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Cancer Technologies

Cancer Technologies - Poster Session D

Poster H3 - Maryam Younis- Decellularizing of Tumor Extracellular Matrix for Tissue Engineering

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Scaffolds are essential in tissue engineering, providing a matrix for cell adhesion and guiding cell proliferation and migration. They can be injected or placed in wounds to mimic the mechanical and biological properties of the extracellular matrix (ECM). The ECM is a network of proteins, carbohydrates, and water to which cells attach to and remodel. By mimicking the ECM, scaffolds instruct cell behavior, promoting invasion, 3D organization, and new matrix formation. Decellularized ECM (dECM) scaffolds mimic the biochemical composition of native tissues and can be derived from various types of tissues. Decellularization removes cellular components while preserving the ECM structure and composition. The decellularized sample can be combined with a crosslinker to create a hydrogel scaffold. This hydrogel allows for in vitro cell examination within a familiar molecular environment. Different tissues yield varied ECM compositions, and the mechanical properties of decellularized ECM can be adjusted by altering crosslinking methods. In this work, we will be using tumors derived from human Pancreatic ductal adenocarcinoma (PDAC) patients which have been transplanted into nude mice for expansion.

Materials and

Methods:  Human PDAC tumors were isolated from patients and grafted to the pancreas of nude mice, as described by Walters et al. Tumors were taken from two different patients, each identified by a number assigned upon isolation. Each tumor was grafted into two mice, which were harvested after ten weeks. Upon harvesting, samples from tumor 738 were combined, as were samples from tumor 450, for the decellularization process (Walters et al., 2013). Tissues were rinsed and cut into small sections while adipose tissue was trimmed from each tumor. Most of the tissue was then decellularized while a fraction was frozen immediately for future testing. Decellularization was accomplished by a stepwise process designed to lyse cells and remove all cellular components from the ECM. The tumors were sequentially immersed in solutions and stirred at 500 rpm: 24-hour wash of hypertonic NaCl (500 mM), 24-hour wash in hypotonic NaCl (20 mM), a 5-day wash in 1% sodium decyl sulfate (SDS) solution, phosphate buffered saline (PBS) rinse, and a 2-hour DNAse wash. Thorough rinsing and freeze-drying to remove cellular components while preserving the ECM structure followed. Following lyophilization, dECM enzymatically digested in a pepsin solution in 0.1 M HCl.(Mesquita et al., 2013). Each wash step was performed in freshly made solutions and transferred to new containers to minimize the amount of cellular debris exposed to newly decellularized samples. Following each wash, samples were rinsed thoroughly in a strainer with 500ml of ultrapure water.

Results, Conclusions, and Discussions: The decellularization process effectively removed cellular components while preserving the ECM structure. The study successfully followed a method for decellularizing pancreatic tumors. The tumor pieces presented a clear and even white color after decellularization. The water was very clear between solution changes. Before freezing the samplees were still elastic and tended to stick together. The pre-gel solution became increasingly dense and cloudy as the samples digested in pepsin. The turbidity of the gel solution will be monitored over time to track gelation kinetics (Výborný et al., 2019), and scanning electron microscopy (SEM) will be performed to assess the gel microstructure. Further characterization of the dECM is underway. Other than collagen content assays, A methylene blue will allow us to assess any remaining SDS in our dECM and a picoGreen assay for double stranded DNA will quantify remaining DNA in the decellularized matrix. The ability of our hydrogels to support cell adhesion and growth will also be monitored by encapsulating PDAC cells, cancer associated fibroblasts (CAFs) and endothelial cells (ECs). In conclusion, this study demonstrates a replicable and effective method for decellularizing PDAC tumors, paving the way for the development of dECM-based hydrogels as versatile scaffolds in tissue engineering.

Acknowledgements (Optional):  Walters DM, Stokes JB, Adair SJ, et al. Clinical, Molecular and Genetic Validation of a Murine Orthotopic Xenograft Model of Pancreatic Adenocarcinoma Using Fresh Human Specimens. 2013. PLoS ONE 8(10): e77065

Výborný, K., Vallová, J., Kočí, Z. et al. Genipin and EDC crosslinking of extracellular matrix hydrogel derived from human umbilical cord for neural tissue repair. 2019, Sci Rep 9, 10674

Mesquita, F., Morrissay, J., Lee, P.F., et al. Cues from a human atrial extracellular matrix enrich the atrial differentiation of Human induced pluripotent stem cell-derived cardiomyocytes. Biomater. Sci., 2021,9, 3737-3749