Developing a novel cancer biomarker blood test by measuring mRNA levels of cell cycle genes in circulating extracellular vesicles

Background: Malignant cancer cells are highly proliferative. These cells actively release extracellular vesicles (EVs) in the tumor environment and into body fluids, such as blood. Cancer-derived EVs are enriched with cell cycle gene-related molecules, such as the target transcripts of the transcription factor family E2F. Quantification of cell cycle gene mRNA in blood-derived EVs of cancer patients can represent an indicator of this proliferation signature, which can be used to diagnose the presence of malignant tumors, predict the prognosis and efficacy of anti-cancer treatment and/or monitor disease progression, representing a potential new useful, minimally invasive, clinically applicable test. Methods: Six highly expressed cell cycle gene transcripts were selected based on the analysis of transcriptome data of cancer tissues available in public databases. The expression levels of these transcripts were then evaluated by qPCR in EVs isolated from cultured canine melanoma cells. To investigate whether the mRNA levels in tumor cells is mirrored into their released EVs, cell proliferation was blocked using a CDK4/6 inhibitor. As a first step to detect the cell cycle gene transcripts in plasmaderived EV, total RNA was extracted from the plasma of canine cancer patients and analyzed for the presence of the selected cell cycle transcripts. Results: The selected transcripts were detected in both large and small melanoma-derived EVs, but were most highly enriched in exosomes. CDK4/6 inhibitor treatment induced a decrease in cellular transcript levels, which were accompanied by a decrease of mRNA levels in EVs. In the plasma of canine cancer patients mRNA of some of the selected genes was detected with overall higher expression levels when compared to plasma of healthy controls. Conclusions: In vitro, cell cycle gene mRNAs are present within cancer cell-derived EVs and their levels reflect the expression levels and proliferation status of donor cells. Furthermore, cell cycle gene transcripts are detectable in the plasma of canine cancer patients. Further investigation will be applied to validate their association to circulating EVs and the correlation with tumor proliferation rate, patient response to therapy, and follow-up data.