BME MS Thesis Defense: Jaya Mills “Effects of Matrix Stiffness on Cell Morphology and Expression of Lymphatic Adhesion Molecules in PANC-1 Pancreatic Ductal Adenocarcinoma Cells”

Master’s Thesis Defense 

“Effects of Matrix Stiffness on Cell Morphology and Expression of Lymphatic Adhesion Molecules in PANC-1 Pancreatic Ductal Adenocarcinoma Cells” 

Jaya Mills 

Abstract: With a five-year survival rate of ~13.7%, Pancreatic Ductal Adenocarcinoma (PDAC) is on track to become the second most deadly cancer within five years. Higher mortality results, in large part, to the majority of PDAC patients being diagnosed with some degree of tumor cell invasion which includes metastasis to the lymph nodes and distant organs facilitated by lymphatic vasculature. PDAC exhibits desmoplastic changes in the tumor tissue that can promote dense fibrosis around the tumor and contribute to epithelial-to-mesenchymal transition (EMT), a sign of metastatic disease progression. Since PDAC is characterized by its dense extracellular matrix (ECM) with a confined ECM microstructure, it is important to understand the disease within the context of these factors. There is a general lack of in vitro PDAC models that account for these ECM changes at a pathological level, and even fewer models investigate desmoplastic effects on tumor cell features that relate to lymphatic metastasis. This research aims to characterize and leverage an in vitro 3D tumor model for assessing changes in PDAC cell morphology and expression of cell adhesion markers involved in lymphatic trafficking during metastasis. Our lab uses methacrylated type I collagen with photo-crosslinking (PhotoCol®) to tune collagen properties to reflect normal and tumor tissue stiffness, and PANC-1 cells (commercial PDAC cell line) are used as a tumor cell population with metastatic potential. Biophysical properties of PhotoCol®, such as stiffness and microstructure (confinement), were evaluated via rheology (shear storage modulus) and confocal microscopy (fibril area fraction). The effects of ECM stiffness and confinement on PANC-1 morphology (compactness, eccentricity, form factor) and expression of lymphatic trafficking markers (CCR7 and CXCR4) were evaluated via fluorescent microscopy and western blotting, respectively. Results showed that 8 mg/mL and 3 mg/mL PhotoCol® formulations could represent pancreatic tissues within the ranges of early tumor ECM (high stiffness, high confinement) and normal ECM (low stiffness, low confinement), respectively. In the lower stiffness and confinement condition, PANC-1 cells exhibited more of a protrusive mesenchymal phenotype (higher compactness and eccentricity; lower form factor) that typically signals potential EMT behavior. Conversely, cells in stiffer, more confined matrices had a more rounded morphology. While the latter result does not have characteristic EMT behavior, it could suggest that cells were not transitioning, or they were undergoing an alternative or secondary transition that is known to occur in solid tumors. When evaluating lymphatic trafficking markers, CXCR4 and CCR7, western blot results showed that CXCR4 expression increased in the high stiffness and confinement condition, while CCR7 expression decreased. Studies have shown that CCR7 is directly related to lymph node metastasis, while CXCR4 is more associated with broad tissue lymphatics and the desmoplastic PDAC microenvironment. This result suggests a more dominant role for stiffness in CXCR4 expression compared to CCR7 and a higher likelihood of metastasis occurring in stiffer conditions via lymphatic vasculature directly surrounding PDAC tumors and in distant organs. However, it does not necessarily show correlation between stiffness and metastatic events at the lymph node specifically.  Overall, this research shows the importance of studying the effects of ECM stiffening and confinement on PDAC progression and metastasis and leverages PhotoCol® as a tunable matrix material for an in vitro PDAC model.    

For a zoom link, please email kharrison@wpi.edu