Investigating toll-like receptor agonists as an immunotherapeutic for metastatic osteosarcoma

This dissertation and the accompanying research is the work of a study designed to determine the plausibility of toll-like receptor agonists as an immunotherapeutic for canine osteosarcoma. From a comparative and translational perspective, the long-term goals of this research include not only the treatment of canine osteosarcoma, but also human osteosarcoma, as this disease has numerous similarities between clinical presentation and progression in both dogs and people. While the translation of our findings into people is far ahead in the future, this understanding facilitated our study approach and helped shape it into not only a systematic research approach, but also an integrated methodology designed to enhance the likelihood of positive outcomes in dogs and possibly even people. Chapter I entails a review of the past and current literature regarding immune interactions in canine osteosarcoma, as well as immunotherapeutic strategies investigated for the treatment of this canine cancer. Here we also briefly detail the clinical presentation and progression of canine osteosarcoma, along with its similarities to human osteosarcoma. The material reviewed in this section create a solid foundation of the current knowledge surrounding canine osteosarcoma and the immune system, including the substantial finding that osteosarcoma is an immunogenic tumor which can be eliminated by the immune system. Chapter II describes the in vitro studies investigating the dichotomy of toll-like receptor expression and functionality between immune and osteosarcoma cell lines of both murine and canine origin. The aims of this study were to identify several toll-like receptor agonists that would maximize an anti-tumor immune response, but minimally stimulate osteosarcoma tumorigenesis. By investigating mRNA expression levels of a diverse repertoire of toll-like receptors and measuring the response of both immune and osteosarcoma cells to treatment with various toll-like receptor agonists, which included read-outs such as cellular viability, cytokine secretion, and pro-tumorigenic protein upregulation, we were able to identify 3 toll-like receptor agonists (Pam3CSK4, Poly(I:C), and CpG ODN 2395) that fit our requirements for immune stimulation while minimizing osteosarcoma tumorigenesis. Chapter III investigates the ability of these 3 agonists to reduce osteosarcoma metastatic lung tumor burden in a clinically-relevant murine model of osteosarcoma, utilizing the highly aggressive K7M2 osteosarcoma cell line. This model employs tail-vein injection of the neoplastic K7M2 cells, which subsequently shower the lungs and consistently replicate the spontaneous occurrence of lung metastasis that is deadly in both dogs and people with osteosarcoma. Here we show that the TLR9 agonist CpG ODN 2395 is highly effective at reducing osteosarcoma lung metastasis in this model and have also detailed the limitations of this therapy. In addition, we investigate a few of the mechanisms that may underlie CpG ODN 2395’s efficacy. Chapter IV explores the safety of single dose CpG ODN 2395, along with its ability to stimulate a measurable immune response in dogs with spontaneous OS in the form of a dose-escalation pilot study. Through measurement of circulating cytokine levels and evaluation of hematologic parameters, we conclude that a single dose of 2 mg CpG ODN 2395 does stimulate an inflammatory response that culminates with evidence of T cell activation, supporting that CpG ODN 2395 is immunologically active in osteosarcoma-bearing dogs. We also report a lack of observable toxicities, making CpG ODN 2395 a strong candidate for future clinical trial exploration in dogs with spontaneous osteosarcoma. Chapter V subsequently concludes the dissertation and discusses future directions in regard to further evaluation of CpG ODN 2395 as a possible osteosarcoma immunotherapeutic.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste