Migration and invasion of cancer stem cells are prevented by low-intensity pulsed ultrasound therapy

Abstract

Background: Ultrasound is considered a safe and non-invasive tool in regenerative medicine. In particular, low-intensity pulsed ultrasound (LIPUS) has been used in the clinic for more than twenty years for applications in bone healing. It has been demonstrated to be an effective tool to treat different chronic diseases. We sought to evaluate the effects produced by LIPUS on the properties of human breast cancer stem cells (bCSCs).

Methods: Cells were stimulated using a traditional ultrasound device with the following parameters: 0.05 W/cm² with 10% duty cycle, frequency of 3 MHz and 8 pulses.

Results: At the parameters used, the ultrasound did not directly affect bCSC proliferation, with no evident changes in morphology. In contrast, the ultrasound protocol affected the migration and invasion ability of bCSCs, limiting their capacity to advance while a major affection was detected on their angiogenic properties. LIPUS-treated bCSCs were unable to transform into aggressive metastatic cancer cells, by decreasing their migration and invasion capacity as well as vessel formation. Finally, RNA-seq analysis revealed major changes in gene expression, with 676 differentially expressed genes after LIPUS stimulation, 578 upregulated and 98 downregulated.

Conclusions: Overall, these results highlight the potential of LIPUS as a promising non-invasive therapy to target bCSCs and attenuate its capacity to drive migration, invasion, angiogenesis and, ultimately, tumor malignancy. Besides, the ability of LIPUS to modulate gene expression points out its capacity to broadly influence the cellular transcriptome. Therefore, the application of LIPUS as an antitumor therapeutic agent targeting bCSCs may offer a promising new approach to treat cancer. In vivo functional experiments will determine in the future the relevance of LIPUS application for the development of metastasis.

Keywords: Angiogenesis; Breast cancer stem cell; Invasion; Low-intensity pulsed ultrasound; Migration.

Fig. 1

Scheme of the LIPUS application and the experimental design. The application of LIPUS was performed with a gel between the transducer and the bottom of the plate where the cells are attached. Ideal LIPUS conditions ( 0.05 W/cm2, 3 MHz, 8 pulses) were applied to a confluent plate of the bCSC line using the Medisound 3000 device, followed by observation and photo-taking. Created by Alba Calero with PowerPoint

Fig. 2

LIPUS stimulation does not affect bCSC proliferation or cell structure. A Representative images of human bCSCs showing control cells and LIPUS-stimulated cells. N = 5. Bar, 100 μm. B bCSC proliferation was evaluated by BrdU incorporation. No significant differences were found between control conditions or treated with LIPUS. N = 5

Fig. 3

LIPUS slows wound closure in bCSCs. A Representative images from wounded bCSCs in both control and LIPUS-stimulated conditions (8 pulses, 0.05 W/cm2 and 3 MHz) at different times (t = 0 h, t = 8 h, t = 24 h, t = 24 h, t = 32 h and t = 48 h). Wound closure is indicated by black dashed lines highlighting the wound edges, and a black horizontal line marks the width of the wound. N = 5. Bar, 100 μm B-C Representation of wound closure in both conditions at different times. LIPUS stimulation significantly slows wound closure at 24 and 32 h (determined by two-way ANOVA with Šídák’s multiple comparisons test). **p < 0.01; ***p < 0.001

Fig. 4

LIPUS stimulation reduces bCSC motility and invasion. A Representative images taken with a 10X objective of the lower part of the Transwell at different times (t = 2 h, t = 4 h and t = 6 h). Control and LIPUS-stimulated (8 pulses, 0.05 W/cm2 and 3 MHz) cells have been stained and fixed to analyse the number of migrated cells. Bar, 50 μm B Representative image of bCSC invasion from the top of the Transwell to the bottom of the filter through the porous membrane. C Representation of the number of cells invaded in both conditions at different times. The number of cells that moved through the Transwell is significantly reduced in LIPUS-stimulated cells at 6 h (two-way ANOVA with Šídák’s multiple comparisons test). ****p < 0,0001. N = 3

Fig. 5

LIPUS decreases angiogenesis in bCSCs within 2 h. A Images of vessel and tubular network formation (panels) in control and LIPUS-stimulated cells (8 pulses, 0.05 W/cm2 and 3 MHz) taken with a 10X objective. White arrows indicate vessels and dash black ovals indicate panels. Bar, 100 μm B Representative images of what have been considered as panels and vessels using a 40X objective. Bar, 50 μm. C Representation of the number of panels and vessels formed in both conditions. Statistical analysis was performed using a Mann-Whitney test. The formation of new vessels and tubes was significantly decreased in LIPUS-treated cells. * p < 0,05. N = 5

Fig. 6

RNA-seq Analysis. A Volcano Plot showing the top 10 differentially expressed genes between control and LIPUS-stimulated human bCSCs. B List of the top 10 differentially expressed genes between control and LIPUS-stimulated human bCSCs. C-D Graphical representation of the upregulated DEGs categorized by Biological Process and Cellular Component using Gen Ontology analysis via the David database. E-F Graphical representation of the downregulated DEGs categorized by Biological Process and Cellular Component using Gen Ontology analysis via the David database