Astrocyte Elevated Gene-1 Promoter-Mediated Imaging of Metastatic Prostate Cancer

We describe a method for detecting prostate cancer, whether localized or widespread and metastatic to soft tissues and bone. This is based on the concept of molecular-genetic imaging that has the potential to guide patient management in a personalized manner. It involves the non-invasive visualization of certain events such as gene expression in vivo in real-time. The technology relies on placing imaging reporters under the control of the astrocyte elevated gene-1 promoter (AEG-Prom), which is selectively active in malignant tissue. AEG-Prom is the promoter of astrocyte-elevated gene-1 (AEG-1), a human gene associated with cancer development and progression. AEG-Prom drives tumor-specific gene expression in a wide spectrum of human cancer cell lines. Based on the promoter’s tumor-specific activity, in part driven by the Ha-ras and c-myc oncogenes, we have developed a systemic, nanoparticle-based delivery method for imaging prostate cancer metastasis. We generated AEG-Prom-driven firefly luciferase (Luc) and Herpes Simplex Virus 1-thymidine kinase (HSV1-tk) reporter constructs, pAEG-Luc and pAEG-HSV1tk, respectively, and delivered them in vivo using a nonviral vehicle, linear polyethyleneimine (l-PEI). Experimental metastasis PC3-ML model of human prostate cancer (PCa) along with controls (Ctrl) were systemically delivered with pAEG-Luc/PEI polyplex followed by serial bioluminescence imaging (BLI). Imaging results were iii confirmed by immunohistochemistry and gross pathology. Similarly, pAEG-HSV1tk/PEI was delivered to PCa and Ctrl animals followed by serial SPECT-CT imaging with [125I]FIAU as the radiotracer. Translational potential of the AEG-Prom-mediated imaging system was assessed through its comparison with the emerging clinical standards, positron emission tomography (PET) with [18F]fluorodeoxyglucose (FDG) and [18F]sodium fluoride (NaF). Both BLI and SPECT-CT imaging demonstrated tumor-specific AEG-Prom activity in the PCa models, but not in the Ctrl. Histological analysis confirmed the localization of reporter gene expression in the metastatic nodules. Systemic metastasis within both soft tissue and bone was detected and localized based on whole body SPECT-CT images, which compared favorably to FDG-PET and NaF-PET imaging results. In conclusion, through systemic, nanoparticle-based delivery of the AEG-Prom driven imaging constructs, lesions can be identified through BLI and SPECT-CT in the PC3-ML murine model at high sensitivity. Ultimately the intent of this technology will be to utilize a radionuclide-based imaging system for clinical translation. Because of its strong promoter activity, tumor-specificity (in part driven by c-Myc oncogene) and systemic delivery using a nonviral vehicle, the AEG-Prom-mediated vehicle may represent a new system to image and even treat a variety of cancers directly and specifically

Astrocyte Elevated Gene-1 Promoter-Mediated Imaging of Metastatic Prostate Cancer

We describe a method for detecting prostate cancer, whether localized or widespread and metastatic to soft tissues and bone. This is based on the concept of molecular-genetic imaging that has the potential to guide patient management in a personalized manner. It involves the non-invasive visualization of certain events such as gene expression in vivo in real-time. The technology relies on placing imaging reporters under the control of the astrocyte elevated gene-1 promoter (AEG-Prom), which is selectively active in malignant tissue. AEG-Prom is the promoter of astrocyte-elevated gene-1 (AEG-1), a human gene associated with cancer development and progression. AEG-Prom drives tumor-specific gene expression in a wide spectrum of human cancer cell lines. Based on the promoter’s tumor-specific activity, in part driven by the Ha-ras and c-myc oncogenes, we have developed a systemic, nanoparticle-based delivery method for imaging prostate cancer metastasis. We generated AEG-Prom-driven firefly luciferase (Luc) and Herpes Simplex Virus 1-thymidine kinase (HSV1-tk) reporter constructs, pAEG-Luc and pAEG-HSV1tk, respectively, and delivered them in vivo using a nonviral vehicle, linear polyethyleneimine (l-PEI). Experimental metastasis PC3-ML model of human prostate cancer (PCa) along with controls (Ctrl) were systemically delivered with pAEG-Luc/PEI polyplex followed by serial bioluminescence imaging (BLI). Imaging results were iii confirmed by immunohistochemistry and gross pathology. Similarly, pAEG-HSV1tk/PEI was delivered to PCa and Ctrl animals followed by serial SPECT-CT imaging with [125I]FIAU as the radiotracer. Translational potential of the AEG-Prom-mediated imaging system was assessed through its comparison with the emerging clinical standards, positron emission tomography (PET) with [18F]fluorodeoxyglucose (FDG) and [18F]sodium fluoride (NaF). Both BLI and SPECT-CT imaging demonstrated tumor-specific AEG-Prom activity in the PCa models, but not in the Ctrl. Histological analysis confirmed the localization of reporter gene expression in the metastatic nodules. Systemic metastasis within both soft tissue and bone was detected and localized based on whole body SPECT-CT images, which compared favorably to FDG-PET and NaF-PET imaging results. In conclusion, through systemic, nanoparticle-based delivery of the AEG-Prom driven imaging constructs, lesions can be identified through BLI and SPECT-CT in the PC3-ML murine model at high sensitivity. Ultimately the intent of this technology will be to utilize a radionuclide-based imaging system for clinical translation. Because of its strong promoter activity, tumor-specificity (in part driven by c-Myc oncogene) and systemic delivery using a nonviral vehicle, the AEG-Prom-mediated vehicle may represent a new system to image and even treat a variety of cancers directly and specifically

Enhanced 5-ALA Induced Fluorescence in Hormone Secreting Pituitary Adenomas

Introduction Cushing’s Disease (CD) is caused by millimeter-sized corticotropinomas (microadenomas) that lead to supraphysiological levels of glucocorticoid. Up to 40% of microadenomas are not visualized on gold-standard MR imaging. Pituitary adenomas metabolize exogenous 5-ALA (an endogenous metabolite) to protoporphyrin IX (PpIX) at rates 20-50 times higher compared with normal tissues. PpIX intensely fluoresces red (635nm) when excited with blue light (375-440nm), enabling its use as an intraoperative fluorescence imaging agent. 5-ALA is now an FDA approved prodrug. We examined the efficacy of ALA-induced-PpIX fluorescence in human derived adenomatous and normal pituitary samples. We explored the modulation of PpIX conversion with CRH or dexamethasone (DEX), and subcellular localization of PpIX. Methods We used flow cytometry for PpIX intensity analysis. A human-derived corticotropinoma, it’s adjacent normal gland, murine normal pituitary cells, and AtT20 cells were incubated with 5-ALA (300 nM) with/without DEX (1µM) or CRH (50nM). For confocal microscopy, live cells imaged for PpIX (405nm/615nm) and mitochondrial (550nm/615nm) fluorescence. Results We found a 10-fold-increase in 5-ALA induced PpIX fluorescence intensity in human-derived adenomatous compared to adjacent normal pituitary tissue (n=1, p\u3c0.05). AtT-20 cell lines (n=6, p\u3c0.05) fluoresced 7-fold more intensely compared to normal murine pituitary tissue (n=3, p\u3c0.05). The addition of DEX, before or after 5-ALA exposure, increased the fluorescence intensity by 31% (n=4, p\u3c0.05). The addition of CRH did not have a significant effect on 5-ALA fluorescence (n=3, p\u3c0.05). We saw localization of 5-ALA to mitochondria, and mitochondrial disruption in 5-ALA treated At-T20s. Conclusions Our results support the use of 5-ALA for fluorescence guided resection in hormone secreting microadenomas. The supraphysiological levels of glucocorticoids, as seen in CD, may enhance the 5-ALA fluorescence in corticotropinomas. We confirm the mitochondrial localization and disruption by 5-ALA, a basis for photodynamic therapy