Graduation Date

Spring 5-9-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Surinder K. Batra Ph.D.

Second Advisor

Sukhwinder Kaur Ph.D.

Third Advisor

Chi lin M.D. Ph.D.

Fourth Advisor

Michel Ouellette Ph.D.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with an estimated 5-year survival rate of less than 9%. The high lethality of PDAC is due to two primary reasons: the discovery of PDAC at later stages, with locally invasive or metastatic disease present at the time of initial diagnosis as well as the lack of efficacious therapeutic interventions that significantly impact survival. In this dissertation, we sought to discover and test novel detection and treatment strategies for PDAC. Firstly, serum EVs were investigated as potential non-invasive liquid biopsy biomarkers, to serve as a means of early cancer detection. Secondly, a recently discovered form of cell death, ferroptosis, was investigated as a means of potentiating radiation therapy.

The investigation into the potential of extracellular vesicles (EVs) as circulating biomarkers began with a label-free analysis of EVs via surface-enhanced Raman Spectroscopy (SERS) and principal component discriminant function analysis (PC-DFA), to identify tumor-specific spectral signatures. This method differentiated EVs originating from PDAC or normal pancreatic epithelial cell lines with 90% overall accuracy. The proof-of-concept application of this method to EVs purified from patient serum exhibited up to 87% and 90% predictive accuracy for healthy control and early PDAC individual samples, respectively. The specific EV surface proteins that may contribute to the observed SERS differences were investigated via surface shaving LC-MS/MS. This analysis provided protein targets that were selected and validated with a combination of bioinformatics, western blot, and immunogold labeling techniques. The first target protein selected for assessment via ELISA, EPHA2, showed elevated expression in complete cancer patient serum as compared to benign controls. Further, EV specific EPHA2 expression was capable of predicting cancer status in 25% (5/20) of the patient samples with 100% specificity. These data suggest a potential role of EV surface profiling for the early detection of PDAC. However, further work is required to increase the overall accuracy.

Additionally, we sought to investigate the involvement of ferroptosis, in radiation-induced cell death. Ferroptosis is a non-apoptotic form of cell death that requires labile ferrous iron (Fe2+) and is caused by the reactive oxygen species (ROS) mediated build-up of lipid hydroperoxides. Further, we tested if the pharmaceutical induction of ferroptosis via the small molecule Erastin can potentiate the lethal effects of radiation in vitro and in vivo. We observed that radiation produces an increase in ROS and free Fe2+ leading to lipid hydroperoxidation, which was enhanced with the addition of Erastin culminating in the likely induction of ferroptosis. The combination of radiation and Erastin synergistically increased cell death in monocultures and patient-derived organoids as well as significantly reduced tumor size in xenograft mouse models. These findings suggest the potential of ferroptosis induction to improve radiation therapy, though specific mechanistic components require further evaluation. Therefore, further studies must be conducted to elucidate the specific role of ferroptosis in radiation-induced cell death.

The combination of early detection and novel therapeutic intervention strategies offers a means of improving the survival of those with this dreaded disease.

Available for download on Monday, September 21, 2020

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