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Cancer Technologies

Cancer Technologies - Poster Session A

Poster H3 - Characterization of Cancer-Associated Fibroblasts In Pancreatic Ductal Adenocarcinoma

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  One of the most distinctive features of pancreatic ductal adenocarcinoma (PDAC) microenvironment is its highly fibrotic stroma (1). Cancer-associated fibroblasts (CAFs) are a key contributor to this stroma and play a crucial role in tumor progression. CAFs are a heterogeneous cell type with different subtypes serving different objectives. In PDAC, there are three subtypes of CAFs: myofibroblasts (myCAFs), inflammatory CAFs (iCAFs) and the rare antigen presenting CAFs (apCAFs). myCAFs are characterized by their myofibroblastic properties and are known to express a high level of alpha-smooth muscle actin (⍺-SMA) (2). iCAFs are characterized by their immunomodulatory functions and are known to express a high level of interleukin 6 (IL-6) (2)(3). apCAFs are characterized by their antigen-presenting capabilities and are known to express a high level of MHC II (2)(3). The PDAC stroma is composed primarily of myCAFs and iCAFs. Biomaterials like PEGDA can provide a basis for in vitro models of the tumor microenvironment, which allow for a detailed study of tumor progression. A PEGDA hydrogel model system allows us the flexibility to change aspects of the matrix while observing cells in conditions that mimic their native environment. PEGDA is a synthetic polymer that resists non-specific protein absorption; however, cell adhesion peptides can be incorporated into the hydrogel to provide sites for cell adhesion and spreading. Therefore, the focus of this project is to characterize CAFs from patient-derived tumors and determine the bioactive properties necessary to culture CAFs in a 3D PEGDA microenvironment, both alone and in co-cultures with PDAC cells.

Materials and

Methods:  Immunostaining was used to visualize and quantify the expression of ⍺SMA and IL-6 to distinguish between CAF subtypes. Three sets of CAFs from different patient tumors (identified by tumor number: 120F, 450F, and 738F )(5), were seeded into two 24-well plates at a seeding density of 0.05 ×10^6 cells/well. After culturing for 48 hrs, both plates were fixed with 4% paraformaldehyde, permeabilized with 0.01% Triton X-100, and blocked with 5% donkey serum. One plate was stained for ⍺SMA using a goat polyclonal ⍺SMA antibody at a 1:500 dilution and the other for IL-6 using a goat polyclonal IL-6 antibody at 5 µg/mL. Both plates were counterstained with DAPI, and the secondary antibody used for each was a donkey anti-goat Alexa Fluor 555 at a 1:200 dilution. Levels of ⍺SMA and IL-6 were assessed by fluorescent microscopy. A minimum of three images were taken in each well and Cell Profiler was used to count nuclei and marker positive cells through thresholding.
To characterize cell adhesion peptides that would best promote CAF adhesion in modified PEGDA surfaces, three different peptides were conjugated to a monoacrylate PEG-succininidyl valerate (PEG-SVA): RGDS, RGDV, and IKVAV. Peptides were crosslinked into 5 wt% PEG hydrogels in the presence of a photoinitiator and UV light (365 nm, 30 s) Gels were swollen in media for 24 hrs and CAFs were seeded on top of each gel. Cell adhesion was assessed by brightfield microscopy and quantified by cell count and % of area cells covered using ImageJ.

Results, Conclusions, and Discussions: Results - Immunofluorescence images were taken of both ⍺SMA and IL-6 plates using a 10x objective with red representing the marker and yellow representing DAPI. Marker (⍺-SMA/IL-6) over DAPI (nuclei count) was analyzed. If the expression of the ⍺SMA is substantially greater than IL-6, the set of CAFs are myCAFs. There was a higher level of expression of ⍺-SMA than IL-6 in all CAFs (Figure 1 A-B). ⍺-SMA: 120F - 9.48% ± 0.948, 450F - 10.61% ± 0.10, 738F - 13.12% ± 0.13. IL-6: 120F – 2.09% ± 0.20, 450F – 2.26% ± 0.22, 738F – 3.14 ± 0.31. Results show all sets of CAFs are likely myCAFs. After 2 days, brightfield images in 10x objective were taken of the adhesion assay to assess the effectiveness of the cells seeding into the gels (Figure 1C). Cell counts from images showed all cells adhered effectively to the gels (Figure 1D): RGDS – 312 ± 31.2, RGDV – 131 ± 13.1, IKVAV – 95 ± 9.5. Cell spreading was also assessed by measuring the area of each cell in all images (Figure 1E): RGDS – 38.12 cells/mm2 ± 3.81, RGDV – 6.69 cells/mm2 ± 0.66, IKVAV 23.47 cells/mm2 ± 2.34. RGDS had both the highest cell count and more spread cells. Though IKVAV had the least cell count, cells did spread on the surface modified with IKVAV. These results indicate that RGDS promotes better adhesion and spreading and is the most suitable cell adhesion peptide for CAFs on PEG hydrogel surfaces.
Discussion/Conclusion - myCAFs are more tumor-promoting than their counterparts as they are responsible for ECM production, playing a key role in depositing the highly fibrotic stroma of the tumor (3). Future directions include studying the factors and signaling pathways myCAFs activate to promote tumor growth and invasion and analyzing how myCAFs interact with other cells in the stroma. Possible future directions also include studying CAFs in varying stiff hydrogels, representing different stages of cancer and normal pancreatic tissue. Understanding how cancer-associated fibroblasts play a role in tumor progression and survival is key to understanding how pancreatic ductal adenocarcinoma tumor metastasizes.

Acknowledgements (Optional):  We would like to thank Sarah Adair and Todd Bauer, MD for the CAFs used in this experiment. Thank you Christopher Highley and the Highley Lab for the use of the LEICA DMi8 Widefield Microscope. We would also like to thank Matthew Lazzara for the IL-6 antibody used in this experiment.

References:
(1) Merali, N. et al. Cancers (Basel). 2022 Feb 17;14(4):1020.
(2) Yang, D. et al. Exp Mol Med 2023, 55 (7), 1322–1332.
(3) Wright, K. et al. Cancers (Basel) 2023, 15 (6), 1899.
(4) Morgan, A., et al. Biology 2023, 12 (8), 1044.
(5) Walters D.M., et al. PLoS ONE 2013, 8(10): e77065