| Popis: |
Cancer remains one of the scourges of modern life despite our radical advances in medical technology and treatment. The need for improved therapies is apparent in the pediatric sarcoma population, including patients with rhabdomyosarcoma, who have a five-year survival of less than thirty-percent when diagnosed with metastatic disease. One emerging anti-cancer approach is the use of oncolytic viruses. Viruses derived from wild-type herpes, but possessing genetic modifications to alter their virulence and render them safer, have been tested in clinical trials and show promise as an anti-cancer treatment. The use of any oncolytic agent is likely impacted by the functions of the patient’s immune system; in theory, the host could mount a beneficial attack against the cancerous cells, or a harmful attack against the treatment agent itself, the latter of which would be expected to limit the anti-cancer activity of oncolytic viruses. I sought to develop a suitable model for exploring the interplay between the tolerogenicregulatory T-cells and the anti-viral and anti-tumor immune effector cells, which could be used to determine their role in the presence of oncolytic virus treatment.To this end, I required a tumor model in animals which possess an intact adaptive immunity for this study, yet human HSV replication is generally attenuated in mouse cells, in contrast to human tumor cells. Thus, I first screened mouse tumors for susceptibility to HSV infection. I used a mouse genetically susceptible to rhabdomyosarcoma due to expression of the hepatocyte growth factor/scatter factor transgene in the INK4a/ARF-/- background. I tested several cell lines derived from tumors to determine their ability to be killed by and support the replication of an oncolytic herpes simplex virus, rRp450. I also tested their tumorigenicity in syngeneic mice and response to intratumoral virus injection. To determine the role of regulatoryT-cells in the anti-tumor immune response I used a Foxp3-DTR transgenic model which expresses the diphtheria toxin receptor under control of the Foxp3 (T-reg selective) promoter. I, along with my collaborators, also established a method to determine the extent of the anti-viral immune response using an immune tetramer. This tetramer incorporates the C57BL/6 MHC class I molecule (H2-Kb haplotype) and the immuno-dominant glycoprotein B peptide fragment (gB498-505) and allows for direct quantitation of the anti-viral cytotoxic T lymphocyte response. Several explanted tumor cell lines were found to support oncolytic virus replication, but only one was found to be tumorigenic in C57BL/6 mice. The Foxp3-DTR animals were found to be quite effective at depleting regulatory T-cells and possess many advantages over traditional antibody-mediated depletion strategies. Finally, the immune tetramer was found to be capable of evaluating the anti-viral immune response, as tetramer-staining was significantly increased in animals pre-treated with HSV versus mock-injected naïve controls. Thus, I have established an explantable mouse model of rhabdomyosarcoma that, similar to human rhabdomyosarcoma cells, is susceptible to oncolytic HSV infection. This model should prove ideal to study the role of regulatory T-cells in regulating the anti-tumor and anti-viral immune responses during oncolytic virotherapy. |
