USMB treatment increases the cell surface abundance of the lysosomal marker LAMP1.

<p>RPE cells grown on glass coverslips were treated with 5.0 μM vacuolin-1 for 60 min, or not treated with this inhibitor (vehicle control). Cells were subsequently treated with USMB or left untreated (control) as indicated. Following treatment, cells were immediately placed on ice to arrest membrane traffic and subjected to immunofluorescence staining to detect cell surface LAMP1 levels. Shown in <b><i>(A)</i></b> are representative epifluorescence micrographs of cell surface LAMP1 levels and in <b><i>(B)</i></b> the mean ± SEM of cell surface TfR fluorescence intensity in each condition (n = 3 independent experiment, each experiment >20 cells per condition). Scale = 20 μm. *, p < 0.05 relative to the control, vehicle-treated condition.</p

Desipramine treatment impairs the reduction in cell surface TfR levels by USMB treatment.

<p>RPE cells grown on glass coverslips were treated with 50 μM desipramine for 60 min, or not treated with this inhibitor (vehicle control). Cells were subsequently treated with USMB or left untreated (control) as indicated. 5 min following USMB treatment, cells were immediately placed on ice to arrest membrane traffic and subjected to immunofluorescence staining to detect cell surface TfR levels. Shown in <b><i>(A)</i></b> are representative epifluorescence micrographs of cell surface TfR levels and in <b><i>(B)</i></b> the mean ± SEM of cell surface TfR fluorescence intensity in each condition (n = 3 independent experiment, each experiment >20 cells per condition). Scale = 20 μm. *, p < 0.05.</p

USMB treatment alters the properties of clathrin-coated pits.

<p>RPE cells stably expressing clathrin light chain fused to green fluorescent protein (RPE GFP-CLC) cells grown on glass coverslips were treated with microbubbles and ultrasound, or left untreated (control), as indicated. Cells were then incubated with A555-Tfn for 3 min to allow labeling of internalizing TfR, and then immediately subjected to fixation and processing for imaging by total internal reflection fluorescence microscopy (TIRF-M). (<b><i>A</i></b>) Shown are representative fluorescence micrographs obtained by TIRF-M. Scale = 5 μm. Images are higher magnification insets of larger images shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156754#pone.0156754.s001" target="_blank">S1 Fig</a>. (<b><i>B-C</i></b>) Images obtained by TIRF-M were subjected to automated detection and analysis of clathrin-coated pits (CCPs), as described in Material and Methods. The mean GFP-CLC (<b><i>B</i></b>) and A555-Tfn (<b><i>C</i></b>) intensity within each detected object (CCP) in each cell are shown. Each diamond symbol represents the mean fluorescence of all objects within a single cell; also shown are the mean of the cellular fluorescence values and interquartile range (red bars). The number of CCPs analyzed (n) and cells (k) from 3 independent experiments for each condition are control: n = 37,762, k = 89; USMB n = 29,897 k = 80.</p

Vacuolin-1 treatment impairs the reduction in cell surface TfR levels by USMB treatment.

<p>RPE cells grown on glass coverslips were treated with 5.0 μM vacuolin-1 for 60 min, or not treated with this inhibitor (vehicle control). Cells were subsequently treated with USMB or left untreated (control) as indicated. 5 min after USMB treatment, cells were placed on ice to arrest membrane traffic and subjected to immunofluorescence staining to detect cell surface TfR levels. Shown in <b><i>(A)</i></b> are representative epifluorescence micrographs of cell surface TfR levels and in <b><i>(B)</i></b> the mean ± SEM of cell surface TfR fluorescence intensity in each condition (n = 3 independent experiments, each experiment >20 cells per condition). Scale = 20 μm. *, p < 0.05.</p

Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

<div><p>Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.</p></div

USMB treatment rapidly reduces cell surface TfR levels.

<p>RPE (<b><i>A</i>, <i>B</i></b>) or MDA-MB-231 <b><i>(C-D)</i></b> cells grown on glass coverslips were treated with microbubbles and/or ultrasound, as indicated. 5 min following USMB treatment, cells were placed on ice to arrest membrane traffic and subjected to immunofluorescence staining to detect cell surface TfR levels. Shown in <b><i>(A</i>, <i>C)</i></b> are representative epifluorescence micrographs of cell surface TfR levels and in <b><i>(B</i>, <i>D)</i></b> the mean ± SEM of cell surface TfR fluorescence intensity in each condition (n = 3 independent experiments, each experiment >20 cells per condition). Scale = 20 μm. *, p < 0.05 relative to the control condition.</p

Average changes in midband-fit parameter.

<p>Each bar represents the mean of midband-fit values of five mouse-borne tumors (n = 5). The error bar indicates the standard error within the sample size. Statistical testing using 2-way ANOVA indicates the effects caused by the changes in both microbubble concentration and dose of radiation to be very significant for every graph (<i>p</i><0.0001). Each graph shows the average changes in midband-fit for varied microbubble concentration and radiation doses at a fixed ultrasound pressure: 250 kPa, 570 kPa, and 750 kPa.</p

High frequency ultrasound B-mode images with ROI parametric overlays of the mid-band fit biomarker for PC-3 xenografts.

<p>(A) Tumors treated with varying radiation doses (0–8 Gy) without ultrasound-microbubble treatment. (B) Tumors treated with varying microbubble concentrations (8–1000 µL/kg) combined with 750 kPa of ultrasound pulse and 8 Gy-radiation. (C) Tumors treated with varying ultrasound pressures (250–750 kPa) combined with 8 µL/kg of microbubbles and 8 Gy-radiation. The scale bar represents 4 mm.</p

High magnification light microscope images of H&E-stained PC-3 xenografts.

<p>In addition to control, tumors treated with ultrasound pulses at 750(A) The panel shows H&E stained cells that exhibit condensed and/or fragmented apoptotic bodies. Each Panel demonstrates a representative region of cell death within tumor. Microbubble concentrations are given as 8 µL/kg, 80 µL/kg, and high 1000 µL/kg. The scale bar represents 25 µm. (B) Quantification of normalized fraction of condensed or fragmented nuclei. Each graph shows the fraction of cell death and disruption for varied microbubble concentration and radiation doses at a fixed ultrasound peak-negative pressure: 250 kPa, 570 kPa, and 750 kPa. Each bar represents the mean value of five samples (n = 5) and the error bar indicates the standard error. Statistical testing using 2-way ANOVA indicates the effects caused by the changes in both microbubble concentration and dose of radiation to be very significant (<i>p</i><0.0001).</p

Summary of growth curve statistics.

<p>Tabulation of p-values for tumor volume differences between groups in the growth experiments. The unbracketed p-values are derived from an unpaired two-tailed Student’s t-test, and the bracketed values indicate the level of significance with a <i>post hoc</i> Bonferroni test (denoted *p). ‘-‘ indicates p-value was >0.1 for the Student t-test and ‘*ns’ within brackets indicates that the <i>post hoc</i> Bonferroni test had a p-value of >0.05. n/a indicates that at least one group was not present in the growth curves at that point (i.e. drop-outs occurred due to tumor burden end-points). By week 3 the combined USMB+DTX group had a significantly (*p<0.01) lower mean tumor volume than the control group and at week 4 and after was also significantly (*p<0.01) lower than the USMB and DTX groups.</p