Engineering silk fibroin scaffolds to model hypoxia in neuroblastoma

Ornell, Kimberly J

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Engineering silk fibroin scaffolds to model hypoxia in neuroblastoma

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Development of novel oncology therapeutics is limited by a lack of accurate pre-clinical models for testing, specifically the inability of traditional 2D culture to accurately mimic in vivo tumors. Neuroblastoma (NB) is a heterogeneous tumor, that in high-risk patients exhibits a 5-year event free survival rate of less than 50%. As such, there is a clinical need for development of novel systems that can mimic the tumor microenvironment and allow for increased understanding of critical pathways as well as be used for preclinical therapeutic testing. In this thesis, lyophilized silk fibroin scaffolds were used to develop 3D neuroblastoma models (scaffolded NB) using multiple neuroblastoma cell lines. Cells grown on scaffolds in low (1%) and ambient (21%) oxygen were compared to traditional 2D (monolayer) cell culture using oxygen-controlled incubators. We hypothesized that scaffolded growth would promote changes in gene expression, cytokine secretion, and therapeutic efficacy both dependent and independent of hypoxia. Monolayer culturing in low oxygen exhibited increased expression of hypoxia related genes such as VEGF, CAIX, and GLUT1, while scaffolded NB exhibited increased expression of hypoxia related genes under both low and ambient oxygen conditions. Pimonidazole staining (hypoxia marker) confirmed the presence of hypoxic regions in the scaffolded NB. Cytokine secretion in monolayer and scaffolded NB suggested differential secretion of cytokines due to both oxygen concentrations (e.g. VEGF, CCL3, uPAR) and 3D culture (e.g. IL-8, GM-CSF, ITAC). Additionally, treatment with etoposide, a standard chemotherapeutic, demonstrated a reduced response in scaffolded culture as compared to monolayer culture regardless of oxygen concentration. However, use of a hypoxia activated therapeutic, tirapazamine exhibited response in low oxygen monolayer culture as well as scaffolded culture in both low and ambient oxygen. To further expand this model into a single culture system capable of generating cell driven oxygen gradients, a stacked culture system was developed. NB scaffolds were stacked using a holder designed based on COMSOL modeling of oxygen tension in the medium. Post-culture, the scaffolds can be separated for analysis on a layer-by-layer basis. Analysis of scaffolds demonstrated a decrease in dsDNA and an increase in hypoxia related genes (VEGF, CAIX, and GLUT1) at the interior of the stack, comparable to that of the scaffolded low oxygen culture. Scaffolds on the periphery of the stack retained gene expression levels similar to that of scaffolded ambient oxygen culture. COMSOL modeling of stacks suggests oxygen gradients present throughout the tumor model similar to that of an in vivo tumor. Gradients of oxygen were confirmed through positive pimonidazole staining. In summary, we developed a system capable of altering critical oxygen-dependent and independent pathways through controlled oxygen levels and 3D culturing. Further, we enhanced this system through the design of a culture system capable of controlling cell driven hypoxic microenvironments to mimic that of an in vivo tumor. This system has the potential to be applied to multiple cancer types, allowing for understanding of key pathway changes and better development of therapeutics.

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