University of Tennessee Health Science Center UTHSC Digital Commons Theses and Dissertations (ETD) College of Graduate Health Sciences 4-2020 MUC13 Modulated Nanomechanical and Biophysical Responses in Pancreatic Cancer Cells Andrew E. Massey University of Tennessee Health Science Center Follow this and additional works at: https://dc.uthsc.edu/dissertations Part of the Pharmacy and Pharmaceutical Sciences Commons Recommended Citation Massey, Andrew E. (https://orcid.org/0000-0001-7688-5501), "MUC13 Modulated Nanomechanical and Biophysical Responses in Pancreatic Cancer Cells" (2020). Theses and Dissertations (ETD). Paper 511. http://dx.doi.org/10.21007/etd.cghs.2020.0496. This Dissertation is brought to you for free and open access by the College of Graduate Health Sciences at UTHSC Digital Commons. It has been accepted for inclusion in Theses and Dissertations (ETD) by an authorized administrator of UTHSC Digital Commons. For more information, please contact [email protected]. MUC13 Modulated Nanomechanical and Biophysical Responses in Pancreatic Cancer Cells Abstract Pancreatic adenocarcinoma is one of the deadliest forms of cancer. Even with recent advances in diagnostic tools, chemotherapeutic regimens, and biomarkers for earlier detection, it still has dismal survival rates. Part of the reason for this is the inherent difficulty in detecting and eatingtr this disease. Recent findings suggest that the altered expression of mucins, including MUC13, may be useful molecular signatures for early disease diagnosis, chemotherapy response and predicting patient survival. MUC13, a recently identified transmembrane glycoprotein, is normally associated with forming a protective barrier on epithelial tissues. However, its overexpression/aberrant subcellular localization has been associated with cancer, disease aggressiveness, poorer patient prognosis and drug resistance via alterations of multiple oncogenic signaling pathways. The main objective of this study is to investigate if MUC13 expression influences nanomechanical and biophysical characteristics of pancreatic cancer cells that might contribute to aggressive nature of this disease. To achieve this goal, we performed innovative nanoindentation analyses using atomic force microscopy in conjunction with standard biochemical assays. To combat this malignant disease, multiple approaches have been considered over the years. Recently, a unique biophysical method of cancer detection has been explored in other cancers, whereby an atomic force microscope (AFM) is used to measure the differences in rigidity and adhesion between normal and cancerous cells. AFMs work by using a thin metallic cantilever with a sharp probe which is brought into contact with a sample. Due to the interactions between the probe and the sample, various data can be extracted. AFMs have been used for decades to acquire high resolution, three-dimensional images of a sample, however it is also possible to acquire other types of data. For cancer research, biophysical data can help to differentiate between cancerous cells and their healthy counterparts. Current evidence suggests that normal cells have a greater rigidity than their cancerous counterparts in most malignancies, however literature dealing with pancreatic cancer is limited. In this report, a comprehensive overview of various pancreatic cell lines (both cancerous and non-cancerous) was physically investigated. We noted that there was a significant trend in the reduction of biophysical characteristics associated with differentiation status - namely, poorly differentiated pancreatic cancer cells (MIA PaCa-2, AsPC-1 and Panc-1) have significantly lower rigidity aluesv as compared to moderately differentiated (BxPC-3), well differentiated (HPAF-II) and normal pancreatic epithelia (HPNE). A positive correlation was seen between the adhesion data and aggregation assays - notably, less aggregation was seen in cancer cells as compared to HPNE cells, and Document Type Dissertation Degree Name Doctor of Philosophy (PhD) Program Pharmaceutical Sciences Research Advisor Subhash C.Chauhan, PhD Keywords Atomic force microscopy, chemotherapeutics, MUC13, nanoindentation, Pancreatic cancer Subject Categories Medicine and Health Sciences | Pharmacy and Pharmaceutical Sciences This dissertation is available at UTHSC Digital Commons: https://dc.uthsc.edu/dissertations/511 UNIVERSITY OF TENNESSEE HEALTH SCIENCE CENTER DOCTOR OF PHILOSOPHY DISSERTATION MUC13 Modulated Nanomechanical and Biophysical Responses in Pancreatic Cancer Cells Author: Advisor: Andrew E. Massey Subhash C. Chauhan, PhD A Dissertation Presented for The Graduate Studies Council of The University of Tennessee Health Science Center in Partial Fulfillment of the Requirements for the Doctor of Philosophy degree from The University of Tennessee in Pharmaceutical Sciences/Pharmaceutics College of Graduate Health Sciences May 2020 Portions of Chapter 4 © 2020 by Elsevier. All other material © 2020 by Andrew E. Massey. All rights reserved. ii DEDICATION I dedicate this dissertation to my parents, Clyde and Nanette Massey. It was first and foremost their constant love and support that gave me the strength to get to this point, and for this I am and will be forever grateful to them. iii ACKNOWLEDGEMENTS This research would not have been possible without the support of those who guided me throughout my studies during the completion of my dissertation work. I would first like to sincerely thank my advisor, Dr. Subhash C. Chauhan, for giving me the opportunity to work in his laboratory. It was due to his guidance that I was introduced to the application of atomic force microscopy (AFM) to biological research at the University of Tennessee Health Science Center. His mentorship and guidance were instrumental in realizing this project. I would also like to thank my other committee members - Drs. Hassan Almoazen, Stephen W. Behrman, Clifton Frilot, and Murali M. Yallapu - for their support and guidance of this project during my time at UTHSC. In addition, I also wish to acknowledge Dr. Bilal Bin Hafeez for assistance with day-to-day experiments during my time in the lab. In addition, I also want to recognize all current and former members of Dr. Chauhan's and Dr. Yallapu's labs for both their camaraderie and their support during my time at UTHSC, especially Kyle Doxtater, for his help on my recent publication as shown in Chapter 4 of this manuscript. I would also like to thank the Herb Kosten Foundation for providing some of the funding for my research, in addition to the National Institutes of Health as well as UTHSC. Lastly, I want to thank the staff at Bruker, who provided assistance and insight onto the use of the AFM for multiple applications. iv ABSTRACT Pancreatic adenocarcinoma is one of the deadliest forms of cancer. Even with recent advances in diagnostic tools, chemotherapeutic regimens, and biomarkers for earlier detection, it still has dismal survival rates. Part of the reason for this is the inherent difficulty in detecting and treating this disease. Recent findings suggest that the altered expression of mucins, including MUC13, may be useful molecular signatures for early disease diagnosis, chemotherapy response and predicting patient survival. MUC13, a recently identified transmembrane glycoprotein, is normally associated with forming a protective barrier on epithelial tissues. However, its overexpression/aberrant subcellular localization has been associated with cancer, disease aggressiveness, poorer patient prognosis and drug resistance via alterations of multiple oncogenic signaling pathways. The main objective of this study is to investigate if MUC13 expression influences nanomechanical and biophysical characteristics of pancreatic cancer cells that might contribute to aggressive nature of this disease. To achieve this goal, we performed innovative nanoindentation analyses using atomic force microscopy in conjunction with standard biochemical assays. To combat this malignant disease, multiple approaches have been considered over the years. Recently, a unique biophysical method of cancer detection has been explored in other cancers, whereby an atomic force microscope (AFM) is used to measure the differences in rigidity and adhesion between normal and cancerous cells. AFMs work by using a thin metallic cantilever with a sharp probe which is brought into contact with a sample. Due to the interactions between the probe and the sample, various data can be extracted. AFMs have been used for decades to acquire high resolution, three- dimensional images of a sample, however it is also possible to acquire other types of data. For cancer research, biophysical data can help to differentiate between cancerous cells and their healthy counterparts. Current evidence suggests that normal cells have a greater rigidity than their cancerous counterparts in most malignancies, however literature dealing with pancreatic cancer is limited. In this report, a comprehensive overview of various pancreatic cell lines (both cancerous and non-cancerous) was physically investigated. We noted that there was a significant trend in the reduction of biophysical characteristics associated with differentiation status - namely, poorly differentiated pancreatic cancer cells (MIA PaCa- 2, AsPC-1 and Panc-1) have significantly lower rigidity values as compared to moderately differentiated (BxPC-3), well differentiated (HPAF-II) and normal pancreatic epithelia (HPNE). A positive correlation was seen between the adhesion data and aggregation