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

The use of ultrasound (US) and microbubbles (MB), usually referred to as sonoporation, has great potential to increase the efficacy of chemotherapy. However, the molecular mechanisms that mediate sonoporation response are not well-known, and recent research suggests that cell stress induced by US + MBs may contribute to the treatment benefit. Furthermore, there is a growing understanding that the effects of US + MBs are beyond only the cancer cells and involves the tumour vasculature and microenvironment. We treated pancreatic cancer cells (MIA PaCa-2) and stromal cells, fibroblasts (BJ) and human umbilical vein endothelial cells (HUVECs), with US ± MB, and investigated the extent of uptake of cell impermeable dye (calcein, by flow cytometry), viability (cell count, Annexin/PI and WST-1 assays) and activation of a number of key proteins in important intracellular signalling pathways immediately and 2 h after sonoporation (phospho flow cytometry). Different cell types responded differently to US ± MBs in all these aspects. In general, sonoporation induces immediate, transient activation of MAP-kinases (p38, ERK1/2), and an increase in phosphorylation of ribosomal protein S6 together with dephosphorylation of 4E-BP1. The sonoporation stress-response resembles cellular responses to electroporation and pore-forming toxins in membrane repair and restoring cellular homeostasis, and may be exploited therapeutically. The stromal cells were more sensitive to sonoporation than tumoural cells, and further efforts in optimising sonoporation-enhanced therapy should be targeted at the microenvironment.

Ultrasound- and Microbubble-Assisted Gemcitabine Delivery to Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death worldwide. Poor drug delivery to tumours is thought to limit chemotherapeutic treatment efficacy. Sonoporation combines ultrasound (US) and microbubbles to increase the permeability of cell membranes. We assessed gemcitabine uptake combined with sonoporation in vitro in three PDAC cell lines (BxPC-3, MIA PaCa-2 and PANC-1). Cells were cultured in hypoxic bioreactors, while gemcitabine incubation ± sonoporation was conducted in cells with operational or inhibited nucleoside membrane transporters. Intracellular active metabolite (dFdCTP), extracellular gemcitabine, and inactive metabolite (dFdU) concentrations were measured with liquid chromatography tandem mass spectrometry. Sonoporation with increasing US intensities resulted in decreasing extracellular gemcitabine concentrations in all three cell lines with inhibited membrane transporters. In cells with inhibited membrane transporters, without sonoporation, dFdCTP concentrations were reduced down to 10% of baseline. Sonoporation partially restored gemcitabine uptake in these cells, as indicated by a moderate increase in dFdCTP concentrations (up to 37% of baseline) in MIA PaCa-2 and PANC-1. In BxPC-3, gemcitabine was effectively inactivated to dFdU, which might represent a protective mechanism against dFdCTP accumulation in these cells. Intracellular dFdCTP concentrations did not change significantly following sonoporation in any of the cell lines with operational membrane transporters, indicating that the gemcitabine activation pathway may have been saturated with the drug. Sonoporation allowed a moderate increase in gemcitabine transmembrane uptake in all three cell lines, but pre-existing nucleoside transporters were the major determinants of gemcitabine uptake and retention.