrocytes and actively dividing IBA1+ mouse microglial cells were clearly identified as among the host-derived tumour infiltrates. Histological quantification of Ki67 protein expression significantly correlated with the mRNA transcripts in the EdU-positive and EdU-negative cell populations.
Conclusion. In summary, our results have shown that EdU is a powerful tool to detect, isolate and characterize actively dividing cells in tumour, forming the basis for differential protein and gene expression profiling of proliferative and non-proliferative cells in a pre-clinical model of cancer.