Cellular characterization of ultrasound-stimulated microbubble radiation enhancement in a prostate cancer xenograft model

Abstract

Tumor radiation resistance poses a major obstacle in achieving an optimal outcome in radiation therapy. In the current study, we characterize a novel therapeutic approach that combines ultrasound-driven microbubbles with radiation to increase treatment responses in a prostate cancer xenograft model in mice. Tumor response to ultrasound-driven microbubbles and radiation was assessed 24 hours after treatment, which consisted of radiation treatments alone (2 Gy or 8 Gy) or ultrasound-stimulated microbubbles only, or a combination of radiation and ultrasound-stimulated microbubbles. Immunohistochemical analysis using in situ end labeling (ISEL) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) revealed increased cell death within tumors exposed to combined treatments compared with untreated tumors or tumors exposed to radiation alone. Several biomarkers were investigated to evaluate cell proliferation (Ki67), blood leakage (factor VIII), angiogenesis (cluster of differentiation molecule CD31), ceramide-formation, angiogenesis signaling [vascular endothelial growth factor (VEGF)], oxygen limitation (prolyl hydroxylase PHD2) and DNA damage/repair (γH2AX). Results demonstrated reduced vascularity due to vascular disruption by ultrasound-stimulated microbubbles, increased ceramide production and increased DNA damage of tumor cells, despite decreased tumor oxygenation with significantly less proliferating cells in the combined treatments. This combined approach could be a feasible option as a novel enhancing approach in radiation therapy.

Keywords: Angiogenesis; Microbubbles; Proliferation; Radiation; Ultrasound.

Fig. 1.

Ceramide labeling of tumor sections. (A) Brown-red labeling of ceramide increased in intensity and distribution with the combined treatments than the single treatments. (B) Labeling analyses using ImageJ indicated a significant difference (*) when comparing the labeling of the different treatment groups to the control (P=0011 with either 2 Gy alone or combined, P=0005 with either US+MB or 8 Gy alone; P=0002 with US+MB+8 Gy. A Mann-Whitney test was used to calculate the P-values. Scale bar: 50 μm.

Fig. 2.

Vessel integrity was detected using immunohistochemical labeling of factor VIII in PC3 xenograft sections. (A) Micrographs of sections from tumors not treated with microbubbles (–MB; upper panels) and from tumors treated with microbubbles (+MB; lower panels). (B) Blood-vessel leakage became more evident and significant when the ultrasound-activated microbubbles were combined with a radiation dose of 8 Gy. Statistical analyses indicated a P<0.029 (*). Scale bar: 50 μm.

Fig. 3.

Angiogenesis assessment with immunohistochemistry labeling of CD31. (A) Micrographs of tumor sections, illustrating labeling of blood-vessel endothelial cells (brown-red) treated with different conditions. (B) Decreased vascularization was observed with the treatments with 8 Gy (*P<0.043), with MB+US+2 Gy (*P<0.032) and with MB+US+8 Gy (*P<0.01). Scale bar: 25 μm.

Fig. 4.

Assessment of angiogenesis signaling using immunohistochemistry labeling of VEGF. (A) Micrographs of tumor sections, illustrating labeling of blood-vessel endothelial cells (brown-red) that were treated with different conditions. (B) A significant signaling increase was observed with combined (MB+US+8 Gy) treatments (*P<0.032). Scale bar: 50 μm.

Fig. 5.

Hypoxia staining using PHD2 immunohistochemistry of tumor sections. (A) Labeled sections illustrated an increased staining with the combined treatments. (B) Statistical analyses revealed a significant change (*) when comparing the controls to 8 Gy (P<0.05), to MB+US+2 Gy (P<0.008) or to MB+US+8 Gy (P<0.012). Scale bar: 25 μm.

Fig. 6.

Staining for DNA damage. (A) Immunostaining of γH2AX (red fluorescence; upper two panels) and overlay with DAPI as a counter stain (blue fluorescence; lower two panels). (B) Increased labeling was observed with all the treatments, with *P<0.029 (MB+US and MB+US+2 Gy) and *P<0.014 (2 Gy, 8 Gy and MB+US+8 Gy). Scale bar: 30 μm.

Fig. 7.

Histopathology, in situ end labeling (ISEL) and clonogenic assays of a PC3 xenograft tumor. (A) H&E staining of whole tumor sections treated with 0, 2 and 8 Gy or with a combination of radiation and ultrasound-stimulated microbubbles (–MB indicates no exposure to ultrasound-stimulated microbubbles; +MB indicates treatment with ultrasound-stimulated microbubbles). (B) Sections adjacent to those in A were labeled with ISEL to illustrate areas of cell death. Scale bars: 1 mm. (C) Quantified analyses of ISEL images, indicating an increased level of cell death with the combined treatments. A Mann-Whitney test was used to calculate the P-values and * symbols indicate where P-values are less than 0.05. (D) Clonogenic assay results illustrated a significant decrease in cellular survival of treated tumor cells when compared to the untreated samples. This was greatest in the combined treatments. A Mann-Whitney test was used to calculate the P-values and * symbols indicate where P-values are less than 0.05.

Fig. 8.

Histopathology and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. The upper panels in A and B represent micro-photographs from sections stained with H&E. These demonstrate very noticeable morphological changes with the combined conditions of ultrasound-stimulated microbubbles and radiation. Cell death retraction artefact is noticeable in addition to condensed appearing pyknotic nuclei. Images in the lower panels in A and B are from sections adjacent to those in the upper panels and were labeled with the TUNEL assay. These illustrate increased nuclear condensation (red) and DNA fragmentation (dark blue) with combined treatment, which was quite obvious in areas of cell death, which were indicated by cellular morphological changes. Scale bar: 25 μm.

Fig. 9.

Detection of cellular proliferation using Ki67 as a marker. (A) An increased number of labeled nuclei were observed in the controls than in the treated samples. This indicated a decreased proliferation specifically with the combined treatment of ultrasound-stimulated microbubbles and radiation, where a significant difference was found P<0.024 (see B, *). (B) The number of positively stained nuclei was counted in whole sections and the number of the labeled cells/mm² was calculated and plotted. A Mann-Whitney test was used to calculate the P-values. Scale bar: 25 μm.

Fig. 10.

Summary model of the response to the combined ultrasound-stimulated microbubble radiation-enhancing treatments. Combining ultrasound-stimulated microbubble therapy with radiation (A) results in the induction of ceramide production (B; green circles) and the potential release of reactive oxygen species (B; ROS, red circles). This microbubble-stimulated damage eventually leads to disruption of vasculature (C) with enhanced secondary tumor cell death. Structural alterations also result in vascular leakage (D) and DNA damage secondary to apoptotic and ischemic tumor cell death (gray cells) (D).