Multifunctional synthetic poly(L-glutamic acid)-based cancer therapeutic and imaging agents

Modern polymer chemistry has led to the generation of a number of biocompatible synthetic polymers that have been increasingly studied as efficient carriers for drugs and imaging agents. Synthetic biocompatible polymers have been used to improve the efficacy of both small-molecular-weight therapeutics and imaging agents. Furthermore, multiple targeted anticancer agents and/or imaging reporters can be attached to a single polymer chain, allowing multifunctional and/or multimodality therapy and molecular imaging. Having both an anticancer drug and an imaging reporter in a single polymer chain allows noninvasive real-time visualization of the pharmacokinetics of polymeric drug delivery systems, which can uncover and explain the complicated mechanisms of in vivo drug delivery and their correlation to pharmacodynamics. This review examines the use of the synthetic biocompatible polymer poly(L-glutamic acid) (PG) as an efficient carrier of cancer therapeutics and imaging agents. This review summarizes and updates our recent research on the use of PG as a platform for drug delivery and molecular imaging, including recent clinical findings with respect to PG-paclitaxel (PG-TXL), the combination of PG-TXL with radiotherapy, mechanisms of action of PG-TXL, and noninvasive visualization of in vivo delivery of polymeric conjugates with contrast-enhanced magnetic resonance imaging, optical imaging, and multimodality imaging.     

Reporter gene imaging of protein-protein interactions in living subjects

In the past few years there has been a veritable explosion in the field of reporter gene imaging, with the aim of determining the location, duration and extent of gene expression within living subjects. An important application of this approach is the molecular imaging of interacting protein partners, which could pave the way to functional proteomics in living animals and might provide a tool for the whole-body evaluation of new pharmaceuticals targeted to modulate protein-protein interactions. Three general methods are currently available for imaging protein-protein interactions in living subjects using reporter genes: a modified mammalian two-hybrid system, a bioluminescence resonance energy transfer (BRET) system, and split reporter protein complementation and reconstitution strategies. In the future, these innovative approaches are likely to enhance our appreciation of entire biological pathway systems and their pharmacological regulation.     

Molecular-genetic imaging: a nuclear medicine-based perspective

Molecular imaging is a relatively new discipline, which developed over the past decade, initially driven by in situ reporter imaging technology. Noninvasive in vivo molecular-genetic imaging developed more recently and is based on nuclear (positron emission tomography [PET], gamma camera, autoradiography) imaging as well as magnetic resonance (MR) and in vivo optical imaging. Molecular-genetic imaging has its roots in both molecular biology and cell biology, as well as in new imaging technologies. The focus of this presentation will be nuclear-based molecular-genetic imaging, but it will comment on the value and utility of combining different imaging modalities. Nuclear-based molecular imaging can be viewed in terms of three different imaging strategies: (1) "indirect" reporter gene imaging; (2) "direct" imaging of endogenous molecules; or (3) "surrogate" or "bio-marker" imaging. Examples of each imaging strategy will be presented and discussed. The rapid growth of in vivo molecular imaging is due to the established base of in vivo imaging technologies, the established programs in molecular and cell biology, and the convergence of these disciplines. The development of versatile and sensitive assays that do not require tissue samples will be of considerable value for monitoring molecular-genetic and cellular processes in animal models of human disease, as well as for studies in human subjects in the future. Noninvasive imaging of molecular-genetic and cellular processes will complement established ex vivo molecular-biological assays that require tissue sampling, and will provide a spatial as well as a temporal dimension to our understanding of various diseases and disease processes.     

Looking and listening to light: the evolution of whole-body photonic imaging

Optical imaging of live animals has grown into an important tool in biomedical research as advances in photonic technology and reporter strategies have led to widespread exploration of biological processes in vivo. Although much attention has been paid to microscopy, macroscopic imaging has allowed small-animal imaging with larger fields of view (from several millimeters to several centimeters depending on implementation). Photographic methods have been the mainstay for fluorescence and bioluminescence macroscopy in whole animals, but emphasis is shifting to photonic methods that use tomographic principles to noninvasively image optical contrast at depths of several millimeters to centimeters with high sensitivity and sub-millimeter to millimeter resolution. Recent theoretical and instrumentation advances allow the use of large data sets and multiple projections and offer practical systems for quantitative, three-dimensional whole-body images. For photonic imaging to fully realize its potential, however, further progress will be needed in refining optical inversion methods and data acquisition techniques.     

Preclinical applications of imaging for cancer gene therapy

Gene therapy is a very attractive strategy in experimental cancer therapy. Ideally, the approach aims to deliver therapeutic genes selectively to cancer cells. However, progress in the improvement of gene therapy formulations has been hampered by difficulties in measuring transgene delivery and in quantifying transgene expression in vivo. In clinical trials, endpoints rely almost exclusively on the analysis of biopsies by molecular and histopathological methods, which provide limited information. Therefore, to ensure the rational development of gene therapy, a crucial issue is the utilisation of technologies for the non-invasive monitoring of spatial and temporal gene expression in vivo upon administration of a gene delivery vector. Such imaging technologies would allow the generation of quantitative information about gene expression and the assessment of cancer gene therapy efficacy. In the past decade, progress has been made in the field of in vivo molecular imaging. This review highlights the various methods currently being developed in preclinical models.     

Tumor-specific imaging through progression elevated gene-3 promoter-driven gene expression

Molecular-genetic imaging is advancing from a valuable preclinical tool to a guide for patient management. The strategy involves pairing an imaging reporter gene with a complementary imaging agent in a system that can be used to measure gene expression or protein interaction or track gene-tagged cells in vivo. Tissue-specific promoters can be used to delineate gene expression in certain tissues, particularly when coupled with an appropriate amplification mechanism. Here we show that the progression elevated gene-3 (PEG-3) promoter, derived from a rodent gene mediating tumor progression and metastatic phenotypes, can be used to drive imaging reporters selectively to enable detection of micrometastatic disease in mouse models of human melanoma and breast cancer using bioluminescence and radionuclide-based molecular imaging techniques. Because of its strong promoter activity, tumor specificity and capacity for clinical translation, PEG-3 promoter-driven gene expression may represent a practical, new system for facilitating cancer imaging and therapy.     

In vivo imaging and tumor therapy with the sodium iodide symporter

There has been great progress in the design of vectors for cancer gene therapy. However, it has been difficult to translate success in the laboratory into clinical practice. A major hurdle in understanding these failures has been the relative difficulty in monitoring repeatedly and non-invasively the biodistribution, gene expression and replication of these viral vector systems. With the advent of molecular imaging technology, this deficiency is being rapidly rectified. A number of reporter genes have been used to monitor gene expression. In this review, we discuss the role of the sodium iodide symporter (NIS) as a reporter and therapeutic gene for cancer gene therapy when combined with various radioactive isotopes.     

Use of bacteria in anti-cancer therapies

While a number of valid molecular targets have been discovered for tumours over the past decade, finding an effective way of delivering therapeutic genes specifically to tumours has proved more problematic. A variety of viral and non-viral delivery vehicles have been developed and applied in anti-cancer gene therapies. However, these suffer from either inefficient and/or short-lived gene transfer to target cells, instability in the bloodstream and inadequate tumour targeting. Recently, various types of non-pathogenic obligate anaerobic and facultative anaerobic bacteria have been shown to infiltrate and selectively replicate within solid tumours when delivered systemically. This has prompted the development of cancer gene therapy protocols that use such bacteria as gene delivery vehicles. Here, we review the evidence for the success of these in pre-clinical models and clinical trials, as single modality treatments and in combination with conventional cancer therapies.     

Comparison of noninvasive fluorescent and bioluminescent small animal optical imaging

Optical imaging is a modality that is cost-effective, rapid, easy to use, and can be readily applied to studying disease processes and biology in vivo. For this study, we used a green fluorescent protein (GFP)- and luciferase-expressing mouse tumor model to compare and contrast the quantitative and qualitative capabilities of a fluorescent reporter gene (GFP) and a bioluminescent reporter gene (luciferase). We describe the relationship between tumor volume, tumor mass, and bioluminescent/fluorescent intensity for both GFP and luciferase. Bioluminescent luciferase imaging was shown to be more sensitive than fluorescent GFP imaging. Luciferase-expressing tumors were detected as early as 1 day after tumor cell inoculation, whereas GFP-expressing tumors were not detected until 7 days later. Both bioluminescent and fluorescent intensity correlated significantly and linearly with tumor volume and tumor weight, as measured by caliper. Compared to bioluminescent imaging, fluorescent imaging does not require the injection of a substrate and may be appropriate for applications where sensitivity is not as critical. Knowing the relative strengths of each imaging modality will be important in guiding the decision to use fluorescence or bioluminescence.     

Local drug and gene delivery through microbubbles and ultrasound

Although gene therapy has great potential as a treatment for diseases, clinical trials are slowed down by the development of a safe and efficient gene delivery system. In this review, we will give an overview of the viral and nonviral vehicles used for drug and gene delivery, and the different nonviral delivery techniques, thereby focusing on delivery through ultrasound contrast agents.The development of ultrasound contrast agents containing encapsulated microbubbles has increased the possibilities not only for diagnostic imaging, but for therapy as well. Microbubbles have been shown to be able to carry drugs and genes, and destruction of the bubbles by ultrasound will result in local release of their contents. Furthermore, ligands can be attached so that they can be targeted to a specific target tissue. The recent advances of microbubbles as vehicles for delivery of drugs and genes will be highlighted.     

Multimodality in vivo molecular-genetic imaging

Multimodality imaging is increasingly being used in molecular-genetic studies in small animals. The coupling of nuclear and optical reporter genes represents the beginning of a far wider application of this technology. Optical imaging and optical reporter systems are cost-effective and time-efficient, they require less resources and space than PET or MRI, and they are particularly well suited for small animal imaging and for in vitro assays to validate different reporter systems. However, optical imaging techniques are limited by depth of light penetration and scatter and do not yet provide optimal quantitative or tomographic information. These issues are not limiting for PET- or MRI-based reporter systems, and PET- and MRI-based animal studies are more easily generalized to human applications. Many of the shortcomings of each modality alone can be overcome by the use of dual- or triple-modality reporter constructs that incorporate the opportunity for PET, fluorescence and bioluminescence imaging. We optimistically expect that some form of tomographic, small animal optical imaging capability will be developed soon, and that this will provide the opportunity for the colocalization of optical signals to anatomical structures provided by tomographic CT and MR imaging.     

Biomaterial-based technologies for brain anti-cancer therapeutics and imaging

Treating malignant brain tumors represents one of the most formidable challenges in oncology. Contemporary treatment of brain tumors has been hampered by limited drug delivery across the blood–brain barrier (BBB) to the tumor bed. Biomaterials are playing an increasingly important role in developing more effective brain tumor treatments. In particular, polymer (nano)particles can provide prolonged drug delivery directly to the tumor following direct intracerebral injection, by making them physiochemically able to cross the BBB to the tumor, or by functionalizing the material surface with peptides and ligands allowing the drug-loaded material to be systemically administered but still specifically target the tumor endothelium or tumor cells themselves. Biomaterials can also serve as targeted delivery devices for novel therapies including gene therapy, photodynamic therapy, anti-angiogenic and thermotherapy. Nanoparticles also have the potential to play key roles in the diagnosis and imaging of brain tumors by revolutionizing both preoperative and intraoperative brain tumor detection, allowing early detection of pre-cancerous cells, and providing real-time, longitudinal, non-invasive monitoring/imaging of the effects of treatment. Additional efforts are focused on developing biomaterial systems that are uniquely capable of delivering tumor-associated antigens, immunotherapeutic agents or programming immune cells in situ to identify and facilitate immune-mediated tumor cell killing. The continued translation of current research into clinical practice will rely on solving challenges relating to the pharmacology of nanoparticles but it is envisioned that novel biomaterials will ultimately allow clinicians to target tumors and introduce multiple, pharmaceutically relevant entities for simultaneous targeting, imaging, and therapy in a unique and unprecedented manner.     

Retrofitting baculoviral vector with Sleeping Beauty transposon system: competent for long-term reporter gene imaging in vivo

Reporter gene imaging is widely used for non-invasively detecting tumorigenesis, trafficking therapeutic cells, and monitoring treatment effect. Baculoviral vectors (BVs) have been utilized as transgenic vectors in the reporter gene imaging systems in recent years. However, BV-mediated report gene imaging can only provide short-term investigation due to its transient transgene expression, which is incompetent for the long-term applications. In the current study, we reconstructed a series of hybrid BVs with several elements, to investigate the feasibility of this hybrid BV-mediated long-term reporter gene imaging in vivo. We showed that with the indispensable assistance of a positive-selection process, hybrid BV containing Sleeping Beauty 100× (SB) transposon system (BV-SB) could significantly prolong the enhanced green fluorescent protein (eGFP) expression for at least 180 days in vitro at nearly 100% eGFP positive percentage and over 10¹¹ arbitrary unit total fluorescence intensity, whereas other hybrid BV-mediated transgene expression gradually faded in only 20 days. Furthermore, BV-SB-mediated eGFP fluorescent reporter gene imaging monitored tumorigenesis in the nude mice for at least 35 days. In addition, we exploited the glucagon-like peptide 1 receptor (glp-1r) gene as a radionuclide reporter gene for in vivo micro-PET imaging. At 50th day post-tumor transplantation, the micro-PET imaging showed considerable radiotracer-receptor-binding in vivo, resulted by stable high level of BV-SB-mediated GLP-1R expression in tumor. In summary, we retrofitted BV with the SB transposon system to make it competent for the long-term reporter gene imaging in vivo, which might broaden the application scopes of BV in the long-term molecular imaging and other biomedicine research fields.

     

Spectroscopic imaging at compact inverse Compton X-ray sources

While K-edge subtraction (KES) imaging is a commonly applied technique at synchrotron sources, the application of this imaging method in clinical imaging is limited although results have shown its superiority to conventional clinical subtraction imaging. Over the past decades, compact synchrotron X-ray sources, based on inverse Compton scattering, have been developed to fill the gap between conventional X-ray tubes and synchrotron facilities. These so called inverse Compton sources (ICSs) provide a tunable, quasi-monochromatic X-ray beam in a laboratory setting with reduced spatial and financial requirements. This allows for the transfer of imaging techniques that have been limited to synchrotrons until now, like KES imaging, into a laboratory environment. This review article presents the first studies that have successfully performed KES at ICSs. These have shown that KES provides improved image quality in comparison to conventional X-ray imaging. The results indicate that medical imaging could benefit from monochromatic imaging and KES techniques. Currently, the clinical application of KES is limited by the low K-edge energy of available iodine contrast agents. However, several ICSs are under development or already in commissioning which will provide monochromatic X-ray beams with higher X-ray energies and will enable KES using high-Z elements as contrast media. With these developments, KES at an ICS has the ability to become an important tool in pre-clinical research and potentially advancing existing clinical imaging techniques.     

Molecular imaging of cell-based cancer immunotherapy

Cell-based cancer immunotherapy represents a new and powerful weapon in the arsenal of anticancer treatments. Non-invasive monitoring of the disposition, migration and destination of therapeutic cells will facilitate the development of cell based therapy. The therapeutic cells can be modified intrinsically by a reporter gene or labeled extrinsically by introducing imaging probes into the cells or on the cell surface before transplant. Various advanced non-invasive molecular imaging techniques are playing important roles in optimizing cellular therapy by tracking cells and monitoring the therapeutic effects of transplanted cells in vivo. This review will summarize the application of multiple molecular imaging modalities in cell-based cancer immunotherapy.     

hNIS-IRES-eGFP dual reporter gene imaging

The human and rodent sodium iodide symporters (NIS) have recently been cloned and are being investigated as potential therapeutic and reporter genes. We have extended this effort by constructing an internal ribosomal entry site (IRES)-linked human NIS (hNIS)-enhanced green fluorescent protein (eGFP) hybrid reporter gene for both nuclear and optical imaging. A self-inactivating retroviral vector, termed pQCNIG, containing hNIS-IRES-eGFP dual reporter gene, driven by a constitutive CMV promoter, was constructed and used to generate RG2-pQCNIG cells and RG2-pQCNIG tumors. 131I-iodide and 99mTcO4-pertechnetate accumulation studies plus fluorescence microscopy and intensity assays were performed in vitro, and gamma camera imaging studies in RG2-pQCNIG and RG2 tumor-bearing athymic rats were performed. RG2-pQCNIG cells expressed high levels of hNIS protein and showed high intensity of eGFP fluorescence compared with RG2 wild-type cells. RG2-pQCNIG cells accumulated Na131I and 99mTcO4- to a 50:1 and a 170:1 tissue/medium ratio at 10 min, compared with 0.8:1.2 tissue/medium ratio in wild-type RG2 cells. A significant correlation between radiotracer accumulation and eGFP fluorescence intensity was demonstrated. RG2-pQCNIG and RG2 tumors were readily differentiated by in vivo gamma camera imaging; radiotracer uptake increased in RG2-pQCNIG but declined in RG2 tumors over the 50-min imaging period. Stomach and thyroid were the major organs of radionuclide accumulation. The IRES-linked hNIS-eGFP dual reporter gene is functional and stable in transduced RG2-pQCNIG cells. Optical and nuclear imaging of tumors produced from these cell lines provides the opportunity to monitor tumor growth and response to therapy. These studies indicate the potential for a wider application of hNIS reporter imaging and translation into patient studies using radioisotopes that are currently available for human use for both SPECT and PET imaging.     

Red-emitting luciferases for bioluminescence reporter and imaging applications

North American firefly Photinus pyralis luciferase, which emits yellow-green light (557 nm), has been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. Luciferase variants with red-shifted bioluminescence and high specific activity can be paired with green-emitting counterparts for use in dual-color reporter assays or can be used alone for in vivo imaging. Beginning with a previously reported red-emitting thermostable mutant and using mutagenesis techniques, we engineered two luciferases with redder emission maxima while maintaining satisfactory specific activities and thermostability. The novel enzymes were expressed in HEK293 cells, where they performed similarly to Promega’s codon-optimized click beetle red luciferase in model reporter assays. When the firefly luciferase variants were codon-optimized and retested using optimized substrate concentrations, they provided 50- to 100-fold greater integrated light intensities than the click beetle enzyme. These results suggest that the novel enzymes should provide superior performance in dual-color reporter and in vivo imaging applications, and they illustrate the importance of codon optimization for assays in mammalian cells.     

Multimodal imaging of stem cell implantation in the central nervous system of mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s). Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting. BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin). MRI on the other hand is a non-invasive technique which is clinically applicable and can be used to precisely locate cellular grafts with very high resolution, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies. In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).