Abstract 4881: A novel method of tumor characterization by protein and microRNA biomarker release using ultrasound
Authors: D'Souza Aloma L. , Chevillet John R. , Kroh Evan M. , Tewari Muneesh , Gambhir Sanjiv S. , Glazer Gary M. 
Published: June 2012

Abstract
Earlier cancer diagnosis and monitoring of therapy could be improved by the earlier detection of circulating biomarkers. Ultrasound at low frequencies is known to permeabilize cell membranes and has been used for delivery of molecules into the cell. We hypothesize and prove that this bioeffect of ultrasound also causes the release of both protein and nucleic acid biomarkers from cells in culture and mice. The ability to focus ultrasonic waves allows for the localization of the potential biomarker source in vivo. This novel strategy could lead to the earlier identification, characterization and localization of incidental lesions, cancer and other disease.
Methods: The colon cancer cell line LS174T that produces protein biomarkers (carcinoembryonic antigen-CEA and cancer antigen 19-9 – CA19-9) and microRNAs (miRNA – miR -16 and miR-141) was exposed to low frequency ultrasound (1 MHz) in culture using varying intensities (0, 0.1, 0.3. 0.5, 0.7, 1.0 W/cm2) and time (0, 10, 30 min). Subcutaneous tumors of LS174T in mice were also exposed to ultrasound (2 W/cm2; 6 min), directly over the tumors or at a non-tumor bearing site. Samples were collected pre and post-ultrasound treatment and compared for changes in biomarker levels. Protein biomarkers were detected using an enzyme-linked immunosorbant assay and miRNAs were detected using quantitative reverse-transcription polymerase chain reaction. Cell death was studied using Trypan blue staining.
Results: LS174T cells treated with 1 MHz ultrasound in culture (n=4) at a low intensity of 0.3 W/cm2 released both CEA and CA19-9 with an increase in time (0, 10, 30 min; p&lt;0.05). Ultrasound treatment increased the abundance of cell-free miRNAs released into the media in a time and intensity dependent manner. This release was seen to increase relative to pre-treatment levels, from 15-fold (0.1 W/cm2) to &gt;1000-fold (1.0 W/cm2) for miR-16 and 100-fold (0.1 W/cm2) to 500-fold (1 W/cm2) for miR-141, within 30 min of ultrasound treatment. Cell death was less than 5% across all conditions. Subcutaneous tumors (n=10) showed an increase release of protein biomarkers when treated with ultrasound at 2 W/cm2 (CEA p&lt;0.04; CA19-9 p&lt;0.002), when comparing pre- and post-ultrasound measurements. Controls treated with no ultrasound or on non-tumor bearing sites of tumor-bearing mice did not show any significant increase in the release of biomarkers.
Conclusions: Increase in protein and miRNA biomarkers were observed when ultrasound was directly applied to cells. We have developed a simple non-invasive method to amplify and spatially localize the biomarker signal from tumors. This method has implications in diagnosis and monitoring of therapy and has a clear pathway into clinical applications uniting the fields of imaging and in vitro diagnostics.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4881. doi:10.1158/1538-7445.AM2011-4881