Date of Award

12-31-2017

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology/Molecular, Cellular, and Organismal Biology

First Advisor

Jonathan P. Celli

Second Advisor

Jill Macoska

Third Advisor

Alexey Veraksa

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a notoriously lethal disease characterized by prominent stromal involvement, which plays complex roles in regulating tumor growth and therapeutic response. The extracellular matrix (ECM)-rich stroma has been implicated as a barrier to drug penetration, although stromal depletion strategies have had mixed clinical success. It remains less clear how biophysical interactions with the ECM regulate invasive progression and susceptibilities to specific therapies. Here, an integrative approach combining 3D cell culture and quantitative imaging techniques is used to evaluate invasive behavior and motility as determinants of response to classical chemotherapy and photodynamic therapy (PDT), in which light activated agents induce site-directed cell death by generating reactive oxygen species. The 3D culture protocol developed for these studies with transplanted multicellular PDAC spheroids in rheologically characterized ECM shows that in invasion-promoting ECM environments, PDT response is markedly enhanced in the most motile populations while the same cells exhibit chemoresistance. Conversely, drug-resistant sublines with characterized increase in invasive potential were generated to compare differential treatment response in identical ECM conditions, monitored by particle-tracking microrheology measurements of matrix remodeling. In both scenarios, ECM infiltrating cells exhibit increased PDT sensitivity, whether invasion is consequent to selection of chemoresistance, or whether chemoresistance is correlated with acquisition of invasive behavior. However, while ECM-infiltrating, chemoresistant cells exhibit mesenchymal phenotype, EMT induction in monolayers lacking ECM is not sufficient to enhance PDT sensitivity, yet does impart chemoresistance as expected. In further experiments seeking to elucidate intertwined roles of mechanical and biochemical interactions with ECM components, invasive progression and response to therapeutics were evaluated using ECM protein admixtures and collagen hydrogels with varying extent of crosslinking. In these studies, increased collagen stiffness or presence of laminin-rich ECM both inhibit invasion of PDAC cells, although cells that do infiltrate into ECM nevertheless exhibit chemoresistance and enhanced PDT sensitivity, independent of their ECM environment. In addition to containing platform development with broader applicability to inform microenvironment-dependent therapeutics, results of this work collectively reveal the efficacy of PDT for targeting the most aggressive, chemoresistant, and invasive PDAC cell populations associated with dismal outcomes for this disease.

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