Design and characterization of tunable hydrogels to examine microenvironmental regulation of breast cancer recurrence
Date
2017
Authors
Journal Title
Journal ISSN
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Publisher
University of Delaware
Abstract
Late recurrence of breast cancer within distant metastatic tissue sites is often
difficult to diagnose and treat, resulting in poor prognosis for patients. It is
hypothesized that cells may go dormant by interactions with or lack of adhesion to the
extracellular matrix (ECM) within these tissues, which differs from native breast
tissue. The metastatic ECM is a complex microenvironment, containing a mixture of
mechanical and chemical cues to which cells respond. To investigate how the ECM
regulates cancer recurrence, two-dimensional (2D, plates) and three-dimensional (3D,
naturally-derived scaffolds) in vitro culture models have been used. However, lack of
complexity (2D), mechanical property control (2D, 3D), and chemical property control
(3D) makes it challenging to identify key factors involved in regulating dormancy or
activation in these systems. The development of synthetic polymer-based scaffolds in
recent years provides an alternate route to investigating cellular response to the
presentation of microenvironmental cues in 3D. Initially bioinert, these scaffolds may
be modified with chemical ligands to permit cell-matrix interactions and their
mechanical properties may be precisely tuned to mimic different tissue sites. The goal
of this dissertation is to develop and characterize a novel synthetic material for cell
culture applications and to examine how physical and chemical factors in this
microenvironment regulate breast cancer activation. ☐ Specifically, we have developed a novel poly(ethylene glycol) (PEG)-based
hydrogel scaffold for in vitro cell culture. PEG modified with thiols and peptides
containing alloxycarbonyl-protected lysines (containing a reactive vinyl) react rapidly
upon the application of light in the presence of a photoinitiator, lithium
acylphosphinate (~minutes). Scaffold mechanical properties are tuned by varying
macromer concentration to mimic soft metastatic site tissue ECMs (Young’s modulus
~ 600 – 6000 Pa). These properties remain stable during long-term culture (~weeks).
We also demonstrate the covalent attachment and spatial presentation of peptides
mimicking proteins found within metastatic tissue ECMs in these scaffolds. All cell
lines remain viable (>70%) after encapsulation, with many at greater than 90%
viability, indicating minimal negative effects of light and radicals on cell survival
post-polymerization. ☐ While initially well-defined, the properties of synthetic hydrogel scaffolds
change as cells secrete soluble factors that permit cell-cell signaling and synthesize
new proteins that provide additional binding sites with which cells may interact. To
investigate these chemical property changes, we developed a shotgun proteomics
technique to isolate and identify large proteins secreted within synthetic, polymerbased
hydrogel scaffolds. Metastatic niche cells (adult human mesenchymal stem
cells, hMSCs) were cultured within hydrogel scaffolds and large proteins, including
fibronectin and collagen VI were identified. Additionally, a bead-based multiplex
assay identified several soluble factors secreted by hMSCs (VEGF, IL-8), which may
play a role in regulating cell function and fate. ☐ Finally, the response and activation of estrogen receptor negative (MDA-MB-
231) and estrogen receptor positive (T-47D) breast cancer cells cultured within
synthetic hydrogels with discrete mechanical and chemical properties was determined.
The highly aggressive MDA-MB-231 cells demonstrated the greatest levels of
activation and spread within these synthetic matrices, while T-47D cells, which have
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been associated with a dormant phenotype, exhibited only minimal response and
formed multicellular spheroids. Specifically, hydrogels with high stiffness and matrix
density restricted cancer cell growth, resulting in decreased spreading and smaller cell
cluster volume. Individual and mixtures of peptides (GFOGER, RGDS, IKVAV)
mimicking ECM proteins found within metastatic tissue sites and targeting cell surface
receptors were also shown to affect response. GFOGER and RGDS, targeting integrin
β1, among others, resulted in the highest levels of activation observed within
microenvironments. Collectively, this work describes the development of a novel
material scaffold with well-defined chemical and physical properties that may be used
to identify critical factors in metastatic microenvironments that regulate breast cancer
activation toward development of new treatments for recurrent cancers.