Intracellular Signaling in Key Pathways Is Induced by Treatment with Ultrasound and Microbubbles in a Leukemia Cell Line, but Not in Healthy Peripheral Blood Mononuclear Cells

Abstract

Treatment with ultrasound and microbubbles (sonoporation) to enhance therapeutic efficacy in cancer therapy is rapidly expanding, but there is still very little consensus as to why it works. Despite the original assumption that pore formation in the cell membrane is responsible for increased uptake of drugs, the molecular mechanisms behind this phenomenon are largely unknown. We treated cancer cells (MOLM-13) and healthy peripheral blood mononuclear cells (PBMCs) with ultrasound at three acoustic intensities (74, 501, 2079 mW/cm2) ± microbubbles. We subsequently monitored the intracellular response of a number of key signaling pathways using flow cytometry or western blotting 5 min, 30 min and 2 h post-treatment. This was complemented by studies on uptake of a cell impermeable dye (calcein) and investigations of cell viability (cell count, Hoechst staining and colony forming assay). Ultrasound + microbubbles resulted in both early changes (p38 (Arcsinh ratio at high ultrasound + microbubbles: +0.5), ERK1/2 (+0.7), CREB (+1.3), STAT3 (+0.7) and AKT (+0.5)) and late changes (ribosomal protein S6 (Arcsinh ratio at low ultrasound: +0.6) and eIF2α in protein phosphorylation). Observed changes in protein phosphorylation corresponded to changes in sonoporation efficiency and in viability, predominantly in cancer cells. Sonoporation induced protein phosphorylation in healthy cells was pronounced (p38 (+0.03), ERK1/2 (−0.03), CREB (+0.0), STAT3 (−0.1) and AKT (+0.04) and S6 (+0.2)). This supports the hypothesis that sonoporation may enhance therapeutic efficacy of cancer treatment, without causing damage to healthy cells.

Low-Intensity Sonoporation-Induced Intracellular Signalling of Pancreatic Cancer Cells, Fibroblasts and Endothelial Cells

“…After treatment with US ± MB, the cells were incubated for 1 h to allow for cell membrane pores to re-seal [4,33], flushed twice with phosphate-buffered saline (PBS), detached using trypsin-EDTA 0.05% and harvested from the Petaka. Following centrifugation and resuspension in PBS, the cells were analysed by flow cytometry on an Accuri C6 flow cytometer (BDBioscience, Franklin Lakes, NJ, USA).…”

“…To investigate changes in intracellular signalling events, cells were harvested from separate Petakas as soon as possible after sonoporation and after 2 h of incubation. Timepoints were selected based on a previous study [4]. Cells cultured in separate Petakas were treated with 1 µM A23187 (calcium ionophore) + 100 nM phorbol myristate acetate (PMA: PKC activator) for 30 min as positive controls for intracellular signalling.…”

“…Cell detachment time varied between cell lines, as shown in Figure S3. Cell culture medium, PBS, and cells were collected and fixed by adding 16% paraformaldehyde (PFA, Alfa Aesar, Haverhill, MA, USA) directly to yield a final concentration of 2%, incubated for 15 min at room temperature and permeabilised by adding ice-cold methanol [4,36]. Before addition of PFA, a sample was taken for cell count (later referred to as cell count 0 h) and counted using a haemocytometer.…”

“…However, there is still no consensus on what the exact mechanisms underlying US + MBs enhanced cancer therapy are. Furthermore, the cellular stress induced by US + MBs themselves has been proposed to contribute to the anti-cancer effects [3,4].…”

“…Our previous study [4] using leukemic cells as a model system of cancer compared to healthy peripheral blood cells demonstrated that different cell types have different sensitivities to sonoporation in terms of molecular uptake, effect on viability and intracellular signalling. This was a simplified system compared to solid tumours, which consist of the complex tumour microenvironment with stromal cells, immune cells, fibroblasts, blood vessels and extracellular matrix (ECM) in addition to, and supporting, the cancer cells [29].…”