Date of Award
Doctor of Philosophy (PhD)
Current immunotherapies are only effective in a fraction of cancer patients. One of the main contributing factors to therapy resistance and overall poor prognosis is the immune status of the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) have been shown to have significant influence on the immune status of the TME. As key players in maintaining tissue homeostasis, macrophages exhibit a broad range of phenotypes and functions, making TAMs a promising target for immunomodulatory therapies. As liaisons between innate and adaptive immunity, TAMs can promote a robust anti-tumor response involving both innate and adaptive immunity. This study investigated the potential of a phagocytic-directed immunomodulatory therapy via zymosan conjugated recombinant adenoviruses encoding constant active forms of interferon regulatory factor 3 (IRF3) and 7 (IRF7), which have been shown to induce a pro-inflammatory, anti-tumor phenotype in TAMs. Conjugation of these recombinant adenoviruses to zymosan particles altered their natural tropism to become targets for phagocytic cells. Derived from the cell wall of yeast, biodegradable zymosan particles can induce pro-inflammatory, anti-tumor responses to some extent due to β-glucans, a known pathogen associated molecular pattern (PAMP). Recombinant adenovirus can also activate pro-inflammatory signaling pathways in host cells. The overall goal of this conjugation was to not only target phagocytic cells but also to generate a synergistic activation signal for pro-inflammatory pathways within the targeted cells. First, this study evaluated the potential of these zymosan-adenovirus conjugate particles to alter TAM phenotype in vitro. The zymosan-adenovirus conjugate particles, regardless of encoding IRF3 or IRF7, induced a shift from M2-like to M1-like phenotype in TAM-like BMDMs in vitro as determined by changes in relative gene expression, cytokine secretion, and transcription factor expression. Of note, zymosan-adenovirus particles increased IL-12 and decreased IL-10, signature cytokines of M1/Th1 and M2/Th2 responses, respectively. Further, M1 transcription factor expression of pSTAT1 and pNF-κBp65 increased. These findings prompted further investigation of the zymosan-adenovirus conjugates as immunomodulatory therapies in vivo in a B16 melanoma double tumor mouse model via intratumoral injection. The TME, which consists of tumor cells, non-tumor stromal cells, immune cells (e.g., macrophages and lymphocytes), and non-cellular components of the extracellular matrix, poses a significant challenge for current immunotherapies. By manipulating the function of cellular and non-cellular components via complex signaling pathways, tumor cells either attract suppressive regulatory cell subtypes or coerce or suppress non-malignant cells to form an immunosuppressive environment. It has been suggested that intratumoral injection of immunomodulatory agents could elicit a non-tumor specific strong immune response within the TME, which in turn creates a favorable environment to ensure subsequent tumor specific antigen presentation and T cell activation to generate cytotoxic CD8 T cells and to form long-term anti-tumor memory responses. In this study, alterations in the immune status of the TME were determined by analysis of TAM phenotype, cytokine and chemokine gene expression, metabolic gene expression, and T cell specific gene expression. TAMs sorted using flow cytometry indicated downregulation of M2-like TAMs, CD11b+F4/80+CD206+, and upregulation of M1-like TAMs, CD11b+F4/80+CD80+. Downregulation of Arg1 and upregulation of Irg1 gene expression suggested a potential shift toward an M1-like metabolic state, which was further sup- ported by upregulation of IFNγ, Trail, and IL-12 and downregulation of CXCL1, CXCL2, and MIF in the TME. A snapshot of lymphocyte composition was derived from tumor tissue gene expression data suggesting an upregulation in CD3, CD4, and CD8 gene expression following all three zymosan-adenovirus treatments. Together, findings suggested that the zymosan-adenovirus particles contributed to upregulation of immune favorable conditions in the TME. Further, induction of systemic tumor immunity was determined by untreated tumor analysis in the double tumor model as well as a splenocyte proliferation assay. Zymosan-adenovirus treatments reduced tumor growth in untreated tumors and their splenocytes displayed enhanced B16 specific lysis. Lastly, constant active IRF3 enhanced zymosan-adenovirus anti-tumor activity as determined by significant reduction in tumor growth of both treated and untreated tumors. Taken together, this work supports the potential of the zymosan-adenovirus encoding constant active IRF3 as an intratumoral immunomodulatory therapy in combination with immune checkpoint inhibitors.
Musick, Margaret, "Immunotherapeutic Effects of Intratumorally Injected Zymosan-Adenovirus Conjugates Encoding Constant Active IRFs in a Melanoma Mouse Model" (2022). All Dissertations. 3074.
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