Graduation Date

Fall 8-24-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Dr. Maneesh Jain

Abstract

Pancreatic cancer (PC) is a lethal malignancy claiming over 49000 lives in the US in 2022. It is established as an immunosuppressive tumor that assists tumor pathogenesis and is associated with poor response to chemotherapy, radiation and immunotherapy. Immunotherapy is hampered by lack of neoantigens, upregulation of checkpoint molecules and obstructive stroma. PC is characterized by aberrant upregulation of mucins, and MUC4 is one of the differentially overexpressed mucins. Due to its overexpression and functional involvement in pathobiology, MUC4 has emerged as a promising therapeutic target. However, the excessive fibrosis and immunosuppression in the pancreatic tumor microenvironment (TME) are challenges to immunotherapy. Therefore, to enhance the efficacy of immunotherapy, we target the Endothelin (ET) axis, which comprises of ET receptors (ETAR and ETBR) and their ligands. It is ubiquitously present in pancreatic tumors and contributes to evolution of a fibrotic and immunosuppressive environment. We utilize a dual ET antagonist Bosentan to enhance the efficacy of a MUC4 nanovaccine for PC.

Firstly, we evaluated the role of MUC4 in immunomodulation of the TME. Our results show that MUC4-expressing tumors are highly aggressive and have increased proliferative potential. MUC4 is highly immunogenic and therefore MUC4-expressing tumors had increased upregulation of genes associated with inflammation etc. and there was an increased infiltration of immune cells in the TME. Previous studies have suggested that MUC4 is a potential therapeutic target for PC, hence we generated a MUC4-based nanovaccine to generate anti-MUC4 responses. We characterized the efficacy of vaccine in the tumor-bearing animals. Our data suggest that MUC4 nanovaccines can robustly activate an adaptive immune system, by generating a humoral response as interpreted by presence of anti-MUC4 antibodies in the sera of vaccinated mice. Next, analysis of the T-cell response showed an overall increase in CD8+ T-cells infiltrating the TME and there was generation of an anti-tumor response. Our study indicated a Th1 type of response, as shown by increase in IL-12, IL-2 and IFN-g. We saw an increase in the mouse survival, but we did not achieve tumor ablation.

In the second part of this research, we targeted the Endothelin axis, to enhance the efficacy of MUC4 nanovaccine. We utilized dual ET antagonist Bosentan and MUC4 nanovaccine to target the implanted tumors in WT animals. Our results indicate increased survival with combination therapy, but we did not observe an enhanced immune response after the combination treatment.

In the third part of this work, we utilized the recombinant MUC4b protein as a therapy for PC. Our in vitro studies indicate exogenous MUC4 acts as a competitive inhibitor for the endogenous MUC4 and inhibits its functions. Furthermore, addition of MUC4 inhibited proliferation, migration, and homotypic cell-cell interaction of MUC4-expressing cells and did not have a significant effect on MUC4-non-expressing cells. Taken together, studies in this dissertation suggest that MUC4 nanovaccine serves as a potential immunotherapeutic agent and its efficacy can be enhanced by employing additional immunomodulators.

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2022 Copyright, the authors

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