Azza Al-Mahrouki

Low-frequency quantitative ultrasound imaging of cell death in vivo.

PURPOSE Currently, no clinical imaging modality is used routinely to assess tumor response to cancer therapies within hours to days of the delivery of treatment. Here, the authors demonstrate the efficacy of ultrasound at a clinically relevant frequency to quantitatively detect changes in tumors in response to cancer therapies using preclinical mouse models. METHODS Conventional low-frequency and corresponding high-frequency ultrasound (ranging from 4 to 28 MHz) were used along with quantitative spectroscopic and signal envelope statistical analyses on data obtained from xenograft tumors treated with chemotherapy, x-ray radiation, as well as a novel vascular targeting microbubble therapy. RESULTS Ultrasound-based spectroscopic biomarkers indicated significant changes in cell-death associated parameters in responsive tumors. Specifically changes in the midband fit, spectral slope, and 0-MHz intercept biomarkers were investigated for different types of treatment and demonstrated cell-death related changes. The midband fit and 0-MHz intercept biomarker derived from low-frequency data demonstrated increases ranging approximately from 0 to 6 dBr and 0 to 8 dBr, respectively, depending on treatments administrated. These data paralleled results observed for high-frequency ultrasound data. Statistical analysis of ultrasound signal envelope was performed as an alternative method to obtain histogram-based biomarkers and provided confirmatory results. Histological analysis of tumor specimens indicated up to 61% cell death present in the tumors depending on treatments administered, consistent with quantitative ultrasound findings indicating cell death. Ultrasound-based spectroscopic biomarkers demonstrated a good correlation with histological morphological findings indicative of cell death (r2=0.71, 0.82; p<0.001). CONCLUSIONS In summary, the results provide preclinical evidence, for the first time, that quantitative ultrasound used at a clinically relevant frequency, in addition to high-frequency ultrasound, can detect tissue changes associated with cell death in vivo in response to cancer treatments.

Bioeffects of Ultrasound-Stimulated Microbubbles on Endothelial Cells: Gene Expression Changes Associated with Radiation Enhancement In Vitro

Ultrasound can be used to target endothelial cells in cancer therapy where the destruction of vasculature leads to tumor cell death. Here, we demonstrate ultrasound bioeffects in which the levels of genes in endothelial cells can be significantly altered by ultrasound-stimulated microbubble exposure. These were compared with established effects of radiation on endothelial cells at a gene level. Human-endothelial cells were exposed to ultrasound and microbubbles, radiation or combinations of ultrasound, microbubbles and radiation. Gene expression analyses revealed an up-regulation of genes known to be involved in apoptosis and ceramide-induced apoptotic pathways, including SMPD2, UGT8, COX6B1, Caspase 9 and MAP2K1 with ultrasound-stimulated microbubble exposure but not SMPD1. This was supported by immunohistochemistry and morphologic changes examined with cell microscopy, which showed changes in SMPD1 gene product in cells with microbubble exposure. This supports the hypothesis that ultrasound-stimulated microbubbles can induce significant bioeffect-related changes in gene expression and can affect ceramide signaling pathways in endothelial cells, leading to apoptosis.

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

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.

EFFECTS OF BIOPHYSICAL PARAMETERS IN ENHANCING RADIATION RESPONSES OF PROSTATE TUMORS WITH ULTRASOUND-STIMULATED MICROBUBBLES

We show here that ultrasound-stimulated microbubbles can enhance cell death within tumors when combined with radiation. The aim of this study was to investigate how different ultrasound parameters, different microbubble concentrations and different radiation doses interact to enhance cell death. Prostate xenograft tumors (PC-3) in severe combined immunodeficiency mice were subjected to ultrasound treatment at various peak negative pressures (250, 570 and 750 kPa) at a center frequency of 500 kHz, different microbubble concentrations (8, 80 and 1000 μL/kg) and different radiation doses (0, 2 and 8 Gy). Twenty-four hours after treatment, tumors were excised and assessed for cell death. Histologic analyses revealed that increases in radiation dose, microbubble concentration and ultrasound pressure promoted apoptotic cell death and disruption within tumors by as much as 21%, 30% and 43%, respectively. Comparable increases in ceramide, a cell death mediator, were identified using immunohistochemistry. We also show here that even clinically used microbubble concentrations combined with ultrasound can induce significant enhancement of cell death.

Sunitinib effects on the radiation response of endothelial and breast tumor cells

BACKGROUND Endothelial cells are suggested regulators of tumor response to radiation. Anti-vascular targeting agents can enhance tumor response by targeting endothelial cells. Here, we have conducted experiments in vitro to discern the effects of radiation combined with the anti-angiogenic Sunitinib on endothelial (HUVEC) and tumor (MDA-MB-231) cells, and further compared findings to results obtained in vivo. METHODS In vitro and in vivo treatments consisted of single dose radiation therapy of 2, 4, 8 or 16 Gy administered alone or in combination with bFGF or Sunitinib. In vitro, in situ end labeling (ISEL) was used to assess 24-hour apoptotic cell death, and clonogenic assays were used to assess long-term response. In vivo MDA-MB-231 tumors were grown in CB-17 SCID mice. The vascular marker CD31 was used to assess 24-hour acute response while tumor clonogenic assays were used to assess long-term tumor cell viability following treatments. RESULTS Using in vitro studies, we observed an enhanced endothelial cell response to radiation doses of 8 and 16 Gy when compared to tumor cells. Administering Sunitinib alone significantly increased HUVEC cell death, while having modest additive effects when combined with radiation. Sunitinib also increased tumor cell death when combined with 8 and 16 Gy radiation doses. In comparison, we found that the clonogenic response of in vivo treated tumor cells more closely resembled that of in vitro treated endothelial cells than in vitro treated tumor cells. CONCLUSION Our results indicate that the endothelium is an important regulator of tumor response to radiotherapy, and that Sunitinib can enhance tumor radiosensitivity. To the best of our knowledge, this is the first time that Sunitinib is investigated in combination with radiotherapy on the MDA-MB-231 breast cancer cell line.

Quantitative Ultrasound Characterization of Tumor Cell Death: Ultrasound-Stimulated Microbubbles for Radiation Enhancement

The aim of this study was to assess the efficacy of quantitative ultrasound imaging in characterizing cancer cell death caused by enhanced radiation treatments. This investigation focused on developing this ultrasound modality as an imaging-based non-invasive method that can be used to monitor therapeutic ultrasound and radiation effects. High-frequency (25 MHz) ultrasound was used to image tumor responses caused by ultrasound-stimulated microbubbles in combination with radiation. Human prostate xenografts grown in severe combined immunodeficiency (SCID) mice were treated using 8, 80, or 1000 µL/kg of microbubbles stimulated with ultrasound at 250, 570, or 750 kPa, and exposed to 0, 2, or 8 Gy of radiation. Tumors were imaged prior to treatment and 24 hours after treatment. Spectral analysis of images acquired from treated tumors revealed overall increases in ultrasound backscatter intensity and the spectral intercept parameter. The increase in backscatter intensity compared to the control ranged from 1.9±1.6 dB for the clinical imaging dose of microbubbles (8 µL/kg, 250 kPa, 2 Gy) to 7.0±4.1 dB for the most extreme treatment condition (1000 µL/kg, 750 kPa, 8 Gy). In parallel, in situ end-labelling (ISEL) staining, ceramide, and cyclophilin A staining demonstrated increases in cell death due to DNA fragmentation, ceramide-mediated apoptosis, and release of cyclophilin A as a result of cell membrane permeabilization, respectively. Quantitative ultrasound results indicated changes that paralleled increases in cell death observed from histology analyses supporting its use for non-invasive monitoring of cancer treatment outcomes.

Molecular cloning of preproinsulin cDNAs from several osteoglossomorphs and a cyprinid

Several preproinsulin cDNAs were isolated and characterized from four members of the Osteoglossomorpha (an ancient teleost group); Osteoglossum bicirrhosum (arawana), Pantodon buchholzi (butterfly fish), Notopterus chitala (feather fin knife fish), Hiodon alosoides (goldeye) and Gnathonemus petersii (elephantnose). In addition, we isolated and characterized the preproinsulin cDNA from Catostomus commersoni (white sucker, as a representative of a generalized teleost). The comparative analysis of the sequences revealed conservation of the cystine residues known to be involved in the formation of the disulfide bridges, as well as residues involved in the hexamer formation, except for B-17 in the butterfly fish, the arawana and the goldeye. However, the N-terminus of the B-chain was very weakly conserved among the species studied. Residues known to be significant for maintaining receptor-binding conformation and those known to comprise the receptor-binding domain were all conserved, except for a conservative substitution at B13, aspartate substituted glutamate in the arawana, goldeye, butterfly fish and white sucker, and at B16, phenylalanine substituted tyrosine in the elephantnose. Phylogenetic analysis of the sequences revealed a monophyletic grouping of the osteoglossomorphs, and showed that they were not the most basal living teleost. Comparative sequence analysis of preproinsulins among the osteoglossomorphs was useful in assessment of intergroup relationship, relating elephantnose with the feather fin knife fish and the arawana, butterfly fish, and goldeye. This arrangement of species is consistent with relationships based on other more classical parameters, except for the goldeye which was assessed as being sister to all the osteoglossomorphs. The white sucker was grouped with the common carp and both are cyprinids.

Quantification of Ultrasonic Scattering Properties of In Vivo Tumor Cell Death in Mouse Models of Breast Cancer

INTRODUCTION: Quantitative ultrasound parameters based on form factor models were investigated as potential biomarkers of cell death in breast tumor (MDA-231) xenografts treated with chemotherapy. METHODS: Ultrasound backscatter radiofrequency data were acquired from MDA-231 breast cancer tumor–bearing mice (n = 20) before and after the administration of chemotherapy drugs at two ultrasound frequencies: 7 MHz and 20 MHz. Radiofrequency spectral analysis involved estimating the backscatter coefficient from regions of interest in the center of the tumor, to which form factor models were fitted, resulting in estimates of average scatterer diameter and average acoustic concentration (AAC). RESULTS: The ∆AAC parameter extracted from the spherical Gaussian model was found to be the most effective cell death biomarker (at the lower frequency range, r2 = 0.40). At both frequencies, AAC in the treated tumors increased significantly (P = .026 and .035 at low and high frequencies, respectively) 24 hours after treatment compared with control tumors. Furthermore, stepwise multiple linear regression analysis of the low-frequency data revealed that a multiparameter quantitative ultrasound model was strongly correlated to cell death determined histologically posttreatment (r2 = 0.74). CONCLUSION: The Gaussian form factor model–based scattering parameters can potentially be used to track the extent of cell death at clinically relevant frequencies (7 MHz). The 20-MHz results agreed with previous findings in which parameters related to the backscatter intensity (i.e., AAC) increased with cell death. The findings suggested that, in addition to the backscatter coefficient parameter ∆AAC, biological features including tumor heterogeneity and initial tumor volume were important factors in the prediction of cell death response.

Multiparametric Monitoring of Chemotherapy Treatment Response in Locally Advanced Breast Cancer Using Quantitative Ultrasound and Diffuse Optical Spectroscopy

Purpose This study evaluated pathological response to neoadjuvant chemotherapy using quantitative ultrasound (QUS) and diffuse optical spectroscopy imaging (DOSI) biomarkers in locally advanced breast cancer (LABC). Materials and Methods The institutions ethics review board approved this study. Subjects (n = 22) gave written informed consent prior to participating. US and DOSI data were acquired, relative to the start of neoadjuvant chemotherapy, at weeks 0, 1, 4, 8 and preoperatively. QUS parameters including the mid-band fit (MBF), 0-MHz intercept (SI), and the spectral slope (SS) were determined from tumor ultrasound data using spectral analysis. In the same patients, DOSI was used to measure parameters relating to tumor hemoglobin and composition. Discriminant analysis and receiver-operating characteristic (ROC) analysis was used to classify clinical and pathological response during treatment and to estimate the area under the curve (AUC). Additionally, multivariate analysis was carried out for pairwise QUS/DOSI parameter combinations using a logistic regression model. Results Individual QUS and DOSI parameters, including the (SI), oxy-hemoglobin (HbO2), and total hemoglobin (HbT) were significant markers for response after one week of treatment (p < 0.01). Multivariate (pairwise) combinations increased the sensitivity, specificity and AUC at this time; the SI + HbO2 showed a sensitivity/specificity of 100%, and an AUC of 1.0. Conclusions QUS and DOSI demonstrated potential as coincident markers for treatment response and may potentially facilitate response-guided therapies. Multivariate QUS and DOSI parameters increased the sensitivity and specificity of classifying LABC patients as early as one week after treatment.

Enhancement of radiation therapy by ultrasonically-stimulated microbubbles in vitro: Effects of treatment scheduling on cell viability and production of ceramide

Microbubbles are used as therapeutic agents to improve drug delivery across blood vessels and cell membranes. Recently, it was shown that the application of ultrasound and microbubbles can enhance the therapeutic effect of radiotherapy using in vitro and in vivo tumour models. This phenomenon depended on ultrasound, microbubble and x-ray exposure parameters. In this study, the effect of ultrasound-microbubble (US+MB) and ionizing radiation (XRT) treatment scheduling on cell viability and production of ceramide, an apoptosis messenger, was investigated in vitro. Human prostate cancer (PC3) and KHT-C murine fibrosarcoma cells in suspension were exposed to US+MB (f=500kHz; Pneg=580kPa; PD=32μs; PRF=3kHz; insonation time 30s; and 1% v/v microbubbles (DA04, Artenga Inc.)) and XRT (3 Gy single fraction). Timing between the treatments — immediately (i.e. within 15 minutes), 3 and 6 hours — and their order were varied. Following treatment, cell viability was assessed using clonogenic assay. Ceramide level within cells was quantified for up to 7.5 hours following treatment using immunohistochemistry and spectrophotometry. Ultrasound and microbubbles improved the therapeutic effect of radiotherapy in KHT-C and PC3 cells; cell death increased by ∼3–5 folds with the combined treatment compared to each treatment alone. Maximal KHT-C cell death (70±2%) was achieved when US+MB was followed in three hours by XRT. KHT-C cell death of 43±2% and 25±2% was achieved with US+MB and XRT treatments, respectively. The maximal PC3 cell death (83±2%) was achieved when US+MB was followed by XRT immediately (i.e. within 15 minutes). PC3 cell death of 15±4% and 48±5% was achieved with US+MB and XRT treatments, respectively. US+MB and XRT treatments increased the level of ceramide in both cell lines compared to untreated controls. A correlation was found between cell death and ceramide at 3 h following treatment for the KHT-C and PC3 cells with correlation coefficients of 0.938 and 0.706, respectively. Ultrasonically-stimulated microbubbles can enhance the therapeutic effect of radiotherapy and the timing between the treatments and their order are important in the optimization of the therapeutic effect of radiotherapy. Future work will investigate this in vivo.