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Tissue Engineering

Tissue Engineering - Poster Session E

Poster BB11 - PDAC cell lines display differential responses to obese adipocyte signaling under fibrotic matrix conditions in vitro

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Pancreatic ductal adenocarcinoma (PDAC) is set to become the third deadliest cancer by 2030, and obesity is a significant risk factor. Knowledge is limited on how obesity sustains PDAC progression but shared fibrotic and inflammatory features of each condition’s extracellular matrix (ECM) and microenvironment could provide insight into obesity’s influence. In both cases, increased collagen deposition and crosslinking raise ECM stiffness and contribute to dysregulated cell phenotypes. Fibrosis creates a complex microarchitecture that forces cells to alter their migration/invasion modes that can result in increased malignancy and chemoresistance, highlighting the need to include fibrotic elements in in vitro models.

Our project objective is to investigate how a fibrotic microenvironment impacts PDAC and adipose cell phenotype and alters paracrine signaling that involves cytokines, growth factors, and exosomes. We hypothesize that initial cell phenotype will differentially influence PDAC cell migration mode in response to adipocyte signaling and ECM stiffness. To model fibrotic ECM conditions, we used a methacrylated type I collagen hydrogel capable of photo-crosslinking to expose PDAC cells to stiffness levels representing either normal or PDAC tissue. Encapsulated PDAC cells were exposed to adipocyte conditioned media and assessed for phenotypic changes to determine if ECM stiffness influences how PDAC cells respond to adipocyte signaling molecules.

Materials and

Methods:  Adipocytes were differentiated from adipose-derived human mesenchymal stem cells with an adipocyte differentiation toolkit (ATCC). Lean adipocytes were differentiated on tissue culture polystyrene and obese phenotype was induced by differentiating cells in a 6 mg/ml type I collagen hydrogel (TeloCol-6, Advanced Biomatrix). Once adipocytes reached maturity (≥ 15 days), conditioned media (CM) was collected. PANC-1 and BxPC-3 PDAC cell lines (ATCC) were maintained in their standard growth media (DMEM or RPMI, 10% FBS, 1x Pen-Strep). Tunable stiffness hydrogels were created by combining 8 mg/ml methacrylated type I collagen (PhotoCol, Advanced Biomatrix) with 2% LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) photoinitiator. PANC-1 and BxPC-3 cells were encapsulated in hydrogels at low (~0.75kPa, uncrosslinked) or high (~3 kPa, crosslinked for 3 minutes with 405 nm light) stiffness that represent normal and pathological tissue stiffness Samples were maintained in standard growth media for 24 hours before exposure to 75% v/v adipocyte CM for an additional 48 hrs. Samples were fixed to visualize F-actin (Phalloidin), epithelial-to-mesenchymal transition (Vimentin), nuclei (DAPI). All samples were imaged on a Keyence BZ-X810 fluorescence microscope (20x) to obtain max projection of 3D z-stacks (100-200 µm stacks; 8 µm/slice). In parallel, adipocyte conditioned media was characterized with a Human Obesity Array C1 (RayBiotech) to detect the presence of key adipokines. Densiometry was used to extract spot intensity which was used to calculate relative protein expression using a manufacturer supplied program. A student’s t-test (p < 0.05) was used to test for significance.

Results, Conclusions, and Discussions: We characterized conditioned media produced by lean and obese adipocytes (Figure 1). Obese adipocytes produced significantly higher levels of obesity-associated proteins (ENA-78, CRP, resistin) and cytokines IL-10 (1498±372.7) and IL-8 (17437±7491) compared to lean adipocytes (IL-10: 607.3±377.3; IL-8: 6689±1554). Both IL-8 and IL-10 increase EMT in PDAC cells.

Exposure to obese CM increased expression of vimentin (mesenchymal marker) in PANC-1 cells (mesenchymal) cultured in low stiffness hydrogels (Figure 2A). These changes in vimentin expression were not seen in BxPC-3 cells (epithelial) under the same conditions (Figure 2B). Obese CM similarly increased vimentin expression in PANC-1 cells at high matrix stiffness and did not induce any changes in vimentin expression for BxPC-3 cells in high stiffness conditions. It is possible that IL-10 and IL-8 present in obese CM caused PANC-1 to increase their vimentin expression. PANC-1 cells are a mesenchymal PDAC cell line while BxPC-3 cells are epithelial. Our results suggest that initial cell phenotype influences response to obese adipocyte signaling. We additionally found that the same was true for PANC-1 in high stiffness gels without adipocyte signaling that exhibited amoeboid phenotype. This phenotype was not observed in BxPC-3 cells. Amoeboid morphology is part of the epithelial-to-mesenchymal transition cascade, wherein mesenchymal cells gain a more rounded morphology with small bleb formation in the actin cytoskeleton. Amoeboid migration is protease-independent and allows cells to navigate complex architecture. The crosslinks present in high stiffness PhotoCol® introduce degradation resistance, and the high collagen density of the gel creates cellular compaction. Both of which force mesenchymal cells into an amoeboid migration mode.

These results contribute to the long-term goal of determining mechanisms of paracrine signaling for improved understanding of how PDAC risk factors contribute to disease progression. Our model contains human cell sources for both PDAC and obesity, to help address an existing gap in obesity-PDAC research, where studies traditionally use murine fat sources. Additionally, our model recapitulates complex environmental changes that occur during fibrosis and produces an amoeboid phenotype that warrants future study on the impact of migratory phenotype of progression and treatment response.

Acknowledgements (Optional):  This work was supported by the Pancreatic Cancer Action Network Career Development Award (831461)