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Replication-competent oncolytic viral therapies have shown great promise preclinically and in clinical trials for the treatment of various cancers. They are able to preferentially and selectively propagate in cancer cells, consequently destroying tumor tissue via cell lysis, while leaving noncancerous tissues unharmed. Currently, biopsy is the gold standard for monitoring of viral tumor colonization and oncolysis. This may be feasible in preclinical or early clinical trials; however, a noninvasive method facilitating ongoing monitoring of viral therapy is needed for human studies. The tracking of viral delivery could give clinicians the ability to assess the biodistribution of oncolytic viruses to ensure safety and correlation with treatment efficacy. This work centers on the construction and testing of a VACV strain, GLV-1h153, carrying the human sodium iodide symporter (hNIS) as a marker gene for non-invasive tracking of virus by imaging. Thus, this project aimed to help develop imaging techniques for use in clinical trials of oncolytic viral therapy. Further, the feasibility and effectiveness of virally induced targeted radiotherapy as an anti-cancer strategy was also investigated. hNIS is an intrinsic plasma membrane protein which mediates the active transport and concentration of iodide in the thyroid gland and some extra-thyroidal tissues. It is also one of several human genes currently being used as reporters in preclinical studies and has already been used in clinical studies for imaging viral replication in prostate cancer. hNIS gene transfer via viral vector may allow infected tumor cells to concentrate several carrier-free radionuclide probes such as Iodide-124 (124I), Iodide-131 (131I), and 99m-Technecium Pertechtenate (99mTcO4), which have long been approved for human use. hNIS also has the advantage of being of human origin thus minimizing immunogenicity, and its transporter based system allows intracellular signal amplification. GLV-1h153 was tested in pancreatic adenocarcinoma cell line PANC-1. GLV-1h153 infected, replicated within, and killed PANC-1 cells in cell culture as efficiently as GLV-1h68 and provided dose-dependent levels of hNIS transgene expression in infected cells. Immunofluorescence detected successful transport of the protein to the cell membrane prior to cell lysis, which enhanced dose and time-dependent intracellular uptake of 131I. In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts. Tumor infection by virus was confirmed via optical imaging and histology. GLV-1h153 further facilitated deep tissue imaging of virus replication in tumors via Iodide-124I positron emission tomography (PET) as well as 99mTcO4-mediated gamma scintigraphy. This was possible with both intratumoral and intravenous injection of the virus with radiouptake retained as long as 24 and 48 hours after radiotracer injection. PET image quantitation of radiouptake in tumors was found to correlate well with tissue radiouptake counts. Autoradiography of GLV-1h153-infected tumors revealed a need for presence of virus (visualized with green fluorescent protein expression), viable tissue, and adequate blood flow to enhance radiouptake in tumors. Dosimetric analysis of uptake in infected tumors displayed potential for therapeutic doses of radiotherapy to be delivered systemically to tumors. When GLV-1h153 was combined with 131I for treatment, a modest additive effect was seen as compared to GLV-1h153 alone. Therefore, GLV-1h153 is a promising new candidate for treating pancreatic cancer and noninvasively imaging viral therapy. These findings warrant further investigation into possible long term monitoring of viral therapy, as well as synergistic or additive effects of radioiodine combined with this novel treatment and imaging modality.
Motivation and Aim:
Cardiovascular disease has been the leading cause of mortality and morbidity throughout the world. In developed countries, cardiovascular diseases are already responsible for a majority of deaths and will become the pre-eminent health problem worldwide (1,2). Rupture of atherosclerotic plaque accounts for approximately 70% of fatal acute myocardial infarction and sudden heart deaths. Conventional criterias for the diagnosis of “vulnerable plaques” are calcified nodules, yellow appearance of plaque, a thin cap, a large lipid core, severe luminal stenosis, intraplaque hemorrhage, inflammation, thrombogenicity, and plaque injury (3-5).
Noninvasive diagnosis of vulnerable plaque still remains a great challenge and a huge research prospect, which triggered us to investigate the feasibility of PET imaging on the evaluation of atherosclerosis. Nuclear imaging of atherosclerosis, especially co-registered imaging modalities, could provide a promising diagnostic tool including both anatomy and activities to identify vulnerable atherosclerotic plaque or early detection of inflammatory endothelium at risk. Furthermore, the development of specific imaging tracers for clinical applications is also a challenging task. The aim of this work was to assess the potential of novel PET imaging probes associated with intra-plaque inflammation on animal models and in human respectively.
Methods
In this work, several molecular imaging modalities were employed for evaluation of atherosclerosis. They included Positron emission tomography / Computed tomography (PET/CT) for human studies, and micro-PET, autoradiography and high-resolution magnetic resonance imaging (MRI) for animal studies. Radiotracers for PET imaging included the glucose analogue 18F-Fluorodeoxyglucose (18F-FDG), the somatostatin receptor avide tracer 68Ga-DOTATATE, and the Gallium-68 labeled fucoidan (68Ga-Fucoidan), which was developed as a PET tracer to detect endothelial P-selectin, which overexpressed at early stage of atherosclerosis and endothelial overlying activated plaque. Tracer’s capabilities were firstly assessed on cellular level in vitro. Subsequently, Animal studies were conducted in two animal models: 1, Apolipoprotein E (ApoE-/-) mice having severe atherosclerotic plaque; 2, Lipopolysaccharide (LPS) -induced mice for receiving acute vascular inflammation. Corresponding analyses on protein and histological level were conducted as well to confirm our results.
In human study, 16 patients with neuroendocrine tumors (NETs) were investigated on imaging vascular inflammation. These patients had undergone both 68Ga-DOTATATE PET/CT and 18F-FDG PET/CT for staging or restaging within 6 weeks. 16 patients were randomized into two groups: high-risk group and low-risk group. Uptake ratio of both tracers from two groups were compared and correlated with common cardiovascular risk factors.
Results and Conclusion
In murine study, the expression of somatostatin receptor 2, which is the main bio-target of 68Ga-DOTATATE on macrophage/monocyte was confirmed by flow cytometry and immunohistochemistry. Prospectively, high specific accumulation of 68Ga-DOTATATE to the macrophage within the plaques was observed in aorta lesions by autoradiography and by micro-PET. In study with 68Ga-fucoidan, a strong expression of P-selectin on active endothelium overlying on inflamed plaque but weaker on inactive plaques was confirmed. Specific focal uptake of 68Ga-fucoidan were detected at aorta segments by micro-PET, and correlated with high-resolution magnetic resonance imaging (MRI), which was used to characterize the morphology of plaques. 68Ga-fucoidan also showed a greater affinity to active inflamed plaque in comparison of inactive fibrous plaque, which was assessed by autoradiography. Specificity of 68Ga-DOTATATE and 68Ga-fucoidan were confirmed by ex-vivo blocking autoradiography and in vivo blocking PET imaging respectively.
In human study, focal uptake of both 18F-FDG and 68Ga-DOTATATE was detected. Analyzing concordance of two tracers’ uptake ratio, Out of the 37 sites with highest focal 68Ga-DOTATATE uptake, 16 (43.2%) also had focal 18F-FDG uptake. Of 39 sites with highest 18F-FDG uptake, only 11 (28.2%) had a colocalized 68Ga-DOTATATE accumulation. Correlated tracers’ uptake and calcium burden and risk factors, Mean target-to-background ratio (TBR) of 68Ga-DOTATATE correlated significantly with the presence of calcified plaques (r=0.52), hypertension (r=0.60), age (r=0.56) and uptake of 18F-FDG (r=0.64). TBRmean of 18F-FDG correlated significantly only with hypertension (r=0.58; p<0.05). Additionally, TBRmean of 68Ga-DOTATATE is significant higher in the high risk group while TBRmean of 18F-FDG is not.
In conclusion, we evaluated vascular inflammation of atherosclerosis non-invasively using the two PET tracers: 68Ga-DOTATATE and 68Ga-Fucoidan. 68Ga-DOTATATE show specific affinity to infiltrated macrophage within the plaques. 68Ga-Fucoidan may hold the potential to discriminate between active and inactive atherosclerotic plaques in terms of variant accumulation on different-types of plaques. PET as leading molecular imaging technique provides superiority in assessing cellular activity, which is pivotal for understanding internal activity of atherosclerotic plaques. Since diagnosis of atherosclerosis is a complex and multi-dimensional task. More integrated imaging technology such as PET/MRI, faster imaging algorithm, more efficient radiotracer are required for further development of atherosclerosis imaging,