Epidermal growth factor receptor-targeted sonoporation with microbubbles enhances therapeutic efficacy in a squamous cell carcinoma model

<div><p>Sonoporation is a drug and gene delivery system using ultrasonication that allows the intracellular delivery of foreign molecules that cannot enter cells under normal conditions. We previously reported that sonoporation with microbubbles (MBs) could achieve effective intracellular drug delivery to human gingival squamous carcinoma Ca9-22 cells. In this study, we developed anti-epidermal growth factor receptor (EGFR) antibody-conjugated MBs (EGFR-MBs) and evaluated their capacity to enhance anti-cancer drug toxicity <i>in vitro</i> and <i>in vivo</i>. We first assessed the effect of sonoporation with EGFR-MBs on Ca9-22 cells by the WST-8 assay, flow cytometry and Hoechst’s staining <i>in vitro</i>. Sonoporation and EGFR-MB had a strong cytotoxic effect on Ca9-22 cells with low-dose bleomycin. Furthermore, bleomycin delivery using sonoporation with EGFR-MBs remarkably increased the number of apoptotic cells. We next examined the effect of EGFR-MBs in a murine squamous cell carcinoma model. Bleomycin delivery by sonoporation with EGFR-MBs exhibited remarkable antitumor activity. Together, our results show that EGFR-MBs and ultrasound treatment increases the efficacy and specificity of intracellular drug uptake, suggesting this could be a novel drug-targeting modality for oral squamous cell carcinoma chemotherapy treatment.</p></div

Growth inhibition of Ca9-22 xenografts after BLM delivery.

<p>KSN/slc nude mice were injected with Ca9-22 cells and divided into four experimental groups: control (n = 4), BLM injection (n = 4), sonoporation with MBs and BLM injection (n = 4) and sonoporation with EGFR-MBs and BLM injection (n = 4). (A) Images of mice from the different groups on days 0, 14 and 28. (B) Tumor volumes of the different treatment groups. Arrows indicate the days mice received treatments. Data are expressed as the means ± s.e.; **P<0.01, *P<0.05.</p

Apoptosis in Ca9-22 cells after BLM delivery <i>in vitro</i>.

<p>(A) Cell cycle distribution analyses by flow cytometry in the no treatment, BLM alone, BLM + sonoporation + MBs, or BLM + sonoporation + anti-EGFR antibody-conjugated MBs groups. The percentage of cells in the sub-G1 phase is indicated. (B) Apoptosis analysis by flow cytometry in the no treatment, BLM alone, BLM + sonoporation + MBs, or BLM + sonoporation + anti-EGFR antibody-conjugated MBs groups. (C) Percentages of apoptotic cells. *P<0.001 (D) Hoechst staining was performed to observe morphological changes in Ca9-22 cells after 48 h BLM delivery treatment. Cells were exposed to no treatment, BLM alone, BLM + sonoporation + MBs, or BLM + sonoporation + anti-EGFR antibody-conjugated MBs. Apoptotic cells (arrows) exhibited characteristic chromatin condensation under fluorescence microscopy. Bar: 20 μm. (E) Percentages of apoptotic cells. *P<0.01.</p

TUNEL analysis of xenografts after <i>in vivo</i> sonoporation with BLM.

<p>Ca9-22 xenograft–bearing mice were exposed to sonoporation <i>in vivo</i>. (a) control, (b) BLM injection, (c) sonoporation with MBs and BLM injection, (d) sonoporation with EGFR-MBs and BLM injection. TUNEL signal was visualized by diaminobezidine (DAB, brown) and cell nuclei were counterstained by methyl green. Magnification, 400×; bar, 100 μm.</p

Specific binding of EGFR-MBs to EGFR on Ca9-22 cells.

<p>(A) Fluorescence intensity was measured by flow cytometry; untreated Ca9-22 cells (a), Ca9-22 cells treated with DIO-labeled MBs (b), DIO-labeled IgG-MBs (c), and DIO-labeled EGFR-MBs (d). (B) Ca9-22 cells were incubated with EGFR-MBs for 30 min at 37°C, fixed and stained as indicated. Stained cells were examined using a fluorescence microscope.</p

<i>In vitro</i> growth inhibition of Ca9-22 cells after BLM delivery.

<p>Ca9-22 cells were treated with BLM by sonoporation using microbubbles (MB+US), IgG-conjugated MBs (IgG-MB+US) or anti-EGFR antibody conjugated MBs (EGFR-MB+US) as indicated. After 48 h incubation, cell proliferation was measured using WST-8 assays. **P<0.001, *P<0.05.</p

Representative MDCT images of odontogenic masses and the GT (arrows) in cases classified as Group 1.

<p><b>(A)</b> Panoramic MDCT image of an AM case classified as Group 1. <b>(B)</b> Cross sectional MDCT image of Fig 2A. <b>(C)</b> Panoramic MDCT image of an OKC case classified as Group 1. <b>(D)</b> Cross sectional MDCT image of Fig 2C. <b>(E)</b> Panoramic MDCT image of a DC case classified as Group 1. <b>(F)</b> Cross sectional MDCT image of Fig 2E.</p

Type and gender distribution of the 313 masses in this study.

<p>Type and gender distribution of the 313 masses in this study.</p

Continuity of each type of mass with the gubernaculum tract.

<p>Continuity of each type of mass with the gubernaculum tract.</p

Relationship between mass size and area of the major axis of continuity on MDCT imaging in AM, DC and OKC cases classified as Group 1 or 2.

<p>In AM (blue diamonds), a strong significant correlation was found between the mass size and the size of the continuity area (r = 0.741, p = 0.0001). In DC (green triangles), a very weak significant correlation was found between the mass size and the size of the continuity area (r = 0.167, p = 0.0028). In OKC (red Xs), there was no correlation between the mass size and the size of the continuity area (r = -0.089, p = 0.557).</p