Date of Graduation
8-2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Engineering (PhD)
Degree Level
Graduate
Department
Biomedical Engineering
Advisor/Mentor
Song, Young Hye
Committee Member
Wolchok, Jeffrey C.
Second Committee Member
Muldoon, Timothy J.
Third Committee Member
Griffin, Robert J.
Keywords
Breast cancer; Cancer neuroscience; Extracellular vesicles; Metabolism; Pancreatic cancer; Tissue engineering
Abstract
Purpose: Within the last 30 years, cancer-nerve interactions have garnered attention for causing patient pain, increasing the risk of recurrence, and decreasing patient prognosis. There are 2 key forms cancer-nerve crosstalk takes: perineural invasion (PNI), in which cancer cells infiltrate nerves as a route for metastasis, and tumor innervation (TI), in which neurites penetrate solid tumors to promote tumor progression. Present in vitro models of cancer-nerve crosstalk primarily include Matrigel® mouse matrix and collagen I hydrogels; whereas collagen I hydrogels neglect tumor extracellular matrix (ECM) complexity, Matrigel® is controversial for its batch-to-batch inconsistencies and inclusion of experiment-altering growth factors. Decellularized ECM (dECM) scaffolds, which utilize clean scaffolds of native tissues, have garnered credibility for their ability to mimic tumors and other diseases. The goal of this dissertation is to fabricate physiomimetic in vitro testbeds for analyzing tumor phenotype and/or neurite outgrowth in PNI and TI. Methodology: A dECM PNI platform to replicate the nerve microenvironment was optimized by examining 3 unique decellularization methods and nerve preparations. Using the selected protocol, hydrogels were fabricated for modeling pancreatic cancer (PDAC) PNI. Tumor and healthy cell-derived extracellular vesicles (EV), which are neglected nanoparticles in cancer-nerve crosstalk research, were harvested, and proteomics and pathway analyses were run. Schwann cells (SC), the support cells of the peripheral nervous system known for perpetrating PNI, were treated with tumor and healthy cell EVs. Invasion assays observed PNI in vitro. Conversely, an adipose dECM hydrogel was created to mimic breast tissue and was combined with collagen I to replicate the desmoplastic breast tumor microenvironment. Using this platform, breast cancer (BC) and healthy cell toxicity, metabolic, and phenotypic responses to Metformin anti-diabetes therapy ± fibroblasts were observed. Neurite outgrowth was measured when PC12 cells were cultured with BC or healthy cells. Results: An SD-based protocol with nerve epineurium removal best removed native cells without compromising ECM composition; the hydrogel digested from these nerves assembled collagen fibrils and maintained mechanical properties similar to nerve tissue. SCs cultured in this hydrogel with tumor EVs were activated in an IL-8-dependent manner, a protein found to be upregulated in tumor EVs over healthy cell EVs, to alter their cytoskeletal protein expression and secretome, including CCL2 upregulation. Invasion studies concluded this signaling pathway facilitated PDAC PNI in vitro. Metabolic cargoes, including those related to glycolysis, gluconeogenesis, and glutathione redox balance, were also altered in tumor EVs; given their association with IL-8 secretion, the exploration of these pathways in regard to PNI was proposed. Additionally, BC cells cultured within the dECM composite scaffold required high doses of Metformin to induce metabolic dysfunction, which also depleted healthy cell health. Fibroblasts cultured with BC cells transformed into cancer-associated fibroblasts with Metformin treatment. Finally, BC cells slightly increased the length of neurite outgrowth. Implications: These findings will help educate future targeted therapies and suggest the negative effects of current treatment options. In summary, the results collected using these platforms will inform future preclinical and clinical work to improve the outcomes of patients with PNI and TI.
Citation
Gregory, E. A. (2024). Tissue Engineered Models of Environmental and Metabolism-Driven Cancer-Nerve Crosstalk. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/5426
