In situ hybridization to detect DNA amplification in extracellular vesicles

Abstract

EVs have emerged as an important component in tumour initiation, progression and metastasis. Although notable progresses have been made, the detection of EV cargoes remain significantly challenging for researchers to practically use; faster and more convenient methods are required to validate the EV cargoes, especially as biomarkers. Here we show, the possibility of examining embedded EVs as substrates to be used for detecting DNA amplification through ultrasensitive in situ hybridization (ISH). This methodology allows the visualization of DNA targets in a more direct manner, without time consuming optimization steps or particular expertise. Additionally, formalin-fixed paraffin-embedded (FFPE) blocks of EVs allows long-term preservation of samples, permitting future studies. We report here: (i) the successful isolation of EVs from liposarcoma tissues; (ii) the EV embedding in FFPE blocks (iii) the successful selective, specific ultrasensitive ISH examination of EVs derived from tissues, cell line, and sera; (iv) and the detection of MDM2 DNA amplification in EVs from liposarcoma tissues, cell lines and sera. Ultrasensitive ISH on EVs would enable cargo study while the application of ISH to serum EVs, could represent a possible novel methodology for diagnostic confirmation. Modification of probes may enable researchers to detect targets and specific DNA alterations directly in tumour EVs, thereby facilitating detection, diagnosis, and improved understanding of tumour biology relevant to many cancer types.

Keywords: EV embedding; EV in situ hybridization; EVs; FISH; extracellular vesicles (EVs); sarcoma; tissue.