High intensity focused ultrasound-induced bubbles stimulate the release of nucleic acid cancer biomarkers

Abstract

Background/introduction Prostate biopsy for prostate cancer (PCa) is invasive with associated morbidity and several diagnostic limitations, suggesting the need for a new approach. Recently, several nucleic acid cancer biomarkers (e.g., microRNA and mutant DNA) have been identified and shown promise for improving cancer diagnostics. However, the abundance of these biomarker classes in the circulation is low, impeding reliable detection and adoption into clinical practice. In order to stimulate the release of these intracellular biomar-kers, the exposures optimized for mechanical disruption of cells may be desirable. Here, two approaches based on HIFU-induced bubble activity were tested for their ability to stimulate release of cancer-associated microRNAs in a heterotopic syngeneic rat prostate cancer model. In the first approach, tumor tissue was locally liquefied with boiling histotripsy (BH) -a HIFU technique utilizing millisecond-long pulses to create boiling bubbles via rapid shockwave heating. The interaction of shocks with the ensuing vapor cavity frac-tionates tissue with negligible thermal effect. In the second approach HIFU-induced inertial cavitation was used for permeabilization of tumor tissue and vasculature. Methods Putative miRNA biomarkers were identified using RT-PCR array profiling of the syngeneic MatLyLu rat PCa cell line. Adult intact male Copenhagen rats were then subcuta-neously grafted with the MatLyLu cells. When the tumors were >1cm, the rats were assigned to one of two HIFU treatment groups: HIFU optimized for inertial cavitation activity (focal peak negative pressure 16 MPa, 1 ms pulses, duty factor 0.001, N=6), BH (intensity ~20kW/cm2, 1% duty factor, N=8) or a to a control group (N=6) that received sham treatment. Treatments were performed in a heated water tank using a single-element 1.5 MHz HIFU transducer (45 mm radius of curvature, 64 mm aperture) under ultrasound image guidance. Blood samples were collected immediately prior to treatment and serially over a 24-hour time course. Specimens were immediately pro-cessed into plasma and miRNAs extracted. Plasma con-centrations of candidate tumor-derived miRNAs were measured via quantitative RT-PCR and compared with ANOVA and the Mann-Whitney test. Results and conclusions Following sham procedure, no significant changes were observed in the relative plasma concentrations of any to the data made available in this article, unless otherwise stated. evaluated miRNA. Conversely, following both cavitation-based and BH treatments, the relative plasma concentra-tions of the putative PCa-associated miRNAs miR-34c and miR-196a increased significantly while the relative concentration of the broadly expressed, non-PCa specific miR-16 was not significantly altered. PCa-associated miRNA concentrations peaked at 0.25 hr (10-23-fold) from initiation of HIFU treatment, remained signifi-cantly elevated for 3 hours, and then returned to base-line within 24 hours. Histologic examination of excised tumor confirmed complete fractionation of targeted tumor by BH and localized areas of intraparenchymal hemorrhage and tissue disruption by cavitation-based treatment. These data suggest a clinically useful applica-tion of HIFU-induced bubbles for non-invasive molecu-lar biopsy. Acknowledgements (Funding) Work supported by NIH 1K01EB015745, R01CA154451, R01DK085714.