Detection and Characterization of Sentinel Lymph Node by Ultrasound Molecular Imaging with B7-H3-Targeted Microbubbles in Orthotopic Breast Cancer Model in Mice.

PURPOSE: Accurate identification and assessment of sentinel lymph node (SLN) using noninvasive imaging methods can play a vital role in tumor staging, surgical planning, and prognostic evaluation. In this study, we assessed the efficacy of B7-H3-targeted molecular-ultrasound imaging for the early SLN detection, and characterization in a mouse model of orthotopic breast cancer. PROCEDURES: We established a mouse breast cancer model with lymph node metastasis by injecting MAD-MB 231 cells which were engineered to express firefly luciferase reporter gene into the fat pad of the right 4th mammary gland in female BALB/c nude mice. The sole lymph node (LN) close to the tumor was regarded as the SLN for imaging investigation, which included metastatic and non-metastatic SLNs. The LN in the right 4th mammary gland from normal mice was used as normal control (normal mice LN). The commercially available preclinical streptavidin-coated, perfluorocarbon-containing lipid-shelled microbubbles (VisualSonics, Toronto, Canada) were used to generate B7-H3-targeted microbubbles (MBB7-H3) and control microbubbles (MBControl). Then, ultrasound molecular imaging (USMI) was performed using a high-resolution transducer (MS250; center frequency, 21 MHz; Vevo 2100; VisualSonics, Toronto, Canada) after intravenous injection of microbubbles. RESULTS: The SLN was clearly detected and located under conventional (B-mode) and contrast-enhanced ultrasonography with microbubble injection. The metastatic SLNs showed a markedly higher signal from B7-H3-targeted microbubbles (MBB7-H3) compared to the non-metastatic SLNs and normal LNs. The metastatic SLN was further confirmed by ex vivo bioluminescence imaging and eventually verified by histological analysis. CONCLUSIONS: Our findings suggest the potential value of USMI using B7-H3 targeted microbubbles in breast cancer and establish an effective imaging method for the non-invasive detection and characterization of SLN.

Photoacoustic Molecular Imaging for the Identification of Lymph Node Metastasis in Head and Neck Cancer Using an Anti-EGFR Antibody-Dye Conjugate.

The presence of lymph node (LN) metastases is an essential prognostic indicator in patients with head and neck squamous cell carcinoma (HNSCC). This study assessed photoacoustic molecular imaging (PAMI) of the antiepidermal growth factor receptor antibody (panitumumab) conjugated to a near-infrared fluorescent dye, IRDye800CW (panitumumab-IRDye800CW; pan800), for the identification of occult metastatic LNs in patients with HNSCC (n = 7). Methods: After in vitro photoacoustic imaging characterization of pan800, PAMI was performed on excised neck specimens from patients infused with pan800 before surgery. Freshly obtained neck specimens were imaged with 3-dimensional, multiwavelength spectroscopic PAMI (wavelengths of 680, 686, 740, 800, 860, 924, and 958 nm). Harvested LNs were then imaged with a closed-field near-infrared fluorescence imager and histologically examined by the pathologist to determine their metastatic status. Results: In total, 53 LNs with a maximum diameter of 10 mm were analyzed with photoacoustic and fluorescence imaging, of which 4 were determined to be metastatic on the final histopathologic report. Photoacoustic signals in the LNs corresponding to accumulated pan800 were spectrally unmixed using a linear least-square-error classification algorithm. The average thresholded photoacoustic signal intensity corresponding to pan800 was 5-fold higher for metastatic LNs than for benign LNs (2.50 ± 1.09 arbitrary units [a.u.] vs. 0.53 ± 0.32 a.u., P < 0.001). Fluorescence imaging showed that metastatic LNs had a 2-fold increase in fluorescence signal compared with benign LNs ex vivo (P < 0.01, 0.068 ± 0.027 a.u. vs. 0.035 ± 0.018 a.u.). Moreover, the ratio of the average of the highest 10% of the photoacoustic signal intensity over the total average, representative of the degree of heterogeneity in the pan800 signal in LNs, showed a significant difference between metastatic LNs and benign LNs (11.6 ± 13.4 vs. 1.8 ± 0.7, P < 0.01) and an area under the receiver-operating-characteristic curve of 0.96 (95% CI, 0.91-1.00). Conclusion: The data indicate that PAMI of IRDye800-labeled tumor-specific antibody may have the potential to identify occult LN metastasis perioperatively in HNSCC patients.

Evaluation of ductal carcinoma in situ grade via triple-modal molecular imaging of B7-H3 expression.

Ductal carcinoma in situ (DCIS) will account for 62,930 cases of breast cancer in 2019. DCIS is a pre-invasive lesion which may not progress to invasive carcinoma, yet surgery remains the mainstay treatment. Molecular imaging of a specific marker for DCIS grade for detection and active surveillance are critically needed to reduce potential overtreatment. First, breast cancer marker B7-H3 (CD276) expression was evaluated by immunohistochemical staining in 123 human specimens including benign epithelium (H-score 10.0 ± 8.2) and low (20.8 ± 17.7), intermediate (87.1 ± 69.5), and high (159.1 ± 87.6) grade DCIS, showing a positive association with DCIS nuclear grade (P < 0.001, AUC 0.96). Next, a murine DCIS model was combined with ultrasound molecular imaging of B7-H3 targeted microbubbles to differentiate normal glands from those harboring DCIS (n = 100, FVB/N-Tg(MMTVPyMT)634Mul, AUC 0.89). Finally, photoacoustic and fluorescence molecular imaging with an anti-B7-H3 antibody-indocyanine green conjugate were utilized for DCIS detection (n = 53). Molecular imaging of B7-H3 expression may allow for active surveillance of DCIS.

Efficacy of Affibody-Based Ultrasound Molecular Imaging of Vascular B7-H3 for Breast Cancer Detection.

PURPOSE: Human B7-H3 (hB7-H3) is a promising molecular imaging target differentially expressed on the neovasculature of breast cancer and has been validated for preclinical ultrasound (US) imaging with anti-B7-H3-antibody-functionalized microbubbles (MB). However, smaller ligands such as affibodies (ABY) are more suitable for the design of clinical-grade targeted MB. EXPERIMENTAL DESIGN: Binding of ABYB7-H3 was confirmed with soluble and cell-surface B7-H3 by flow cytometry. MB were functionalized with ABYB7-H3 or anti-B7-H3-antibody (AbB7-H3). Control and targeted MB were tested for binding to hB7-H3-expressing cells (MS1hB7-H3) under shear stress conditions. US imaging was performed with MBABY-B7-H3 in an orthotopic mouse model of human MDA-MB-231 coimplanted with MS1hB7-H3 or control MS1WT cells and a transgenic mouse model of breast cancer development. RESULTS: ABYB7-H3 specifically binds to MS1hB7-H3 and murine-B7-H3-expressing monocytes. MBABY-B7-H3 (8.5 ± 1.4 MB/cell) and MBAb-B7-H3 (9.8 ± 1.3 MB/cell) showed significantly higher (P < 0.0001) binding to the MS1hB7-H3 cells compared with control MBNon-targeted (0.5 ± 0.1 MB/cell) under shear stress conditions. In vivo, MBABY-B7-H3 produced significantly higher (P < 0.04) imaging signal in orthotopic tumors coengrafted with MS1hB7-H3 (8.4 ± 3.3 a.u.) compared with tumors with MS1WT cells (1.4 ± 1.0 a.u.). In the transgenic mouse tumors, MBABY-B7-H3 (9.6 ± 2.0 a.u.) produced higher (P < 0.0002) imaging signal compared with MBNon-targeted (1.3 ± 0.3 a.u.), whereas MBABY-B7-H3 signal in normal mammary glands and tumors with B7-H3 blocking significantly reduced (P < 0.02) imaging signal. CONCLUSIONS: MBABY-B7-H3 enhances B7-H3 molecular signal in breast tumors, improving cancer detection, while offering the advantages of a small size ligand and easier production for clinical imaging.

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research.

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

Affibody-Indocyanine Green Based Contrast Agent for Photoacoustic and Fluorescence Molecular Imaging of B7-H3 Expression in Breast Cancer.

Spectroscopic photoacoustic (sPA) molecular imaging has high potential for identification of exogenous contrast agents targeted to specific markers. Antibody-dye conjugates have recently been used extensively for preclinical sPA and other optical imaging modalities for highly specific molecular imaging of breast cancer. However, antibody-based agents suffer from long circulation times that limit image specificity. Here, the efficacy of a small protein scaffold, the affibody (ABY), conjugated to indocyanine green (ICG), a near-infrared fluorescence dye, as a targeted molecular imaging probe is demonstrated. In particular, B7-H3 (CD276), a cellular receptor expressed in breast cancer, was imaged via sPA and fluorescence molecular imaging to differentiate invasive tumors from normal glands in mice. Administration of ICG conjugated to an ABY specific to B7-H3 (ABYB7-H3-ICG) showed significantly higher signal in mammary tumors compared to normal glands of mice. ABYB7-H3-ICG is a compelling scaffold for molecular sPA imaging for breast cancer detection.

Ultrasound molecular imaging as a non-invasive companion diagnostic for netrin-1 interference therapy in breast cancer.

In ultrasound molecular imaging (USMI), ligand-functionalized microbubbles (MBs) are used to visualize vascular endothelial targets. Netrin-1 is upregulated in 60% of metastatic breast cancers and promotes tumor progression. A novel netrin-1 interference therapy requires the assessment of netrin-1 expression prior to treatment. In this study, we studied netrin-1 as a target for USMI and its potential as a companion diagnostic in breast cancer models. Methods: To verify netrin-1 expression and localization, an in vivo immuno-localization approach was applied, in which anti-netrin-1 antibody was injected into living mice 24 h before tumor collection, and revealed with secondary fluorescent antibody for immunofluorescence analysis. Netrin-1 interactions with the cell surface were studied by flow cytometry. Netrin-1-targeted MBs were prepared using MicroMarker Target-Ready (VisualSonics), and validated in in vitro binding assays in static conditions or in a flow chamber using purified netrin-1 protein or netrin-1-expressing cancer cells. In vivo USMI of netrin-1 was validated in nude mice bearing human netrin-1-positive SKBR7 tumors or weakly netrin-1-expressing MDA-MB-231 tumors using the Vevo 2100 small animal imaging device (VisualSonics). USMI feasibility was further tested in transgenic murine FVB/N Tg(MMTV/PyMT634Mul) (MMTV-PyMT) mammary tumors. Results: Netrin-1 co-localized with endothelial CD31 in netrin-1-positive breast tumors. Netrin-1 binding to the surface of endothelial HUVEC and cancer cells was partially mediated by heparan sulfate proteoglycans. MBs targeted with humanized monoclonal anti-netrin-1 antibody bound to netrin-1-expressing cancer cells in static and dynamic conditions. USMI signal was significantly increased with anti-netrin-1 MBs in human SKBR7 breast tumors and transgenic murine MMTV-PyMT mammary tumors compared to signals recorded with either isotype control MBs or after blocking of netrin-1 with humanized monoclonal anti-netrin-1 antibody. In weakly netrin-1-expressing human tumors and normal mammary glands, no difference in imaging signal was observed with anti-netrin-1- and isotype control MBs. Ex vivo analysis confirmed netrin-1 expression in MMTV-PyMT tumors. Conclusions: These results show that USMI allowed reliable detection of netrin-1 on the endothelium of netrin-1-positive human and murine tumors. Significant differences in USMI signal for netrin-1 reflected the significant differences in netrin-1 mRNA & protein expression observed between different breast tumor models. The imaging approach was non-invasive and safe, and provided the netrin-1 expression status in near real-time. Thus, USMI of netrin-1 has the potential to become a companion diagnostic for the stratification of patients for netrin-1 interference therapy in future clinical trials.

Intraoperative Resection Guidance with Photoacoustic and Fluorescence Molecular Imaging Using an Anti-B7-H3 Antibody-Indocyanine Green Dual Contrast Agent.

Purpose: Breast cancer often requires surgical treatment including breast-conserving surgical resection. However, with current postsurgical histologic margin analysis, one quarter of breast cancer patients undergo reexcision to achieve negative margins corresponding to decreased local recurrence and better outcomes. Therefore, a method with high resolution and specificity for intraoperative margin assessment is needed.Experimental Design: First, quantitative immunofluorescence staining of B7-H3 expression was assessed in four pathologic stages of breast cancer progression of the MMTV-PyMT transgenic murine model. Next, an antibody-dye contrast agent, B7-H3-ICG, was injected into mice prior to surgical resection of breast cancer. Anatomic ultrasound, spectroscopic photoacoustic (sPA), and fluorescence imaging were used to guide resection of mammary glands suspected of containing cancer. Resected tissues were processed for H&E staining and pathologic assessment and compared with sPA and fluorescence imaging signals.Results: Tissue containing DCIS (46.0 ± 4.8 a.u.) or invasive carcinoma (91.7 ± 21.4 a.u.) showed significantly higher (P < 0.05) B7-H3 expression than normal and hyperplastic tissues (1.3 ± 0.8 a.u.). During image-guided surgical resection, tissue pieces assessed as normal or hyperplastic (n = 17) showed lower average sPA (3.17 ± 0.48 a.u.) and fluorescence signal [6.83E07 ± 2.00E06 (p/s)/(µW/cm²)] than DCIS and invasive carcinoma tissue (n = 63) with an average sPA signal of 23.98 ± 4.88 a.u. and an average fluorescence signal of 7.56E07 ± 1.44E06 (p/s)/(µW/cm²) with AUCs of 0.93 [95% confidence interval (CI), 0.87-0.99] and 0.71 (95% CI, 0.57-0.85), respectively.Conclusions: It was demonstrated that sPA and fluorescence molecular imaging combined with B7-H3-ICG agent can assess the disease status of tissues with high diagnostic accuracy, intraoperatively, with high resolution, sensitivity, and specificity. Clin Cancer Res; 24(15); 3572-82. ©2018 AACR.

Spectroscopic Photoacoustic Molecular Imaging of Breast Cancer using a B7-H3-targeted ICG Contrast Agent.

Purpose: Breast cancer imaging methods lack diagnostic accuracy, in particular for patients with dense breast tissue, and improved techniques are critically needed. The purpose of this study was to evaluate antibody-indocyanine green (ICG) conjugates, which undergo dynamic absorption spectrum shifts after cellular endocytosis and degradation, and spectroscopic photoacoustic (sPA) imaging to differentiate normal breast tissue from breast cancer by imaging B7-H3, a novel breast cancer associated molecular target. Methods: Quantitative immunohistochemical staining of endothelial and epithelial B7-H3 expression was assessed in 279 human breast tissue samples, including normal (n=53), benign lesions (11 subtypes, n=129), and breast cancers (4 subtypes, n=97). After absorption spectra of intracellular and degraded B7-H3-ICG and Isotype control-ICG (Iso-ICG) were characterized, sPA imaging in a transgenic murine breast cancer model (FVB/N-Tg(MMTVPyMT)634Mul) was performed and compared to imaging of control conditions [B7-H3-ICG in tumor negative animals (n=60), Iso-ICG (n=30), blocking B7-H3+B7-H3-ICG (n=20), and free ICG (n=20)] and validated with ex vivo histological analysis. Results: Immunostaining showed differential B7-H3 expression on both the endothelium and tumor epithelium in human breast cancer with an area under the ROC curve of 0.93 to differentiate breast cancer vs non-cancer. Combined in vitro/in vivo imaging showed that sPA allowed specific B7-H3-ICG detection down to the 13 nM concentration and differentiation from Iso-ICG. sPA molecular imaging of B7-H3-ICG showed a 3.01-fold (P<0.01) increase in molecular B7-H3-ICG signal in tumors compared to control conditions. Conclusions: B7-H3 is a promising target for both vascular and epithelial sPA imaging of breast cancer. Leveraging antibody-ICG contrast agents and their dynamic optical absorption spectra allows for highly specific sPA imaging of breast cancer.

Ultrasound Molecular Imaging of the Breast Cancer Neovasculature using Engineered Fibronectin Scaffold Ligands: A Novel Class of Targeted Contrast Ultrasound Agent.

Molecularly-targeted microbubbles (MBs) are increasingly being recognized as promising contrast agents for oncological molecular imaging with ultrasound. With the detection and validation of new molecular imaging targets, novel binding ligands are needed that bind to molecular imaging targets with high affinity and specificity. In this study we assessed a novel class of potentially clinically translatable MBs using an engineered 10(th) type III domain of human-fibronectin (MB-FN3VEGFR2) scaffold-ligand to image VEGFR2 on the neovasculature of cancer. The in vitro binding of MB-FN3VEGFR2 to a soluble VEGFR2 was assessed by flow-cytometry (FACS) and binding to VEGFR2-expressing cells was assessed by flow-chamber cell attachment studies under flow shear stress conditions. In vivo binding of MB-FN3VEGFR2 was tested in a transgenic mouse model (FVB/N Tg(MMTV/PyMT634Mul) of breast cancer and control litter mates with normal mammary glands. In vitro FACS and flow-chamber cell attachment studies showed significantly (P<0.01) higher binding to VEGFR2 using MB-FN3VEGFR2 than control agents. In vivo ultrasound molecular imaging (USMI) studies using MB-FN3VEGFR2 demonstrated specific binding to VEGFR2 and was significantly higher (P<0.01) in breast cancer compared to normal breast tissue. Ex vivo immunofluorescence-analysis showed significantly (P<0.01) increased VEGFR2-expression in breast cancer compared to normal mammary tissue. Our results suggest that MBs coupled to FN3-scaffolds can be designed and used for USMI of breast cancer neoangiogenesis. Due to their small size, stability, solubility, the lack of glycosylation and disulfide bonds, FN3-scaffolds can be recombinantly produced with the advantage of generating small, high affinity ligands in a cost efficient way for USMI.

Photoacoustic Imaging in Oncology: Translational Preclinical and Early Clinical Experience.

Photoacoustic imaging has evolved into a clinically translatable platform with the potential to complement existing imaging techniques for the management of cancer, including detection, characterization, prognosis, and treatment monitoring. In photoacoustic imaging, tissue is optically excited to produce ultrasonographic images that represent a spatial map of optical absorption of endogenous constituents such as hemoglobin, fat, melanin, and water or exogenous contrast agents such as dyes and nanoparticles. It can therefore provide functional and molecular information that allows noninvasive soft-tissue characterization. Photoacoustic imaging has matured over the years and is currently being translated into the clinic with various clinical studies underway. In this review, the current state of photoacoustic imaging is presented, including techniques and instrumentation, followed by a discussion of potential clinical applications of this technique for the detection and management of cancer. (©) RSNA, 2016.

Breast Cancer Detection by B7-H3-Targeted Ultrasound Molecular Imaging.

Ultrasound complements mammography as an imaging modality for breast cancer detection, especially in patients with dense breast tissue, but its utility is limited by low diagnostic accuracy. One emerging molecular tool to address this limitation involves contrast-enhanced ultrasound using microbubbles targeted to molecular signatures on tumor neovasculature. In this study, we illustrate how tumor vascular expression of B7-H3 (CD276), a member of the B7 family of ligands for T-cell coregulatory receptors, can be incorporated into an ultrasound method that can distinguish normal, benign, precursor, and malignant breast pathologies for diagnostic purposes. Through an IHC analysis of 248 human breast specimens, we found that vascular expression of B7-H3 was selectively and significantly higher in breast cancer tissues. B7-H3 immunostaining on blood vessels distinguished benign/precursors from malignant lesions with high diagnostic accuracy in human specimens. In a transgenic mouse model of cancer, the B7-H3-targeted ultrasound imaging signal was increased significantly in breast cancer tissues and highly correlated with ex vivo expression levels of B7-H3 on quantitative immunofluorescence. Our findings offer a preclinical proof of concept for the use of B7-H3-targeted ultrasound molecular imaging as a tool to improve the diagnostic accuracy of breast cancer detection in patients.

Multiparametric spectroscopic photoacoustic imaging of breast cancer development in a transgenic mouse model.

OBJECTIVE: To evaluate the potential of multiparametric spectroscopic photoacoustic imaging using oxygen saturation, total hemoglobin, and lipid content to differentiate among four different breast histologies (normal, hyperplasia, ductal carcinoma in situ (DCIS), and invasive breast carcinoma) in a transgenic mouse model of breast cancer development. MATERIALS AND METHODS: Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mammary glands (n=251) of a transgenic mouse model of breast cancer development (FVB/N-Tg(MMTV-PyMT)634Mul) were imaged using B-mode ultrasound and spectroscopic photoacoustic imaging, analyzed for oxygen saturation, total hemoglobin, and lipid content, and processed for histological analysis. Statistical analysis was performed using one-way ANOVA, two-sample t-tests, logistic regression, and ROC analysis. RESULTS: Eighty-two normal, 12 hyperplastic, 96 DCIS, and 61 invasive breast carcinoma mammary glands were analyzed. Based on spectroscopic photoacoustic imaging, the oxygen saturation of hyperplasia (50.6%), DCIS (43.0%), and invasive carcinoma (46.2%) significantly increased compared to normal glands (35.5%, P <0.0001), while both total hemoglobin (P<0.01), and lipid content (P<0.0008) significantly decreased with advancing histology. In differentiating normal and hyperplasia from DCIS and invasive breast carcinoma, multiparametric imaging of oxygen saturation, lipid content, and raw photoacoustic signal at 750 nm provided an AUC value of 0.770. CONCLUSION: Multiparametric spectroscopic photoacoustic imaging is feasible and allows detection of differences in concentration of tissue chromophores among different histologies in a transgenic mouse model of breast cancer development.

Indocyanine green-loaded photoacoustic nanodroplets: dual contrast nanoconstructs for enhanced photoacoustic and ultrasound imaging.

Recently, perfluorocarbon (PFC) nanodroplets were introduced as contrast agents for imaging and image-guided therapy. For example, in sonography, high-intensity ultrasound pulses were used to phase-transition liquid perfluorocarbon to produce gas microbubbles. More recently, perfluorocarbon nanodroplets with encapsulated gold nanorods were used as dual ultrasound/photoacoustic contrast agents. To expedite clinical translation, we synthesized and characterized ICG-loaded perfluorocarbon nanodroplets, i.e., constructs comprising biocompatible, nontoxic and biologically safe materials. We then demonstrated enhanced photoacoustic contrast through optically triggered phase transition of PFC nanodroplets and ultrasound contrast from the resulting PFC bubbles. We assessed the quality enhancement of photoacoustic and ultrasound images through analysis of contrast and contrast-to-noise ratio. We further investigated the changes in image contrast due to increased ambient temperature. Our studies suggest that ICG-loaded perfluorocarbon nanodroplets may become a valuable tool for various imaging modalities, and have promising therapeutic applications.

Ultrasound and microbubble guided drug delivery: mechanistic understanding and clinical implications.

Ultrasound mediated drug delivery using microbubbles is a safe and noninvasive approach for spatially localized drug administration. This approach can create temporary and reversible openings on cellular membranes and vessel walls (a process called "sonoporation"), allowing for enhanced transport of therapeutic agents across these natural barriers. It is generally believed that the sonoporation process is highly associated with the energetic cavitation activities (volumetric expansion, contraction, fragmentation, and collapse) of the microbubble. However, a thorough understanding of the process was unavailable until recently. Important progress on the mechanistic understanding of sonoporation and the corresponding physiological responses in vitro and in vivo has been made. Specifically, recent research shed light on the cavitation process of microbubbles and fluid motion during insonation of ultrasound, on the spatio-temporal interactions between microbubbles and cells or vessel walls, as well as on the temporal course of the subsequent biological effects. These findings have significant clinical implications on the development of optimal treatment strategies for effective drug delivery. In this article, current progress in the mechanistic understanding of ultrasound and microbubble mediated drug delivery and its implications for clinical translation is discussed.

Acoustic and photoacoustic molecular imaging of cancer.

Ultrasound and combined optical and ultrasonic (photoacoustic) molecular imaging have shown great promise in the visualization and monitoring of cancer through imaging of vascular and extravascular molecular targets. Contrast-enhanced ultrasound with molecularly targeted microbubbles can detect early-stage cancer through the visualization of targets expressed on the angiogenic vasculature of tumors. Ultrasonic molecular imaging can be extended to the imaging of extravascular targets through use of nanoscale, phase-change droplets and photoacoustic imaging, which provides further molecular information on cancer given by the chemical composition of tissues and by targeted nanoparticles that can interact with extravascular tissues at the receptor level. A new generation of targeted contrast agents goes beyond merely increasing imaging signal at the site of target expression but shows activatable and differential contrast depending on their interactions with the tumor microenvironment. These innovations may further improve our ability to detect and characterize tumors. In this review, recent developments in acoustic and photoacoustic molecular imaging of cancer are discussed.

Biomedical photoacoustics beyond thermal expansion using triggered nanodroplet vaporization for contrast-enhanced imaging.

Since being discovered by Alexander Bell, photoacoustics may again be seeing major resurgence in biomedical imaging. Photoacoustics is a non-ionizing, functional imaging modality capable of high contrast images of optical absorption at depths significantly greater than traditional optical imaging techniques. Optical contrast agents have been used to extend photoacoustics to molecular imaging. Here we introduce an exogenous contrast agent that utilizes vaporization for photoacoustic signal generation, providing significantly higher signal amplitude than that from the traditionally used mechanism, thermal expansion. Our agent consists of liquid perfluorocarbon nanodroplets with encapsulated plasmonic nanoparticles, entitled photoacoustic nanodroplets. Upon pulsed laser irradiation, liquid perfluorocarbon undergoes a liquid-to-gas phase transition generating giant photoacoustic transients from these dwarf nanoparticles. Once triggered, the gaseous phase provides ultrasound contrast enhancement. We demonstrate in phantom and animal studies that photoacoustic nanodroplets act as dual-contrast agents for both photoacoustic and ultrasound imaging through optically triggered vaporization.

Advances in Clinical and Biomedical Applications of Photoacoustic Imaging.

IMPORTANCE OF THE FIELD: Photoacoustic imaging is an imaging modality that derives image contrast from the optical absorption coefficient of the tissue being imaged. The imaging technique is able to differentiate between healthy and diseased tissue with either deeper penetration or higher resolution than other functional imaging modalities currently available. From a clinical standpoint, photoacoustic imaging has demonstrated safety and effectiveness in diagnosing diseased tissue regions using either endogenous tissue contrast or exogenous contrast agents. Furthermore, the potential of photoacoustic imaging has been demonstrated in various therapeutic interventions ranging from drug delivery and release to image-guided therapy and monitoring. AREAS COVERED IN THIS REVIEW: This article reviews the current state of photoacoustic imaging in biomedicine from a technological perspective, highlights various biomedical and clinical applications of photoacoustic imaging, and gives insights on future directions. WHAT THE READER WILL GAIN: Readers will learn about the various applications of photoacoustic imaging, as well as the various contrast agents that can be used to assist photoacoustic imaging. This review will highlight both pre-clinical and clinical uses for photoacoustic imaging, as well as discuss some of the challenges that must be addressed to move photoacoustic imaging into the clinical realm. TAKE HOME MESSAGE: Photoacoustic imaging offers unique advantages over existing imaging modalities. The imaging field is broad with many exciting applications for detecting and diagnosing diseased tissue or processes. Photoacoustics is also used in therapeutic applications to identify and characterize the pathology and then to monitor the treatment. Although the technology is still in its infancy, much work has been done in the pre-clinical arena, and photoacoustic imaging is fast approaching the clinical setting.