Anna G. Sorace

A Triple-Targeted Ultrasound Contrast Agent Provides Improved Localization to Tumor Vasculature

Actively targeting ultrasound contrast agents to tumor vasculature improves contrast‐enhanced sonography of tumor angiogenesis. This report summarizes an evaluation of multitargeted microbubbles, comparing single‐, dual‐, and triple‐targeted motifs.

Quantitative mapping of tumor vascularity using volumetric contrast-enhanced ultrasound

Objective:The goal of this research project was to develop a volumetric strategy for real-time monitoring and characterization of tumor blood flow using microbubble contrast agents and ultrasound (US) imaging. Materials and Methods:Volumetric contrast-enhanced US (VCEUS) imaging was implemented on a SONIX RP US system (Ultrasonix Medical Corp, Richmond, BC) equipped with a broadband 4DL14–5/38 probe. Using a microbubble-sensitive harmonic imaging mode (transducer transmits at 5 MHz and receives at 10 MHz), acquisition of postscan-converted VCEUS data was achieved at a volume rate of 1 Hz. After microbubble infusion, custom data processing software was used to derive microbubble time-intensity curve-specific parameters, namely, blood volume (IPK), transit time (T1/2PK), flow rate (SPK), and tumor perfusion (AUC). Results:Using a preclinical breast cancer animal model, it is shown that millimeter-sized deviations in transducer positioning can have profound implications on US-based blood flow estimators, with errors ranging from 6.4% to 40.3% and dependent on both degree of misalignment (offset) and particular blood flow estimator. These errors indicate that VCEUS imaging should be considered in tumor analyses, because they incorporate the entire mass and not just a representative planar cross-section. After administration of an antiangiogenic therapeutic drug (bevacizumab), tumor growth was significantly retarded compared with control tumors (P > 0.03) and reflects observed changes in VCEUS-based blood flow measurements. Analysis of immunohistologic data revealed no differences in intratumoral necrosis levels (P = 0.70), but a significant difference was found when comparing microvessel density counts in control with therapy group tumors (P = 0.05). Conclusions:VCEUS imaging was shown to be a promising modality for monitoring changes in tumor blood flow. Preliminary experimental results are encouraging, and this imaging modality may prove clinically feasible for detecting and monitoring the early antitumor effects in response to cancer drug therapy.

Molecular Ultrasound Imaging Using a Targeted Contrast Agent for Assessing Early Tumor Response to Antiangiogenic Therapy

Contrast‐enhanced ultrasound (US) and targeted microbubbles have been shown to be advantageous for angiogenesis evaluation and disease staging in cancer. This study explored molecular US imaging of a multitargeted microbubble for assessing the early tumor response to antiangiogenic therapy.

Microbubble-mediated ultrasonic techniques for improved chemotherapeutic delivery in cancer

Background: Ultrasound (US) exposed microbubble (MB) contrast agents have the capability to transiently enhance cell membrane permeability. Using this technique in cancer treatment to increase the efficiency of chemotherapy through passive, localized delivery has been an emerging area of research. Purpose: Investigation of the influence of US parameters on MB-mediated drug delivery in cancer. Methods: The 2LMP breast cancer cells were used for in vitro experiments and 2LMP tumor-bearing mice were used during in vivo experiments. Changes in membrane permeability were investigated after the influence of MB-mediated US therapy parameters (i.e. frequency, mechanical index, pulse repetition period, US duration, and MB dosing and characteristics) on cancer cells. Calcein, a non-permeable fluorescent molecule, and Taxol, chemotherapeutic, were used to evaluate membrane permeability. Tumor response was also assessed histologically. Results: Combination chemotherapy and MB-mediated US therapy with optimized parameters increased cancer cell death by 50% over chemotherapy alone. Discussion: Increased cellular uptake of chemotherapeutic was dependent upon US system parameters. Conclusion: Optimized MB-mediated US therapy has the potential to improve cancer patient response to therapy via increased localized drug uptake, which may lead to a lowering of chemotherapeutic drug dosages and systemic toxicity.

Microbubble Therapy Enhances Anti-tumor Properties of Cisplatin and Cetuximab In Vitro and In Vivo

Objective. To determine if microbubble-mediated ultrasound therapy (MB-UST) can improve cisplatin or cetuximab cytotoxicity of head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo by increasing tumor-specific drug delivery by disruption of tumor cell membranes and enhancing vascular permeability. Study Design. In vitro and in vivo study. Setting. University medical center. Subjects. Immunodeficient mice (6 weeks old) and 4 HNSCC cell lines. Methods. Changes to cell permeability were assessed in vitro after MB-UST. Cellular apoptosis resulting from adjuvant MB-UST with subtherapeutic doses of cisplatin or cetuximab was assessed by cell survival assays in vitro. The in vivo effect of adjuvant MB-UST in flank tumors was assessed in vivo with histological analysis and diffusion-weighted magnetic resonance imaging (DW-MRI). Results. In vitro results revealed that MB-UST can increase cell permeability and enhance drug uptake and apoptosis in 4 HNSCC cell lines. In vivo adjuvant MB-UST with cetuximab or cisplatin showed a statistically significant reduction in tumor size when compared with untreated controls. TUNEL analysis yielded a larger number of cells undergoing apoptosis in tumors treated with cetuximab and adjuvant MB-UST than did cetuximab alone but was not significantly greater in tumors treated with cisplatin and adjuvant MB-UST compared with cisplatin alone. DW-MRI analysis showed more free water, which corresponds to increased cell membrane disruption, in tumors treated with MB-UST. Conclusion. MB-UST promotes disruption of cell membranes in tumor cells in vitro, which may be leveraged to selectively improve the uptake of conventional and targeted therapeutics in vivo.

Nondestructive tissue analysis for ex vivo and in vivo cancer diagnosis using a handheld mass spectrometry system

A handheld and biocompatible mass spectrometry system provides nondestructive molecular diagnosis of cancer tissue samples. Is the pen mightier than the scalpel? Although a surgeon’s goal is to remove cancer in its entirety during excision surgery, achieving negative margins (absence of cancer cells at the outer edge of the excised tumor specimen) can be challenging. To facilitate intraoperative diagnosis, Zhang et al. developed a handheld pen-like device that rapidly identifies the molecular profile of tissues using a small volume water droplet and mass spectrometry analysis. After 3 s of gentle physical contact with a tissue surface, the water droplet is transported to a mass spectrometer, which characterizes diagnostic proteins, lipids, and metabolites. The pen could be used to rapidly distinguish tumor from healthy tissue during surgery in mice, without requiring specific labeling or imaging and without evidence of tissue destruction. Conventional methods for histopathologic tissue diagnosis are labor- and time-intensive and can delay decision-making during diagnostic and therapeutic procedures. We report the development of an automated and biocompatible handheld mass spectrometry device for rapid and nondestructive diagnosis of human cancer tissues. The device, named MasSpec Pen, enables controlled and automated delivery of a discrete water droplet to a tissue surface for efficient extraction of biomolecules. We used the MasSpec Pen for ex vivo molecular analysis of 20 human cancer thin tissue sections and 253 human patient tissue samples including normal and cancerous tissues from breast, lung, thyroid, and ovary. The mass spectra obtained presented rich molecular profiles characterized by a variety of potential cancer biomarkers identified as metabolites, lipids, and proteins. Statistical classifiers built from the histologically validated molecular database allowed cancer prediction with high sensitivity (96.4%), specificity (96.2%), and overall accuracy (96.3%), as well as prediction of benign and malignant thyroid tumors and different histologic subtypes of lung cancer. Notably, our classifier allowed accurate diagnosis of cancer in marginal tumor regions presenting mixed histologic composition. Last, we demonstrate that the MasSpec Pen is suited for in vivo cancer diagnosis during surgery performed in tumor-bearing mouse models, without causing any observable tissue harm or stress to the animal. Our results provide evidence that the MasSpec Pen could potentially be used as a clinical and intraoperative technology for ex vivo and in vivo cancer diagnosis.

Antibody-based imaging strategies for cancer

Although mainly developed for preclinical research and therapeutic use, antibodies have high antigen specificity, which can be used as a courier to selectively deliver a diagnostic probe or therapeutic agent to cancer. It is generally accepted that the optimal antigen for imaging will depend on both the expression in the tumor relative to normal tissue and the homogeneity of expression throughout the tumor mass and between patients. For the purpose of diagnostic imaging, novel antibodies can be developed to target antigens for disease detection, or current FDA-approved antibodies can be repurposed with the covalent addition of an imaging probe. Reuse of therapeutic antibodies for diagnostic purposes reduces translational costs since the safety profile of the antibody is well defined and the agent is already available under conditions suitable for human use. In this review, we will explore a wide range of antibodies and imaging modalities that are being translated to the clinic for cancer identification and surgical treatment.

Volumetric Contrast-Enhanced Ultrasound Imaging to Assess Early Response to Apoptosis-Inducing Anti-Death Receptor 5 Antibody Therapy in a Breast Cancer Animal Model

The objective of this study was to determine whether volumetric contrast‐enhanced ultrasound (US) imaging could detect early tumor response to anti–death receptor 5 antibody (TRA‐8) therapy alone or in combination with chemotherapy in a preclinical triple‐negative breast cancer animal model.

Systemic delivery of a breast cancer-detecting adenovirus using targeted microbubbles

One of the major limitations of cancer gene therapy using recombinant human adenovirus (Ad) is rapid Ad inactivation from systemic delivery. To eliminate this, biotin-coated ultrasound contrast agents, or microbubbles (MBs), were streptavidin-coupled with biotinylated antibodies to three distinct tumor vasculature-associated receptors (αVβ3 integrin, P-selectin and vascular endothelial growth factor receptor-2) for systemic targeting of a previously generated vector Ad5/3-Id1-SEAP-Id1-mCherry. This cancer-specific, dual-reporter vector was loaded in the targeted MBs and confirmed by confocal microscopy. MB loading capacity was estimated by functional assays as 4.72±0.2 plaque forming unit (PFU) per MB. Non-loaded (free) Ad particles were effectively inactivated by treatment with human complement. The Ad-loaded, targeted-MBs were injected systemically in mice bearing MDA-MB-231 tumors (Grp 1) and compared with two control groups: Ad-loaded, non-targeted MBs (Grp 2) and free Ad (Grp 3) administered under the same conditions. Two days after administration the blood levels of secreted embryonic alkaline phosphatase (SEAP) reporter in Grp 1 mice (16.1 ng ml–1±2.5) were significantly higher (P<0.05) than those in Grp 2 (9.75 ng ml–1±1.5) or Grp 3 (4.26 ng ml–1±2.5) animals. The targeted Ad delivery was also confirmed by fluorescence imaging. Thus, Ad delivery by targeted MBs holds potential as a safe and effective system for systemic Ad delivery for the purpose of cancer screening.

Ultrasound-stimulated drug delivery for treatment of residual disease after incomplete resection of head and neck cancer.

Microbubbles triggered with localized ultrasound (US) can improve tumor drug delivery and retention. Termed US-stimulated drug delivery, this strategy was applied to head and neck cancer (HNC) in a post-surgical tumor resection model. Luciferase-positive HNC squamous cell carcinoma (SCC) was implanted in the flanks of nude athymic mice (N = 24) that underwent various degrees of surgical tumor resection (0%, 50% or 100%). After surgery, animals received adjuvant therapy with cetuximab-IRDye alone, or cetuximab-IRDye in combination with US-stimulated drug delivery or saline injections (control) on days 4, 7 and 10. Tumor drug delivery was assessed on days 0, 4, 7, 10, 14 and 17 with an in vivo fluorescence imaging system, and tumor viability was evaluated at the same times with in vivo bioluminescence imaging. Tumor caliper measurements occurred two times per week for 24 d. Optical imaging revealed that in the 50% tumor resection group, US-stimulated drug delivery resulted in a significant increase in cetuximab delivery compared with administration of drug alone on day 10 (day of peak fluorescence) (p = 0.03). Tumor viability decreased in all groups that received cetuximab-IRDye in combination with US-stimulated drug delivery, compared with the group that received only the drug. After various degrees of surgical resection, this novel study reports positive improvements in drug uptake in the residual cancer cells when drug delivery is stimulated with US.