Date of Award

Fall 12-12-2022

Embargo Period

12-15-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell and Molecular Pharmacology and Experimental Therapeutics

College

College of Graduate Studies

First Advisor

Richard Drake

Second Advisor

Peggi Angel

Third Advisor

Anand Mehta

Fourth Advisor

Russell Norris

Fifth Advisor

Denis Guttridge

Abstract

The severity of pancreatic ductal adenocarcinoma (PDAC) is largely attributed to a failure to detect the disease before metastatic spread has occurred. CA19-9, a carbohydrate biomarker, is used clinically to surveille disease progression, but due to specificity challenges is not suitable for early discovery. As CA19-9 and other prospective markers are glycan epitopes, there is great clinical interest in understanding the glycobiology of pancreatic cancer. Unfortunately, few studies have been able to link glycosylation changes directly to pancreatic tumors and instead have focused on peripheral glycan alterations in the serum of PDAC patients. To address this gap in our understanding, we applied an imaging mass spectrometry (IMS) approach with complementary enzymatic and chemical isomer separation techniques to spatially assess the PDAC N-glycome in a cohort of pancreatic cancer patients. Orthogonally, we characterized the expression of CA19-9 and a new biomarker, sTRA, by multi-round immunofluorescence (IF) in the same cohort. These analyses revealed increased sialylation, fucosylation and branching amongst other structural themes in areas of PDAC tumor tissue. CA19-9 expressing tumors were defined by multiply branched, fucosylated bisecting N-glycans while sTRA expressing tumors favored tetraantennary N-glycans with polylactosamine extensions. IMS and IF-derived glycan and biomarker features were used to build classification models that detected PDAC tissue with an AUC of 0.939, outperforming models using either dataset individually. While studying sialylation isomers in our PDAC cohort, we saw an opportunity to enhance the chemical derivatization protocol we were using to address its shortcomings and expand its functionality. Subsequently, we developed a set of novel amidation-amidation strategies to stabilize and differentially label 2,3 and 2,6-linked sialic acids. In our alkyne-based approach, the differential mass shifts induced by the reactions allow for isomeric discrimination in imaging mass spectrometry experiments. This scheme, termed AAXL, was further characterized in clinical tissue specimens, biofluids and cultured cells. Our azide-based approach, termed AAN3, was more suitable for bioorthogonal applications, where the azide tag installed on 2,3 and 2,8-sialic acids could be reacted by click chemistry with a biotin-alkyne for subsequent streptavidin-peroxidase staining. Furthering the use of AAN3, we developed two additional techniques to fluorescently label (SAFER) and preferentially enrich (SABER) 2,3 and 2,8-linked sialic acids for more advanced glycomic applications. Initial experiments with these novel approaches have shown successful fluorescent staining and the identification of over 100 sialylated glycoproteins by LC-MS/MS. These four bioorthogonal strategies provide a new glycomic tool set for the characterization of sialic acid isomers in pancreatic and other cancers. Overall, this work furthers our collective understanding of the glycobiology underpinning pancreatic cancer and potentiates the discovery of novel carbohydrate biomarkers for the early detection of PDAC.

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