Focused Ultrasound Treatment of a Spheroid In Vitro Tumour Model

Abstract

Focused ultrasound (FUS) is a non-invasive technique producing a variety of biological effects by either thermal or mechanical mechanisms of ultrasound interaction with the targeted tissue. FUS could bring benefits, e.g., tumour sensitisation, immune stimulation, and targeted drug delivery, but investigation of FUS effects at the cellular level is still missing. New techniques are commonly tested in vitro on two-dimensional (2D) monolayer cancer cell culture models. The 3D tumour model-spheroid-is mainly utilised to mimic solid tumours from an architectural standpoint. It is a promising method to simulate the characteristics of tumours in vitro and their various responses to therapeutic alternatives. This study aimed to evaluate the effects of FUS on human prostate and glioblastoma cancer tumour spheroids in vitro. The experimental follow-up enclosed the measurements of spheroid integrity and growth kinetics, DNA damage, and cellular metabolic activity by measuring intracellular ATP content in the spheroids. Our results showed that pulsed FUS treatment induced molecular effects in 3D tumour models. With the disruption of the spheroid integrity, we observed an increase in DNA double-strand breaks, leading to damage in the cancer cells depending on the cancer cell type.

Keywords: FUS; focused ultrasound; glioblastoma; in vitro experiments; prostate cancer; spheroid.

Figure 1

FUS treatment system and generation process of tumour spheroids. (a) Scheme of the in vitro FUS transducer and spheroid treatment. (b) The FUS in vitro system was specially designed for 96-well cell culture plates, with 32 transducers at a frequency of 1.1 MHz and water cooling. (c) The layer of the spheroid formation process for dedicated cancer cell lines. The prostate cancer cell line PC-3 and glioblastoma U87 were cultured in a dedicated medium using a liquid overlay technique to generate tumour spheroids.

Figure 2

Assessment of the single-transducer pressure distribution in 2D and 1D. (a,b) show two 2D sound fields (Peak-to-Peak pressure in dB is plotted) acquired in a water tank measurement to assess the extent of the focal area. (c) Shows a lateral cross-section through the depth of highest pressure.

Figure 3

Assessment of the applicator performance. An XY sound field scan was performed in front of the cell applicator. For (a), the acoustic intensity I_SPTA was averaged over the surface of one well of the 96-well plate in front of each transducer element. (b) A histogram allowing assessing the homogeneity of the intensity output. (a,b) were performed with a power setting of 1% and were made to compare the relative performance of the different wells, not the absolute intensities. (c) The I_SPTA as a function of the power setting that can be user-defined using the “Cell Therapy Planning Tool”.

Figure 4

FUS reduced spheroid size and led to a loss of integrity. (a,b) Representative microscopy images showing alterations in spheroid morphology and (c,d) brightfield images of FUS-treated spheroids; the corresponding 3D reconstructions were obtained using ReViSP, http://sourceforge.net/p/revisp/ (accessed on 8 May 2020). (e/f) Bar chart representation of changes in the spheroid area before, immediately, 48, and 96 h after FUS treatment at an intensity of 2.95 and 5.9 W/cm², + control: +5% DMSO. Data analysis was carried out by one-way ANOVA. * Significantly different from the untreated group. (p ≤ 0.05). # 4th day spheroid formation; * in the ‘untreated’ group only, well-to-well (re-)transfer of spheroid was carried out to equalise the impact. PC-3 cancer cell line: Scale bar = 200 μm. U87 cancer cell line: Scale bar = 100 μm. n = 9.

Figure 5

FUS diminished spheroid metabolic activity. ATP content of PC-3 (a) and U87 (b) spheroids was assessed using CellTiter-Glo^® 3D Cell Viability assay 48 and 96 h after treatment, showing the reduction of cell metabolic activity. Data sets were normalised to the untreated control group (100%), while data analysis was carried out by one-way ANOVA. * Significantly different from the untreated group (p ≤ 0.05). n = 9.

Figure 6

FUS treatment enhanced the number of DNA double-strand breaks after 24 h. (a) Representation of γH2A.X percentages as a function of cell count determined by gating histograms derived from dissociated PC-3 spheroids with flow cytometric analysis 1 and 24 h post-treatment. (b) Overlayed flow cytometry images (c) and quantified results show an increasing number of γH2A.X positive cells 24 h after 5.9 W/cm². Data analysis was carried out by one-way ANOVA. * Significantly different from negative control (p ≤ 0.05).