HTERT Promoter-targeted Optical Imaging In Tumor Diagnosis And Therapy Response Evaluation

Background and ObjectiveMalignant tumors are among the most severe diseases that threaten human health. Early detection of malignant tumors and their metastasis is a vital factor for patients'prognosis. In the past several decades, the world has seen a dramatic improvement in tumor detection by medical imaging techniques. Ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) are among the most common and effective tools that are used to locate and image tumors. However, these tools are unable to differentiate malignant tumors from benign ones, and also have limits in their targets size. Positron emission tomography (PET), when combined with proper target agent, is better to identify malignant tumors than the above methodologies. However, the choosing of PET target reporter and injection of matched marker agent can be a hurdle for its application. For this reason, tumor-specific imaging that allows for the definitive identification of malignant tissues and the differentiation between tumor and their surrounding normal tissues is of considerable value in the diagnosis and treatment of human cancers. Meanwhile, the tumor-specific imaging is also needed for the evaluation of tumor therapies, especially for the recently explored tumor gene therapy.Reporter gene mediated tumor specific optical iamging is a recently developed technology for tumor research. Following the intensive development of bioengineering and super sensitive optical device that can catch light signal that is out of the visible spectrum, this new imaging strategy employ variours of gene tranfering technique, to genetically label the tumor cells with collor coding reporter genes, such as fluorescent proteins genes and luciferase genes. Special super-sensitive charge-coupled device (CCD) camera is used to capture the fluorescent signals or bioluminescence signals from the labelled cells in the living intact animal in a real-time, noninvasive manner. This technology draws the attentions of tumor researchers as soon as its emerging, because of the following advantages. 1. Using special tumor biomarker targeting elements, tumor cells can be specifically tagged with reporter gene. For this first time, malignant tisssues can be differentiated with benign ones. 2. By optimized gene transfering tool, such as lentivirus, not only tumor cells, but also tumor stem cells, stem-like cells, and other silent cells can be labelled and imaged or treated. 3. This technolopgy involves no invasiveness, no contrast agent and can be achieved in real-time manner.How to target the reporter gene in malignant tumors cells is the most vital part of this technology. Hence, selecting a tumor biomarker element is of immense importance for the imaging model. In the past, many tumor-associated antigen promoters, including CEA, AFP and PSA promoters were used as tumor specific promoter to conduct diagnostic or therapeutic experiment, some of which were very strictly tissue-selective. However, these tissue-specific promoters are restricted to very few tumor types, thus limited the scope of their application as tumor specific promoters.Telomerase was commonly recognized as a unique and most effective anticancer target and was widely studied in tumor diagnosis and treatment. The human telomerase catalytic subunit (hTERT), as telomerase's rate-limiting component, is expressed in most malignant tumors while was not found in normal somatic cells. This universal tumor specificity of hTERT provides an opportunity of using it in tumor diagnosis and treatment. Promoter deletion analysis has identified a 260-bp region, containing binding sites of other transcriptive factors, as the core region of hTERT promoter. Within this core region, a transcription factor-binding sites (E-box) was identified that can bind a basic helix-loop-helix zipper (bHLHZ) encoded by the Myc family, and up-regulate hTERT expression. This finding makes it possible to modify hTERT promoter as a high efficient tumor-specific promoter, which may be utilized in tumor-targeted diagnosis and therapy.Combining a tumor targeting element with an in vivo imaging platform, in the current study we created a tumor specific imaging model that allowing us to visualize tumors in living organism, and exlore its use in early stage diagnosis and therapy reaction evaluation.Material and methods1. hTERT promoter designing and function testWe optimized the promoter sequence based on preivous studies. The modified hTERT promoter sequence was synthesized and subcloned into a standard promoter function test vector pGL3-basic. Plasmids that contain CMV, SV40, and original hTERT promoter were also constructed as controls. A dual luciferase assay was conducted to test the optimized hTERT promoter function in hTERT-positive and–negative tumor cells, as well as in primarily cultured human fibroblast cells. The hTERT promoter's target effect will be analyzed.2. Study of optimized hTERT promoter-based tumor-targetting optical imaging in tumor diagnosisThe third generation lentivirus system was used as a gene transfering back bone plasmid, and a GFP gene was cloned into its multiple clone sites, downstream of a CMV promoter, as a control lentiviral vector (pLenti-CMVp-GFP). Then the CMV promoter was replaced by the optimized hTERT promoter, forming a vector containing optimized hTERT promoter driving the expression of GFP (pLenti-hTERTp-GFP). A four-plasmids packaging system was used to produce high-titre lentiviru and a fluorescent quantitative PCR strategy was adopted to determine the viral titre. For in vitro study, lentivirus was used to infect hTERT-positive and–negative tumor cells, as well as in primarily cultured human fibroblast cells. Expression of GFP was observed by laser confocal microscopy. For in vivo study, nude mice model subcutaneously injetcted with telomerase-positive and -negative tumor were constructed and the above two kinds of lentivirus were used to intratumorally infect mice. An in vivo optical imaging system was used to observe the targetted tumor imaging effect of lentivirus. The GFP protein was furtherly examined by postmortem biopsy.3. Study of optimized hTERT promoter-based tumor-targetting optical imaging in real-time tumor therapy evaluationA cytosine deaminase (CD) gene was cloned into pLenti-hTERTp-CD/GFP, downstream of hTERT promoter, as a therapeutic vector (pLenti-hTERTp-CD/GFP), the pLenti-hTERTp-GFP was used as negative comtrol. A four-plasmids packaging system was used to produce high-titre lentiviru and a fluorescent quantitative PCR strategy was adopted to determine the viral titre. For in vitro study, the above lentivirus were used to infect telomerase-positive and -negative tumor cells, and the CD and GFP expression were confirmed. Then the CD prodrug, 5-FC, was added into the lentivirus-infected cells, and a MTT study was carried out to examine cell survival. This lentivirus'therapeutic effect on hTERT-positive tumor cells was confirmed bu in vitro study. In vivo study was conducted using therapeutic lentiviru to infect tumor-bearing mice, folowed by 30 days of tumor growing pattern observation, both by measuring tumor volum and observing GFP signals on a whole-body optical imaging system. Postmortem biopsy was conducted to confirm the tumor-specific expression of suicidal gene CD and reporter gene GFP.ResultsA 295-bp optimized hTERT promoter was synthesized and comfirmed. Promoter function was tested in a pGL3-baic system by dual luciferase assay. Results indicated that optimized hTERT promoter can drive the expression of luciferae strictly in hTERT-positive tumor cells. It showed equal activity to CMV and SV40 promoters, and even slightly higher activity, compared with the original hTERT promoter.For the tumor diagnosis study, lentivirus containing optimized hTERT promoter expressed GFP strictly in telomerase-positive tumor cell lines, with intensive fluorescence stably lasting for 30 days. While the control lentivirus containing CMV promoter expressed GFP both in hTERT-positive and -negative cell lines. In vivo study indicated that after infected with lentivirus, all the hTERT-positive tumors showed intensive fluorescence, which increased in scale and intensity as tumors grow. While all the in the control lentivirus infected tumors, both hTERT-positive and -negative tumors showed high level fluorescence.For the therapy evaluztion study, in the therapeutic lentivirus infected cell lines, RT PCR, western blot and laser confocal microscope indicated that only hTERT-positive tumor cells had CD and GFP expression. MTT cell survival assay indicated that therapeutic lentivirus-infected hTERT-positive tumor cells showed significantly decreased cell survival, compared with blank control lentivirus infected cells, which showed no decrease in cell survival. In vivo study indicated that during the 30 days observation, hTERT-positive tumors exhibited significant growth supression, compared with control tumors that were infected with blank lentivirus. This growth supression was confirmed both by optical in vivo imaging and by tumor volume measurement. Postmortem biopsy further confirmed the expression of GFP and CD in supressed tumors.Conclusions1. By inserting several E-boxes into the original hTERT promoter sequence, the modified hTERT promoter was proved to be strictly hTERT specific, and have equal activity to CMV, SV40 promoter, and slightly higher activity than original hTERT promoter.2. The GFP-coding diagnostic lentivirus can strictly infect and express GFP in hTERT-positive cell lines or in vivo tumors. Tumors and its growing patterns can be visualized in a real time noninvasive manner.3. The CD and GFP-coding therapeutic lentivirus can express CD and GFP strictly in hTERT-positive cell lines or in vivo tumors. CD protein is able to convey significant lethal effect to hTERT-positive tumor cells in vitro and in vivo. The long term tumor supression process can be visualized by in vivo optical imaging. And the fluorescence images precisely indecated the substantial size volum of the tumors, as well as the tumor growing patterns.The above conclusions first elucidated a lentivirus-based, hTERT-targeted tumor specific imaging and therapy model. It provided data for preclinical use of virus-mediated tumor-specific diagnosis and gene therapy in a real-time and non-invasive method through optical imaging system.