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Tissue Engineering

Tissue Engineering - Poster Session C

Poster L3 - Influence of extracellular matrix stiffness on exosome production from PDAC line BxPC-3.

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Pancreatic Ducal Adenocarcinoma Cancer (PDAC) remains one of the deadliest cancers in the United States due to poor early detection and high metastasis rates, resulting in a five-year survival rate of 12.8%. Therefore, developing new diagnostic biomarkers is critical for improving patients' outcomes. As PDAC advances, the extracellular matrix (ECM) stiffens due to excess type I collagen deposition and increased ECM crosslinking that influences tumor cell proliferation and changes in intercellular communication, including exosome signaling.

Exosomes are small extracellular nanovesicles that contain biomolecules (e.g. lipids, RNA, metabolites, etc.) that facilitate intercellular signaling under normal and disease conditions. Recently, exosomes have emerged as potential biomarkers for diagnosing PDAC and understanding how tumor and surrounding stromal cells interact. Therefore, it is important to understand how exosomes change as the tumor ECM stiffens during PDAC progression.

This study aims to determine if ECM stiffness alters exosome yield from PDAC cells cultured in vitro. Epithelial BxPC-3 cells (PDAC cell line) were seeded onto methacrylated type I collagen hydrogels capable of on-demand stiffening via photo-crosslinking, and exosomes were isolated from cell media. We hypothesized that stiffer photo-crosslinked collagen gels would yield a higher exosome concentration than lower-stiffness collagen gels.

Materials and

Methods:  Methacrylated type I collagen (PhotoCol®, Advanced Biomatrix) was combined with 8% v/v manufacturer-supplied neutralization solution and 2% v/v LAP (Lithium phenyl (2,4,6-trimethyl benzoyl phosphonate) photoinitiator and incubated at 37°C/5% CO2 for 30 minutes for gel formation. Post gelation, the high stiffness group was photo-crosslinked with 405 nm light for 3 minutes while low stiffness gels were left uncrosslinked. BxPC-3 cells (ATCC) were seeded on top of the hydrogel (30,000 cells/mL) and cultured for 3 days. At approximately 80% cell confluency, samples were washed and switched to fetal bovine serum (FBS) free media in preparation for exosome collection. After 24 hours, media containing exosomes was collected, mixed with Total Exosome Isolation Reagent (ThermoFisher Scientific), and incubated overnight at 4°C to promote precipitation and concentration of exosomes in cell culture media. Samples were centrifuged, and isolated exosomes were resuspended in PBS for analysis with a Bradford Assay to determine exosome concentration. Hydrogel samples seeded with cells were fixed with 4% formaldehyde and stained with phalloidin (F-actin), DAPI (4′,6-diamidino-2-phenylindole; nucleus), and Ki67 (proliferation). A study was also performed to compare exosome concentrations in samples collected from culture media with and without FBS to identify potential interference from exosomes already present in FBS.

Results, Conclusions, and Discussions: Results from the Bradford Assay show a higher concentration of exosomes (M=843.75 ± 192.69 µg/mL) on stiffer photo-crosslinked gels (~3 kPa) compared to exosome concentration (605.7, SD ± 291.01 µg/mL) from BxPC-3 on low-stiffness gels (~0.75 kPa) (Figure 1A). Moreover, culture media supplemented with FBS exhibited increased concentrations over FBS-free media in the Bradford Assays (Figure 1B).
We also observed that BxPC-3 cultured on high-stiffness collagen gels grew to confluency faster than cells on low-stiffness gels. Fluorescence staining of the actin cytoskeleton and cell nucleus shows that cells grown on high-stiffness collagen are spread out and grow as multicellular clusters while cells on low-stiffness gels are rounded and grow as individual cells (Figure 2A). The confluency on high-stiffness gels exceeded 100% after 5 days of growth. During the same time interval, low-stiffness collagen gels had a lower cell confluency of 75-80%. This finding also aligned with proliferation results using Ki-67 that that showed reduced Ki67 expression and cell proliferation in low-stiffness gels compared to high-stiffness gels (Figure 2B).
Future studies will involve performing a Calcein-AM/ethidium homodimer viability assay on BxPC-3 cells grown on low and high-stiffness collagen gels and further characterizing exosome production from cells on these substrates (size and exosome content). Cancer is heterogeneous with different cell lines from the same pathology displaying different behaviors, therefore we will use our system to expose different PDAC cell lines to varying ECM stiffness to evaluate how exosome production may change from these cells. Additionally, we will evaluate exosome production from normal pancreatic epithelial cells, to determine if matrix stiffness alters exosome yield or cargo to promote cancer initiation.

Acknowledgements (Optional):  Thank you to Brian Ruliffson (PhD candidate), and Stephen Larson (PhD student) for their invaluable support.
This work was funded by the Pancreatic Cancer Action Network Career Development Award (831461).
This work was supported by NSF REU grant EEC2150076.