Enhanced Detection of Cancer Biomarkers in Blood-Borne Extracellular Vesicles Using Nanodroplets and Focused Ultrasound

Abstract

The feasibility of personalized medicine approaches will be greatly improved by the development of noninvasive methods to interrogate tumor biology. Extracellular vesicles shed by solid tumors into the bloodstream have been under recent investigation as a source of tumor-derived biomarkers such as proteins and nucleic acids. We report here an approach using submicrometer perfluorobutane nanodroplets and focused ultrasound to enhance the release of extracellular vesicles from specific locations in tumors into the blood. The released extracellular vesicles were enumerated and characterized using micro flow cytometry. Only in the presence of nanodroplets could ultrasound release appreciable levels of tumor-derived vesicles into the blood. Sonication of HT1080-GFP tumors did not increase the number of circulating tumor cells or the metastatic burden in the tumor-bearing embryos. A variety of biological molecules were successfully detected in tumor-derived extracellular vesicles, including cancer-associated proteins, mRNAs, and miRNAs. Sonication of xenograft HT1080 fibrosarcoma tumors released extracellular vesicles that contained detectable RAC1 mRNA with the highly tumorigenic N92I mutation known to exist in HT1080 cells. Deep sequencing serum samples of embryos with sonicated tumors allowed the identification of an additional 13 known heterozygous mutations in HT1080 cells. Applying ultrasound to HT1080 tumors increased tumor-derived DNA in the serum by two orders of magnitude. This work is the first demonstration of enhanced extracellular vesicle release by ultrasound stimulation and suggests that nanodroplets/ultrasound offers promise for genetic profiling of tumor phenotype and aggressiveness by stimulating the release of extracellular vesicles.

Low‐intensity pulsed ultrasound affects growth, differentiation, migration, and epithelial‐to‐mesenchymal transition of colorectal cancer cells

“…Moreover, analyzing the gene expression profiles of microarray datasets on US‐treated cells, Yang et al (2017) showed that LIPUS enhanced EVs biogenesis and internalization. D'Souza et al also showed that US can be used to amplify and localize biomarkers in blood with a relatively simple and noninvasive strategy (D'Souza et al, 2009; Paproski, Jovel, Wong, Lewis, & Zemp, 2017). We observed an effective increased release of EVs upon LIPUS exposure.…”

3D Cellular Architecture Affects MicroRNA and Protein Cargo of Extracellular Vesicles

“…[19] The information conveyed by tumor-derived EVs may functionally influence recipient cells and foster their reprogramming, [20] thus contributing to the regulation of different stages of tumor progression. [21][22][23][24][25] Therefore, once released into the circulation, EVs and EV-associated molecules can be used as biomarkers in diagnosis, [26][27][28] prognosis, [29,30] and therapeutic follow-up of cancer patients. [31,32] The role of EVs in tumor progression has been addressed using mostly in vivo and conventional 2D in vitro models.…”

Focused Ultrasound-enabled Brain Tumor Liquid Biopsy

“…Building on this initial work, in vitro cell culture studies showed the feasibility of enhancing mRNA biomarker release from a breast cancer cell line using microbubble-enhanced ultrasound 11 , and the feasibility of releasing a combination of ovarian cancer biomarkers using ultrasound 12 . Two recent in vivo studies further demonstrated that ultrasound-mediated release of biomarkers into the bloodstream is a promising approach for detecting tumor biomarkers via blood sampling 13 , 14 . In one of the studies, pulsed HIFU with high acoustic pressures was used to induce histotripsy ( i.e.…”

“…, a technique for mechanical tissue fractionation) in a rat model of prostate cancer, and this enhanced release of cell-free tumor microRNA into the blood circulation 13 . In the other study, a chicken embryo tumor model was used to show the feasibility of amplifying the release of extracellular vesicles using high-intensity focused ultrasound (HIFU) in combination with phase-change nanodroplets which changed to microbubbles upon HIFU sonication 14 . Although promising, these preliminary findings cannot be readily extended to applications in the brain, given challenges inherent to the brain: first, delivery of acoustic energy to the brain is impeded by attenuation and distortion of acoustic waves by the skull; second, biomarker release from the brain is inherently limited by the presence of the BBB.…”