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Cancer Technologies

Tumor Microenvironment

Investigating the combined effects of matrix stiffness and pancreatic-adipose crosstalk in directing pancreatic cancer initiation and progression

Thursday, October 12, 2023
9:15 AM – 9:30 AM PDT
Location: 343 (Center, Level 3)
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is set to become the third deadliest cancer by 2030, and obesity is a significant risk factor for developing PDAC. Knowledge is limited on how obesity contributes to PDAC initiation, but shared features of each condition’s extracellular matrix (ECM) and microenvironment could provide insight into obesity’s causal influence. PDAC and obesity are both fibrotic and inflammatory conditions, and during fibrotic progression, increased collagen deposition and ECM crosslinking increase ECM stiffness and contribute to dysregulated cell phenotypes. Fibrosis also contributes to malignant cell behaviors in advanced stages of PDAC, but less is known about its role in disease initiation, particularly when risk factors like obesity are present. 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. To model fibrotic ECM conditions, we use a methacrylated type I collagen hydrogel capable of photo-crosslinking, to expose PDAC and adipose cells to stiffness levels that represent either normal or diseased pancreatic tissue. Encapsulated, cells will be exposed to adipocyte conditioned media and assessed for phenotypic changes to determine if ECM stiffness influences how PDAC cells respond to adipocyte signaling molecules. We hypothesize that both matrix stiffness and conditioned media exposure will induce phenotypic changes in PDAC cells. Study results will contribute to the long-term goal of determining mechanisms of paracrine signaling for improved mechanistic understanding of how PDAC risk factors contribute to disease initiation and progression.

Materials and Methods: Adipocytes were differentiated from adipose-derived human mesenchymal stem cells with an adipocyte differentiation toolkit (ATCC). Once adipocytes reached maturity (≥ 15 days), conditioned media was collected for use in experiments. In parallel, exosomes were isolated from conditioned media using a total exosome isolation reagent (ThermoFisher), and relative protein levels from isolated exosomes were quantified with a BCA assay. For 2D studies, PDAC cells (Panc-1) were grown on tissue culture polystyrene for 24 hours in their standard growth media (DMEM, 10% FBS, 1x Pen-Strep) then exposed to different volume ratios of DMEM growth media to adipocyte conditioned media for an additional 48 hours. For 3D studies, tunable stiffness hydrogels were created by combining 8 mg/ml methacrylated type I collagen (ColMA, Advanced Biomatrix) with 2% LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) photoinitiator. Panc-1 cells were encapsulated in hydrogels at a low (~0.75kPa) or high (~3 kPa) stiffness that represent normal and pathological tissue stiffness, respectively. High stiffness hydrogels were photo-crosslinked with 3 minutes of light (405 nm). Samples were maintained in DMEM growth media for 24 hours before exposure to conditioned media ratios. Cell viability was assessed in all samples with Calcein-AM/Ethidium homodimer and AlamarBlueTM metabolic assay. Samples were fixed for F-actin fluorescent staining (Phalloidin) with a nuclear counterstain (DAPI) All samples were imaged on a Keyence BZ-X810 fluorescence microscope (10x or 20x) to obtain max projection of 3D z-stacks (100 µm image stacks; 10 µm between image slices).

Results, Conclusions, and Discussions: Immunofluorescence microscopy showed no significant changes in 2D cell morphology after conditioned media exposure (Fig. 1A). In 2D culture, viability and metabolic activity results showed no significant decrease in cell viability with conditioned media, although1:2 DMEM to conditioned media displayed the lowest viability (~80%) (Fig. 1B). In 3D studies, 1:1 conditioned media was used because it supported the highest cell survival. Qualitative assessment of cell morphology showed that Panc-1 cells in low and high stiffness hydrogels had smoother, more defined borders when grown in DMEM alone (Fig. 2A). Adipocyte conditioned media appeared to introduce border irregularities in a subset of cells in low stiffness hydrogels. Stiffer hydrogels appeared to have a higher number of cells with morphological irregularities regardless of media formulation. Quantitative morphological assessment is on-going. All 3D groups showed higher metabolic activity following conditioned media exposure regardless of hydrogel stiffness when compared to DMEM alone (Fig. 2B). Also, exosomes were detected in adipocyte conditioned media via BCA assay with little difference between the amount of exosomes produced over time as cells mature from pre-adipocytes (8 days) to adipocytes (15 days) (Fig. 2B). Although this work is conducted with cells that are already malignant, results show that PDAC cells show some responsiveness to matrix conditions and signaling molecules secreted from adipocytes. The observed changes in border irregularity suggest that stiffness and paracrine signals from adipocytes promote invasive phenotypes in Panc-1 cells, which is indicative of disease progression. Future studies will be performed with non-malignant pancreatic epithelial cells to determine how the biophysical and biochemical environment can guide PDAC initiation. PDAC is deadly, and we do not fully understand how risk factors increase the likelihood of developing the disease. Our model encompasses two key contributors to disease development—fibrosis via ECM stiffness and paracrine signaling from adipocytes—and we show that PDAC cells change in response to matrix conditions and paracrine signaling. We demonstrate the importance of both factors in improving our understanding of pancreatic-adipose crosstalk in disease initiation and progression, as well as future development of our model as a tool to identify disease biomarkers and potential therapeutic targets.

Acknowledgements (Optional): I would like to acknowledge the Pancreatic Cancer Action Network Career Development Award (831461) for providing funding for this project.

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