Transcriptionally targeted gene therapy to detect and treat cancer

The greatest challenge in cancer treatment is to achieve the highest levels of specificity and efficacy. Cancer gene therapy could be designed specifically to express therapeutic genes to induce cancer cell destruction. Cancer-specific promoters are useful tools to accomplish targeted expression; however, high levels of gene expression are needed to achieve therapeutic efficacy. Incorporating an imaging reporter gene in tandem with the therapeutic gene will allow tangible proof of principle that gene expression occurs at the correct location and at a sufficient level. Gene-based imaging can advance cancer detection and diagnosis. By combining the cancer-targeted imaging and therapeutic strategies, the exciting prospect of a 'one-two punch' to find hidden, disseminated cancer cells and destroy them simultaneously can potentially be realized.     

Targeted photodynamic therapy agent with a built-in apoptosis sensor for in vivo near-infrared imaging of tumor apoptosis triggered by its photosensitization in situ

Imaging apoptotic cells or tissues after cancer therapy in situ would be a very useful tool for assessing proper treatment conditions and therapeutic outcome. By combining therapeutic and imaging functions, we have designed a multifunctional, membrane-permeable, and cancer-specific agent that triggers and images apoptosis in targeted cells. We chose photodynamic therapy (PDT) as an appropriate cancer treatment modality and caspase 3 as an apoptosis-specific imaging target. This targeted photodynamic therapy agent with a built-in apoptosis sensor (TaBIAS) induces photodamage only to target cells and simultaneously identifies those that are apoptotic by its near-infrared fluorescence. It contains a fluorescent photosensitizer used as an anticancer drug and a cancer-associated folate receptor homing molecule connected to a caspase 3 cleavable peptide linker that has a fluorescence quencher on the opposing site. We demonstrated that PDT-triggered cleavage of the peptide linker by caspase 3, one of the key executioner caspases, results in a detectable increase in fluorescence in folate receptor-overexpressing cancer cells and tumors. The presence of apoptosis was confirmed in vitro by flow cytometry and ex vivo by Apoptag assay, supporting the ability of TaBIAS to specifically induce and image apoptosis in situ.     

Computed tomography in the diagnosis of head and neck diseases]

A total of 106 patients suffering from tumors and inflammatory diseases of the head and neck were investigated. CT was performed in axial and frontal projections using a method of intravenous contrast image enhancement. The purpose of CT was to reveal an abnormal lesion, to detect its initial site, spreading, to make differential diagnosis, and to evaluate therapeutic results. Some limitations of CT in differential diagnosis of small-size tumors were observed. CT can be used for differential diagnosis of tumors and inflammatory processes in the accessory nasal sinuses. CT results were important for a choice of therapeutic methods, radiation therapy design, assessment of efficacy of surgical and radiation therapy, and the detection of tumor recurrences.     

Theranostics: combining imaging and therapy

Employing theranostic nanoparticles, which combine both therapeutic and diagnostic capabilities in one dose, has promise to propel the biomedical field toward personalized medicine. This review presents an overview of different theranostic strategies developed for the diagnosis and treatment of disease, with an emphasis on cancer. Herein, therapeutic strategies such as nucleic acid delivery, chemotherapy, hyperthermia (photothermal ablation), photodynamic, and radiation therapy are combined with one or more imaging functionalities for both in vitro and in vivo studies. Different imaging probes, such as MRI contrast agents (T(1) and T(2) agents), fluorescent markers (organic dyes and inorganic quantum dots), and nuclear imaging agents (PET/SPECT agents), can be decorated onto therapeutic agents or therapeutic delivery vehicles in order to facilitate their imaging and, in so doing, gain information about the trafficking pathway, kinetics of delivery, and therapeutic efficacy; several such strategies are outlined. The creative approaches being developed for these classes of therapies and imaging modalities are discussed, and the recent developments in this field along with examples of technologies that hold promise for the future of cancer medicine are highlighted.     

影像学在肾癌靶向治疗中的临床应用价值-现状与展望

肾癌为最常见的肾脏恶性肿瘤,其易复发及易出现远隔转移,靶向治疗已逐渐成为肾癌治疗中不可或缺的治疗手段。由于RECIST标准评价实体瘤疗效具有明显的局限性,临床实践中常使用电子计算机断层扫描(CT)、磁共振成像(MRI)评价疗效。随着技术的不断发展,很多新颖的影像学手段如动态增强超声(DCE-US)、正电子发射断层显像(PET-CT)等可以更准确更早期地评价及预测靶向治疗的疗效。本文对肾癌靶向治疗的影像学现状及展望进行综述。     

Biomarkers in molecular medicine: cancer detection and diagnosis

In spite of advances in diagnostics and therapeutics, cancer remains the second leading cause of death in the U.S. Successful cancer treatment depends not only on better therapies but also on improved methods to assess an individual's risk of developing cancer and to detect cancers at early stages when they can be more effectively treated. Current cancer diagnostic imaging methods are labor-intensive and expensive, especially for screening large asymptomatic populations. Effective screening strategies depend on methods that are noninvasive and detect cancers in their early stages of development. There is increasing interest and enthusiasm in molecular markers as tools for cancer detection and prognosis. It is hoped that newly discovered cancer biomarkers and advances in high-throughput technologies would revolutionize cancer therapies by improving cancer risk assessment, early detection, diagnosis, prognosis, and monitoring therapeutic response. These biomarkers will be used either as stand-alone tests or to complement existing imaging methods.     

Synthesis of a Novel IR-822-Met near-infrared probe for in vivo tumor diagnosis

Objective

Methionine is a valid target for the treatment of cancer and to achieve in vivo imaging and early diagnosis of tumors, we have synthesized near-infrared (NIR) fluorochrome IR-822-labeled methionine (IR-822-Met).

Results

NIR fluorescent dye IR-822 was conjugated with methionine through its amide bond. It had low toxicity to normal cell/tissues. In vitro and in vivo studies demonstrated its high targeting capability to tumors. The results support the potential of using ligand-modified methionine probe for tumor diagnosis and targeted therapy. The probe also exhibited good photostability, and excellent cell membrane permeability.

Conclusion

IR-822-Met is a promising imaging agent for tumor diagnosis, especially in their early stage.

     

In vivo fluorescence lifetime imaging monitors binding of specific probes to cancer biomarkers

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the "image and treat" concept, especially for early evaluation of the efficacy of the therapy.     

Molecular imaging of gene expression and efficacy following adenoviral-mediated brain tumor gene therapy

Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity. Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer gene therapy protocols by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules. In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (yCD) along with an optical reporter gene (luciferase). Following intratumoral injection of the vector into orthotopic 9 L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill, respectively. In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression. MR images revealed significant reduction in tumor growth rates associated with yCD/5-fluorocytosine (5FC) gene therapy. Significant increases in mean tumor diffusion values were also observed during treatment with 5FC. Moreover, spatial heterogeneity in tumor diffusion changes were also observed revealing that diffusion magnetic resonance imaging could detect regional therapeutic effects due to the nonuniform delivery and/or expression of the therapeutic yCD transgene within the tumor mass. In addition, in vivo bioluminescence imaging detected luciferase gene expression, which was found to decrease over time during administration of the prodrug providing a noninvasive surrogate marker for monitoring gene expression. These results demonstrate the efficacy of the yCD/5FC strategy for the treatment of brain tumors and reveal the feasibility of using multimodality molecular and functional imaging for assessment of gene expression and therapeutic efficacy.     

磁性纳米技术在癌症基因治疗中的应用

癌症基因组的最新进展使直接针对癌症基因进行治疗具有极大的可能性。然而,需要新的基因传递方法使这种潜力向临床应用转化,为治疗病人服务。磁性纳米技术是通过外部磁场选择性地高效传递治疗基因,还能同时用影像监测体内的传递过程。相比传统的基因传递方法,这种技术能明显提高人类移植肿瘤和不同的内脏器官如肝、肾及中枢神经系统的基因传递效率。因此,磁性纳米技术使活体内癌症的基因治疗进入到新的前沿领域。     

Abnormal alternative splicing promotes tumor resistance in targeted therapy and immunotherapy

Abnormally alternative splicing events are common hallmark of diverse types of cancers. Splicing variants with aberrant functions play an important role in cancer development. Most importantly, a growing body of evidence has supported that alternative splicing might play a significant role in the therapeutic resistance of tumors. Targeted therapy and immunotherapy are the future directions of tumor therapy; however, the loss of antigen targets on the tumor cells surface and alterations in drug efficacy have resulted in the failure of targeted therapy and immunotherapy. Interestingly, abnormal alternative splicing, as a strategy to regulate gene expression, is reportedly involved in the reprogramming of cell signaling pathways and epitopes on the tumor cell surface by changing splicing patterns of genes, thus rendering tumors resisted to targeted therapy and immunotherapy. Accordingly, increased knowledge regarding abnormal alternative splicing in tumors may help predict therapeutic resistance during targeted therapy and immunotherapy and lead to novel therapeutic approaches in cancer. Herein, we provide a brief synopsis of abnormal alternative splicing events in cancer progression and therapeutic resistance.     

In vivo molecular target assessment of matrix metalloproteinase inhibition

A number of different matrix metalloproteinase (MMP) inhibitors have been developed as cytostatic and anti-angiogenic agents and are currently in clinical testing. One major hurdle in assessing the efficacy of such drugs has been the inability to sense or image anti-proteinase activity directly and non-invasively in vivo. We show here that novel, biocompatible near-infrared fluorogenic MMP substrates can be used as activatable reporter probes to sense MMP activity in intact tumors in nude mice. Moreover, we show for the first time that the effect of MMP inhibition can be directly imaged using this approach within hours after initiation of treatment using the potent MMP inhibitor, prinomastat (AG3340). The developed probes, together with novel near-infrared fluorescence imaging technology will enable the detailed analysis of a number of proteinases critical for advancing the therapeutic use of clinical proteinase inhibitors.     

单克隆抗体ND-1-量子点荧光探针对大肠癌细胞的特异成像研究

目的制备抗人大肠癌单克隆抗体ND-1的量子点荧光探针,实现对大肠癌细胞的靶向成像。方法采用共价偶联方法,以1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)和N-羟基硫代琥珀酰亚胺(NHS)为缩合剂,通过在反应体系中加入不同摩尔比例的单克隆抗体ND-1和游离量子点QD605进行条件优化,制备偶联产物ND-1-QD605荧光探针;利用荧光光谱扫描技术对ND-1-QD605进行光学特性表征,并检测其抗光漂白能力;利用免疫荧光方法检测ND-1-QD605对大肠癌细胞的靶向结合能力。结果在量子点QD605与单克隆抗体ND-1摩尔比1:40条件下,可实现二者的高效偶联;荧光光谱分析显示ND-1-QD605保留了游离量子点QD605优良的荧光特性;在激发光照射1h内,ND-1-QD605荧光强度未发生明显改变;荧光显微镜观察可见该探针能够与表达有相应抗原LEA的人大肠癌CCL187细胞特异性结合,呈现高灵敏度、特异性荧光成像。结论制备的单克隆抗体ND-1的量子点荧光探针具有大肠癌细胞靶向成像能力,有望为大肠癌的体内靶向成像研究和临床诊断提供新方法。     

In vivo imaging of siRNA delivery and silencing in tumors

With the increased potential of RNA interference (RNAi) as a therapeutic strategy, new noninvasive methods for detection of siRNA delivery and silencing are urgently needed. Here we describe the development of dual-purpose probes for in vivo transfer of siRNA and the simultaneous imaging of its accumulation in tumors by high-resolution magnetic resonance imaging (MRI) and near-infrared in vivo optical imaging (NIRF). These probes consisted of magnetic nanoparticles labeled with a near-infrared dye and covalently linked to siRNA molecules specific for model or therapeutic targets. Additionally, these nanoparticles were modified with a membrane translocation peptide for intracellular delivery. We show the feasibility of in vivo tracking of tumor uptake of these probes by MRI and optical imaging in two separate tumor models. We also used proof-of-principle optical imaging to corroborate the efficiency of the silencing process. These studies represent the first step toward the advancement of siRNA delivery and imaging strategies, essential for cancer therapeutic product development and optimization.     

SELEX技术在胰腺癌分子诊断及靶向治疗中应用的研究进展

胰腺癌由于起病隐匿,早期诊断率较低,临床治疗效果差,是目前预后最差的恶性肿瘤之一。目前,临床上尚缺乏有效的非创伤早期筛查胰腺癌的手段,因而胰腺癌的早期诊断和治疗显得尤为重要。近年来,指数富集配基的系统进化(SELEX)技术以其在其他疾病中所表现的应用价值为疾病的诊治提供了一个新的途径。对于缺乏有效确诊手段,发病隐匿且病死率高的胰腺癌而言,SELEX技术基于胰腺癌发病的分子机制,可以筛选出特异结合于胰腺癌分子靶标的适配体,对筛选所得适配体进一步化学修饰,可以实现分子水平成像及靶向治疗,进而达到胰腺癌早期诊治的目的,具有重要的临床意义。本文就SELEX技术在胰腺癌分子诊断及靶向治疗中的应用研究进展进行了综述。     

Nanotechnology and tumor imaging: seizing an opportunity

Nanoparticles, labeled with a signaling moiety for in vivo imaging, and one or more ligands for molecularly targeted specificity, hold considerable promise in oncology. Nanoparticles can serve as modular platforms, from which a wide variety of highly sensitive and specific imaging agents can be created. For example, many hundreds or thousands of atoms that provide imaging signals, such as radioisotopes, lanthanides, or fluorophores, can be attached to each nanoparticle, to form imaging agents that would provide higher sensitivity that can be obtained from agents based on small molecules. Similarly, many copies of targeted ligands can be attached to nanoparticles to markedly increase specific binding. Drugs or therapeutic isotopes can be added to create multifunctional nanoparticles. Appropriately labeled and targeted nanoparticles could lead to a paradigm change in which cancer detection, diagnosis, and therapy are combined in a single molecular complex.     

Quantitative monitoring of mouse lung tumors by magnetic resonance imaging

Primary lung cancer remains the leading cause of cancer-related death in the Western world, and the lung is a common site for recurrence of extrathoracic malignancies. Small-animal (rodent) models of cancer can have a very valuable role in the development of improved therapeutic strategies. However, detection of mouse pulmonary tumors and their subsequent response to therapy in situ is challenging. We have recently described MRI as a reliable, reproducible and nondestructive modality for the detection and serial monitoring of pulmonary tumors. By combining respiratory-gated data acquisition methods with manual and automated segmentation algorithms described by our laboratory, pulmonary tumor burden can be quantitatively measured in approximately 1 h (data acquisition plus analysis) per mouse. Quantitative, analytical methods are described for measuring tumor burden in both primary (discrete tumors) and metastatic (diffuse tumors) disease. Thus, small-animal MRI represents a novel and unique research tool for preclinical investigation of therapeutic strategies for treatment of pulmonary malignancies, and it may be valuable in evaluating new compounds targeting lung cancer in vivo.     

Improved targeting of ligand-modified adenovirus as a new near infrared fluorescence tumor imaging probe

E1/E3-deleted Adenovirus 5 (Ad.5) possesses a great potential in gene therapy because of its high efficacy in gene transfer and low toxicity. Studies have shown that Coxsackie-Adenovirus receptor (CAR) is the determinant factor for the targeting of Adenovirus vectors. To extend the natural targeting of Ad to low CAR expressing tumors, we covalently attached folic acid (FA) to E1/E3-deleted Ad.5 capsids. Near-infrared (NIR) fluorescent dye ICG-Der-02 was subsequently conjugated with FA-Ad particles for in vivo imaging. The cell experiments and acute toxicity studies demonstrated the low toxicity of FA-Ad-ICG02 to normal cell/tissues. The dynamic behavior and targeting ability of FA-Ad-ICG02 to different tumors were investigated by NIR fluorescence imaging. In vitro and in vivo studies demonstrated its high targeting capability to CAR or FR positive tumors. The results support the potential of using ligand-modified Ad probe for tumor diagnosis and targeted therapy.     

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.     

Using genetically engineered mouse models to validate candidate cancer genes and test new therapeutic approaches

Genetically engineered mouse models (GEMMs) have contributed greatly to the field of cancer research. In contrast to tumor cell transplantation models, GEMMs have the potential to capture both the cell-intrinsic and cell-extrinsic factors that drive de novo formation of autochthonous tumors and their progression toward metastatic disease. In addition, GEMMs provide experimentally tractable in vivo platforms for validating candidate cancer genes, determining therapy efficacy, and defining mechanisms of drug resistance. Studies in GEMMs of human cancer provide new insight in the molecular biology of cancer and contribute to development of novel therapeutic strategies that may ultimately lead to more cures rather than temporal remissions.