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Background: Gene transfer to malignant sites using human adenoviruses (hAds) has been limited because of their immunogenic nature and host specificity. Murine cells often lack some of the receptors needed for hAds attachment, thus murine cells are generally non-permissive for human adenoviral infection and replication, which limits translational studies., Methods: We have developed a gene transfer method that uses a combination of lipid-encapsulated perfluorocarbon microbubbles and ultrasound to protect and deliver hAds to a target tissue, bypassing the requirement of specific receptors., Results: In an in vitro model, we showed that murine TRAMP-C2 and human DU145 prostate cancer cells display a comparable expression pattern of receptors involved in hAds adhesion and internalization. We also demonstrated that murine and human cells showed a dose-dependent increase in the percentage of cells transduced by hAd-GFP (green fluorescent protein) after 24 h and that GFP transgene was efficiently expressed at 48 and 72 h post-transduction. To assess if our image-guided delivery system could effectively protect the hAds from the immune system in vivo, we injected healthy immunocompetent mice (C57BL/6) or mice bearing a syngeneic prostate tumor (TRAMP-C2) with hAd-GFP/MB complexes. Notably, we did not observe activation of innate (TNF-α and IL-6 cytokines), or adaptive immune response (neutralizing antibodies, INF-γ+ CD8 + T cells)., Conclusions: This study brings us a step closer to demonstrating the feasibility of murine cancer models to investigate the clinical translation of image guided site-specific adenoviral gene therapy mediated by ultrasound-targeted microbubble destruction.

Administration of ineffective anticancer therapy is associated with unnecessary toxicity and development of resistant clones. Cancer stem-like cells (CSLCs) resist chemotherapy, thereby causing relapse of the disease. Thus, development of a test that identifies the most effective chemotherapy management offers great promise for individualized anticancer treatments. We have developed an ex vivo chemotherapy sensitivity assay (ChemoID), which measures the sensitivity of CSLCs as well as the bulk of tumor cells to a variety of chemotherapy agents. Two patients, a 21-year old male (patient 1) and a 5-month female (patient 2), affected by anaplastic WHO grade-III ependymoma were screened using the ChemoID assay. Patient 1 was found sensitive to the combination of irinotecan and bevacizumab, which resulted in a prolonged disease progression free period of 18 months. Following recurrence, the combination of various chemotherapy drugs was tested again with the ChemoID assay. We found that benzyl isothiocyanate (BITC) greatly increased the chemosensitivity of the ependymoma cells to the combination of irinotecan and bevacizumab. After patient 1 was treated for two months with irinotecan, bevacizumab and supplements of cruciferous vegetable extracts containing BITC, we observed over 50% tumoral regression in comparison with pre-ChemoID scan as evidenced by MRI. Patient 2 was found resistant to all treatments tested and following 6 cycles of vincristine, carboplatin, cyclophosphamide, etoposide, and cisplatin in various combinations, the tumor of this patient rapidly progressed and proton beam therapy was recommended. As expected animal studies conducted with patient derived xenografts treated with ChemoID screened drugs recapitulated the clinical observation. This assay demonstrates that patients with the same histological stage and grade of cancer may vary considerably in their clinical response, suggesting that ChemoID testing which measures the sensitivity of CSLCs as well as the bulk of tumor cells to a variety of chemotherapy agents could lead to more effective and personalized anticancer treatments in the future.

Combining radiation therapy and direct intratumoral (IT) injection of adenoviral vectors has been explored as a means to enhance the therapeutic potential of gene transfer. A major challenge for gene transfer is systemic delivery of nucleic acids directly into an affected tissue. Ultrasound (US) contrast agents (microbubbles) are viable candidates to enhance targeted delivery of systemically administered genes. Here we show that p53, pRB, and p130 gene transfer mediated by US cavitation of microbubbles at the tumor site resulted in targeted gene transduction and increased reduction in tumor growth compared to DU-145 prostate cancer cell xenografts treated intratumorally with adenovirus (Ad) or radiation alone. Microbubble-assisted/US-mediated Ad.p53 and Ad.RB treated tumors showed significant reduction in tumor volume compared to Ad.p130 treated tumors (p<0.05). Additionally, US mediated microbubble delivery of p53 and RB combined with external beam radiation resulted in the most profound tumor reduction in DU-145 xenografted nude mice (p<0.05) compared to radiation alone. These findings highlight the potential therapeutic applications of this novel image-guided gene transfer technology in combination with external beam radiation for prostate cancer patients with therapy resistant disease.

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas.