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    Cancer Technologies

    Investigating of the effects of pancreatic tumor-adipose cell cross talk in a 2D in vitro model

    (F-236) Investigating of the effects of pancreatic tumor-adipose cell cross talk in a 2D in vitro model

    Saturday, October 14, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row F - Poster # 236
    Introduction:: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers currently, with only a 12.5% 5-year relative survival rate. Obesity is a known risk factor to PDAC carcinogenesis, however, the mechanism for how obesity contributes is unclear. Fibrosis and inflammation are key components to the disease states of both PDAC and obesity, with changes to the collagen deposition and extracellular matrix (ECM) crosslinking influencing the stiffness of the microenvironment in both conditions. These changes to the ECM contribute to cell dysregulation and may explain the role of obesity as a risk factor of PDAC. However, pancreatic-adipose paracrine signaling through cytokines, growth factors, exosomes and other molecules may play a significant role in PDAC development even without the additional effects of a fibrotic microenvironment. This project aims to investigate the role of adipocyte paracrine signaling on the viability and morphology of pancreatic cancer cells in a 2D environment, in preparation for investigations into the combined effect of paracrine signaling and variable ECM stiffness in future work with 3D microenvironments. We hypothesize that applying adipocyte-derived conditioned media to BxPC-3 cells will influence cell viability and result in morphological changes that deviate from the BxPC-3 epithelial phenotype. Results from these experiments will contribute to the mechanistic understanding of the PDAC microenvironment and paracrine signaling with adipocytes to better understand how obesity contributes to PDAC tumor initiation and progression.

    Materials and Methods:: Adipocyte conditioned media used in the experiments was collected from mature adipocytes ( >14 days) that were differentiated from adipose-derived human mesenchymal stem cells using an adipocyte differentiation toolkit (ATCC). 2D sample studies were conducted by growing BxPC-3 cells, PDAC tumor cell line with epithelial phenotype, on a tissue culture polystyrene 96 well plate for 24 hours in RPMI (FBS, Pen-Strep) seeded at 10,000 cells/well. At 24 hrs after BxPC-3 seeding, adipocyte-derived conditioned media was applied to 2D samples at variable ratios of adipocyte conditioned media to RPMI (100%, 75%, 50%). Control groups included cells treated with non-conditioned adipocyte media at the same ratios and 100% RPMI. On Day 3 (72 hrs) samples were stained for viability using Calcein-AM and Ethidium Homodimer and fixed for morphological evaluation. Additional morphological characteristics–cell area, elongation, and aspect ratio–were assessed using a F-actin stain  (Phalloidin) and a nuclear counterstain (DAPI). All samples were imaged on the Keyence BZ-X810 fluorescent microscope at 10x and 20x magnification, and images were analyzed using CellProfiler.

    Results, Conclusions, and Discussions:: Results revealed that adipocyte conditioned media had no significant effect on cell viability (Figure 1-B). Non-conditioned adipocyte media controls exhibited viability consistent with these findings. As seen in Figure 1-A, qualitative observation of cultures suggested that adipocyte-conditioned medium may produce an altered cell morphology. However, adipocyte-derived conditioned media had no significant effect on the four evaluated metrics:  Area (Figure 2-B), Eccentricity (the ratio of the distance between 2 foci of an ellipse to the major axis length, where 0 is a perfect circle and 1 is a line segment) (Figure 2-C), Form factor (4*π*Area/Perimeter2 , where 1 is a perfect circle) (Figure 2-D), and Median Radius (Figure 2-E) of both the cell and nuclei detected. Future studies will utilize BxPC-3 cells grown within 3D collagen gels with tunable stiffness to model the effects of adipocyte-derived conditioned media on cells at normal stiffness levels (~0.75 kPA) as well as a higher disease-state stiffness (~3 kPa). Additional studies will investigate the quantification and characterization of exosomes isolated from adipocyte-derived media. While the presence of adipocyte-derived media may not appear to have a significant effect on cell viability or morphology in 2D samples, cell-ECM interactions within 3D collagen gels may have a different result. Overall, this work contributes to our understanding of how adipocyte paracrine signaling affects pancreatic cancer cells could be a key to understanding the mechanisms of obesity as a PDAC risk factor.

    Acknowledgements (Optional): : We would like to acknowledge the Pancreatic Cancer Action Network Career Development Award (#831461) and WPI Women's Impact Network for funding.

    References (Optional): :