J20 - Developing a Novel 3D In-Vitro Model to Mimic Mechanical Compression in Pancreatic Tumors

Introduction: : Pancreatic cancer is classified as one of the most aggressive and lethal forms of cancer worldwide. The most common form of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), which accounts for about 90% of cases of pancreatic cancer. PDAC is characterized by a severely altered tumor microenvironment marked by mechanical dysregulation. Uncontrollable cell proliferation and excessive deposition of extracellular matrix components (ECM) simultaneously occur, resulting in mechanical compression, tissue stiffness and fibrosis. These phenotypic changes are vital in the characteristic drug resistance and metastasis of PDAC. Mechanical compression is extremely relevant to PDAC progression as it plays a role in altering gene expression, specifically through an epithelial-mesenchymal transition (EMT). EMT markers are associated with increased metastatic potential, so investigation into this transition is vital. Fibrosis contributes significantly to the structure of PDAC stroma, indicating a need to examine the role of fibrosis on PDAC progression. There have been several in vitro models developed to study the impact of mechanical compression in PDAC, however they lack a fibrotic context, especially when fibrosis is significantly present in PDAC microenvironments. In fact, many of these existing models ignore the potential for multiple variables to simultaneously impact metastasis and drug resistance. To fill this gap in knowledge, we developed a model that investigates the combined variables of mechanical compression and fibrosis conditions in 3D. The development of a more precise, pathologically accurate model of pancreatic cancer enables further investigation into the roles of multiple variables in the mechanisms of metastasis and drug resistance.

Materials and

Methods: : To validate the impact of mechanical compression on PDAC, a 2D in vitro model was designed to observe the morphological changes and dysregulation of protein expression. Suit-2 pancreatic cells were plated onto 6-well transwell inserts and covered by an agarose gel cushion. These cells were mechanically compressed at 0 Pa, ~150 Pa and ~300 Pa, and incubated over a period of 5 days (120 hours). The cells were then fixed and prepared for immunofluorescence staining, and imaged.
Recreating in vivo-like conditions for pancreatic tumor cells was accomplished through the use of tumor spheroids and engineered collagen architectures. PDAC cell lines (Suit-2) were used to generate fibrotic and non-fibrotic spheroids (3.2 x 106 cells/mL, 1000 cells/well in low adhesion 96 well plates for 96-120 hrs). Fibrotic collagen bundles were generated utilizing a novel macromolecular crowding-based matrix fabrication method. These engineered bundles were plated into a low adhesion 96-well plate with PDAC cells to form fibrotic spheroids. Once extracted, the spheroids were mechanically compressed (~300 Pa), by applying a weight atop a layer of low gelling temperature agarose.The pressure was evenly distributed across the surface area of the agarose. The applied pressure mimics conditions present within the body as tumor cells rapidly divide and grow against the adjacent tissue. These transwell membranes were incubated with media over the span of 5 days, then fixed and stained for immunofluorescence staining.

Results, Conclusions, and Discussions:: Analysis of in vitro mechanical compression of Suit-2 pancreatic cancer cells displayed survival responses when increasing mechanical compression. In our 2D model, Suit-2 cells that were exposed to increasing mechanical pressure exhibited an increase in cell area, morphological changes (changes in cell shape) and a drastic decrease in intercellular space. Following immunofluorescence staining, mechanically compressed cells displayed mechanical dysregulation. Most notably, the cells experienced a loss of E-cadherin expression, and upregulated expression of vimentin with increasing mechanical compression. In addition, mechanical compression led to the formation of actin stress fibers within the cell cytoskeleton. These findings are extremely relevant to the creation of a 3D in vitro model. The lack of E-cadherin expression indicates a loss of cell to cell adhesion which explains PDACs highly invasive and metastatic nature. Additionally, the increased expression of vimentin under high compression indicates that mechanical pressure invokes an epithelial to mesenchymal transition in cell phenotype. This contributes to the idea that PDAC undergoes a phenotypic change under mechanical pressure, promoting survival behaviors within the cells. With the 3D model spheroids, cells exhibited morphological changes as well as cytoskeleton rearrangement around surrounding fibroids. By embedding and mechanically compressing fibrotic spheroids, we can successfully develop a model that explores mechanical compression in a fibrotic context. Our novel model has the potential to drive further investigation into the features that drive metastasis and drug resistance, and has applications in the development of a clinical model for PDAC screening.

Acknowledgements and/or References (Optional):: I would like to express my gratitude to several individuals and institutions who have supported me throughout this research endeavor. First and foremost, I extend my deepest thanks to Xiangyu Gong for his guidance, insights and support throughout this project. His contributions have been vital to the success of this work. I am also grateful to Michael Mak for his guidance and mentorship that shaped this research. This research was made possible by the resources of the Mak Research Lab. I am extremely grateful to have the opportunity to work alongside such talented and dedicated researchers. Finally, I would like to acknowledge Stony Brook University for providing me with the academic support and resources necessary to conduct this research project.