The development of novel targeted tools for use in the early detection and treatment of metastatic breast cancer is critical for reducing mortality associated with this disease. In this current study, a transcriptionally targeted, recombinant adenovirus served as a diagnostic tool by delivering imaging reporter genes to tumor cells, enabling the direct detection and visualization of tumor metastasis in lymph nodes and liver. This system made use of the tumor-specific MUC1 promoter in conjunction with the TSTA system, a two-tiered amplification scheme that has been applied successfully to modify the prostate-specific antigen promoter (10
), the VEGF promoter (29
), pancreatic-specific cholecystokinin type A receptor promoter (30
), and the survivin promoter (31
). Through adaptation of TSTA, mucin-1 promoter activity was amplified up to 260-fold, greatly enhancing its sensitivity and efficiency.
Systemic application of Ad vectors has been limited by their strong tropism for the liver, which diminishes gene delivery to other organs (6
). Over the years much work has focused on abolishing liver transduction of adenovirus through various strategies. Some include modifications to the adenoviral structure (32
), retargeting the virus through use of adapters (34
), and most recently through ablation of FX-facilitated transduction (35
). However, strategies to detarget the adenovirus away from the liver have not shown enhanced transduction to extrahepatic target sites. We and others have shown that transcriptionally targeted adenoviral vectors could be a means to safeguard against unwanted liver transduction (11
). Although a transcriptional targeting strategy will not prevent adenoviral liver infection, it can eliminate viral gene expression in the liver. In this study, we showed that the mucin-1 promoter – driven AdMUC1-TSTA-fl remained transcriptionally silent in the liver due to weak mucin-1 expression in normal liver tissue. Conversely, this cancer-selective imaging Ad can successfully target and produce optical signals to detect breast tumor lesions in the liver following i.v. administration. To localize the metastatic lesions more precisely we did PET-CT scans that enabled us to visualize the heightened glucose metabolism of tumors by the FDG tracer. By combining two molecular imaging techniques that provide metabolic and gene expression information, conferred by FDG-PET and bioluminescence imaging respectively, the specificity of detecting metastases can be greatly enhanced.
Besides strong liver tropism, human adenoviruses exhibit preferential distribution into the lymphatic system (7
). Its lymphotropic properties can be gleaned from historical evidence that the virus was isolated from adenoids, which collect the majority of lymph drainage of upper respiratory tracts (38
). In a recently published report, we were able to successfully target and express PET imaging reporter genes in metastatic prostate cancer tumor cells within draining lymph nodes with a prostate-specific TSTA Ad (8
). This current study further verifies the feasibility of exploiting the inherent lymphotropic properties of Ad to query the sentinel lymph node status in a breast tumor model. Employing an approach that parallels clinical lymphoscintigraphy, the AdMUC1-TSTA-fl viral particles were injected peritumorally. We observed that the tumor-selective firefly luciferase signals correlated directly with the Renilla luciferase signals of the marked tumor cells in the sentinel lymph nodes of the breast tumor – bearing cohort. This finding indicates that AdMUC1-TSTA-fl is specific and efficient at detecting sentinel lymph node metastases. Compared with conventional lymphoscintigraphy and the sentinel lymph node biopsy method, this transcriptionally targeted adenoviral-based method of lymphangiography has the advantage of being a noninvasive imaging modality, obviating the need to harvest draining nodes for histologic analyses, thereby eliminating the risk of side effects associated with sentinel lymph node biopsy.
A recent insightful review by Punglia et al. (39
) examined the findings of 78 randomized clinical trials in the Early Breast Cancer Trialists' Collaborative Group evaluating the extent of surgery and the use of radiation therapy (40
). The definitive message from this thorough review is that improved local control, such as excision to negative margins and the use of additional radiation doses, provided a highly statistically significant improvement in long-term breast cancer survival. The most significant indicators for disease recurrence and poor outcome as reported in this study and others include the number of lymph nodes involved, tumor size and grade, lymphovascular invasion, mitotic index, and Her2 positivity (39
). According to these perspectives, the ability of AdMUC1-TSTA-fl to target lymph node metastasis might provide an additional tool to achieve better local control. Not only can the tumor-selective Ad be used to express various imaging genes for tumor staging, but the same strategy can be applied to express cytotoxic genes to eradicate lymph node metastasis, offering the potential to reduce systemic tumor spread. The TSTA expression system is particularly amenable to simultaneously express multiple transgenes (43
). In particular, we have succeeded in expressing an imaging gene and a therapeutic gene simultaneously using the survivin tumor-specific promoter (31
), the MUC1 promoter (44
), and the prostate-specific antigen promoter.7
We anticipate that a significant challenge to the delivery of gene expression – targeted interventions to the clinic, such as the one described here, would be the immunogenicity of the adenoviral vector (45
). To overcome this limitation, we and others have undertaken several approaches to modify the surface properties of the viral capsid in order to reduce its antigenicity and to enhance its stability in circulation (37
). Other steps taken include transiently dampening the host immune system to prolong vector gene expression (47
). Recently, we have adapted a method to synthesize block copolymers with precise control of chain lengths and compositions (48
). In turn, these synthetic polymers are used to coat the adenovirus by noncovalent electrostatic interactions. Coated adenoviruses have shown significant improvement in their transductional efficiency in cell culture and in animals.8
This polymer coating approach in conjunction with further transductional targeting modifications (34
) could greatly enhance the specificity and performance of our MUC1-expression–targeted Ad in living subjects. Combination use of this system with various transductional targeting strategies would be a vast improvement upon currently available technologies.
In this study, we illustrated in principle the ability of the Ad-based TSTA gene transfer technology to deliver and express imaging reporter genes specifically in breast tumor metastases in living subjects. Due to the low energy of visible light, optical imaging approaches, such as bioluminescence imaging employed in this study, are unable to detect signals beyond a few centimeters of depth as the photons are scattered or absorbed by tissue. We envision the use of PET-CT to be much more appropriate in the clinics and we recently reported the feasibility of employing a parallel Ad-mediated functional PET imaging method to detect prostate cancer nodal metastasis (8
). To continue refining this technology for extension into human studies, we are focused on expanding its targeting capabilities with additional promoters, combining with transductional modifications to increase specificity, and integrating clinically relevant PET-CT approaches. We believe that this system holds great promise and has the potential to significantly impact diagnostic and treatment options for breast cancer patients.