Molecular imaging is revolutionizing the way we study the inner workings of the human body, diagnose diseases, approach drug design, and assess therapies. The field as a whole is making possible the visualization of complex biochemical processes involved in normal physiology and disease states, in real time, in living cells, tissues, and intact subjects. In this review, we focus specifically on molecular imaging of intact living subjects. We provide a basic primer for those who are new to molecular imaging, and a resource for those involved in the field. We begin by describing classical molecular imaging techniques together with their key strengths and limitations, after which we introduce some of the latest emerging imaging modalities. We provide an overview of the main classes of molecular imaging agents (i.e., small molecules, peptides, aptamers, engineered proteins, and nanoparticles) and cite examples of how molecular imaging is being applied in oncology, neuroscience, cardiology, gene therapy, cell tracking, and theranostics (therapy combined with diagnostics). A step-by-step guide to answering biological and/or clinical questions using the tools of molecular imaging is also provided. We conclude by discussing the grand challenges of the field, its future directions, and enormous potential for further impacting how we approach research and medicine.

A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications

Molecular Imaging: Current Status and Emerging Strategies

Molecular ultrasound imaging: current status and future directions

Ultrasound imaging is clinically established for routine screening examinations of breast, abdomen, neck, and other soft tissues, as well as for therapy monitoring. Microbubbles as vascular contrast agents improve the detection and characterization of cancerous lesions, inflammatory processes, and cardiovascular pathologies. Taking advantage of the excellent sensitivity and specificity of ultrasound for microbubble detection, molecular imaging can be realized by binding antibodies, peptides, and other targeting moieties to microbubble surfaces. Molecular microbubbles directed against various targets such as vascular endothelial growth factor receptor-2, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, selectins, and integrins were developed and were shown in preclinical studies to be able to selectively bind to tumor blood vessels and atherosclerotic plaques. Currently, the first microbubble formulations targeted to angiogenic vessels in prostate cancers are being evaluated clinically. However, microbubbles can be used for more than diagnosis: disintegrating microbubbles emit acoustic forces that are strong enough to induce thrombolysis, and they can also be used for facilitating drug and gene delivery across biologic barriers. This review on the use of microbubbles for ultrasound-based molecular imaging, therapy, and theranostics addresses innovative concepts and identifies areas in which clinical translation is foreseeable in the near future.

https://jnm.snmjournals.org/content/jnumed/53/3/345.full.pdf

Ultrasound Microbubbles for Molecular Diagnosis, Therapy, and Theranostics

Molecular imaging for cancer diagnosis and surgery

Targeted contrast-enhanced ultrasound imaging with gas-filled echogenic microbubbles as contrast agents is increasingly being recognized as a promising and powerful molecular imaging tool (1). Ultrasound contrast agents can be targeted to specific molecular markers through the attachment of appropriate ligands to the surface of the microbubbles. After intravenous injection, these functionalized microbubbles attach at tissue sites expressing the target molecular marker, leading to a local increase in the ultrasonic imaging signal. Because of their size (several micrometers), currently available contrast microbubbles stay predominantly within the vascular system (2). This feature makes targeted contrast-enhanced ultrasound imaging exquisitely attractive as a noninvasive imaging tool for detecting and tracking biologic processes on vascular endothelial cells, such as tumor angiogenesis.

In tumor angiogenesis, which is the formation and recruitment of new blood vessels from the surrounding host tissue, various molecular markers are overexpressed on tumor vascular endothelial cells (3,4). These molecular markers are involved in the regulation and maintenance of an angiogenic phenotype that is needed in many tumors to maintain a sufficient supply of oxygen, nutrients, and other growth factors. Among others, integrins are well-characterized molecular markers of tumor angiogenesis. Integrins are a class of heterodimeric cell surface adhesion receptors that are overexpressed on tumor endothelial cells during tumor angiogenesis. They bind to extracellular matrix proteins such as fibronectin, fibrinogen, von Willebrand factor, vitronectin, and proteolysis-generated forms of collagen and laminin, which promote cell adhesion to the extracellular matrix and activation of signaling pathways involved in tumor cell growth, invasion, and metastasis (5). In particular, αvβ3 integrin is highly expressed on activated angiogenic endothelial cells in tumor vessels, resulting in cancers that are more invasive, more migratory, and better able to survive in different microenvironments (6). Therefore, noninvasive imaging strategies for the detection and quantification of αvβ3 integrin may be particularly helpful for diagnosing cancer at early stages, developing anticancer therapeutic agents in preclinical animal models, and monitoring antiangiogenic and tumoricidal treatments in cancer patients.

Recent studies showed the feasibility of targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with different commercially available integrin-targeting ligands, such as peptidomimetic agents, monoclonal antibodies, or echistatin (7–9). In the present proof-of-principle study, we tested whether engineered cystine knot (knottin) peptides could be coupled to the surface of contrast microbubbles and used as a new class of targeting ligands for targeted contrast-enhanced ultrasound imaging of tumor angiogenesis. Knottin peptides are small peptides (30–40 amino acids) containing conformationally constrained loop structures that naturally bind to a wide variety of targets (10). Previously, directed evolution was used to engineer the surface-exposed trypsin-binding loop of the Ecballium elaterium trypsin inhibitor II knottin peptide (11) to bind to tumor-specific integrin receptors with a low nanomolar affinity (12). Here, we chemically coupled this integrin-binding knottin peptide to the surface of contrast micro-bubbles and assessed their ability to bind to tumor cells expressing αvβ3 integrin and their ability to be used as ultrasound contrast agents for human subcutaneous ovarian cancer xenograft tumors in living mice. We compared our results with those obtained with microbubbles conjugated to an integrin-binding peptidomimetic agent or an antiintegrin monoclonal antibody. Our study validates knottin peptides as a general class of ligands for targeted ultrasound with contrast microbubbles and demonstrates that knottin peptides engineered against other molecular targets besides integrins hold much promise for molecular imaging applications.

https://jnm.snmjournals.org/content/jnumed/51/3/433.full.pdf

Targeted Contrast-Enhanced Ultrasound Imaging of Tumor Angiogenesis with Contrast Microbubbles Conjugated to Integrin-Binding Knottin Peptides

The results of our study suggest that expression levels of different tumor angiogenic markers vary during tumor growth in subcutaneous human breast, ovarian, and pancreatic cancer xenografts in mice and that targeted contrast-enhanced US imaging allows longitudinal noninvasive assessment of the temporal tumor angiogenic molecular marker expression levels in vivo.

/pdf/tumor-angiogenic-marker-expression-levels-during-tumor-2ffujewovq.pdf

Tumor angiogenic marker expression levels during tumor growth: longitudinal assessment with molecularly targeted microbubbles and US imaging.

Angiogenesis, the growth of new blood vessels, plays a critical role in progression of tumor growth and metastasis, making it an attractive target for both cancer imaging and therapy. Several molecular markers, including those that are involved in the angiogenesis signaling pathway and those unique to tumor angiogenic vessels, have been identified and can be used as targets for molecular imaging of cancer. With the introduction of ultrasound contrast agents that can be targeted to those molecular markers, targeted contrast-enhanced ultrasound (molecular ultrasound) imaging has become an attractive imaging modality to non-invasively assess tumor angiogenesis at the molecular level. The advantages of molecular ultrasound imaging such as high temporal and spatial resolution, non-invasiveness, real-time imaging, relatively low cost, lack of ionizing irradiation and wide availability among the imaging community will further expand its roles in cancer imaging and drug development both in preclinical research and future clinical applications.

Molecular ultrasound assessment of tumor angiogenesis.

Targeted contrast-enhanced US with microbubbles targeted to P-selectin enables accurate and reliable quantification and monitoring of inflammation at the molecular level in a chemically induced murine model of inflammatory bowel disease.

/pdf/quantification-and-monitoring-of-inflammation-in-murine-2zjp6urzb0.pdf

Nirupama Deshpande

Papers

Molecular ultrasound imaging: current status and future directions

Targeted Contrast-Enhanced Ultrasound Imaging of Tumor Angiogenesis with Contrast Microbubbles Conjugated to Integrin-Binding Knottin Peptides

Tumor angiogenic marker expression levels during tumor growth: longitudinal assessment with molecularly targeted microbubbles and US imaging.

Molecular ultrasound assessment of tumor angiogenesis.

Quantification and Monitoring of Inflammation in Murine Inflammatory Bowel Disease with Targeted Contrast-enhanced US