Microsystems are a broad category of engineered technologies in the micro and nano scale
that have a diverse range of applications. They are emerging as a powerful tool in the field
of biomedical research, drug discovery, as well as clinical diagnostics and prognostics, especially
with regards to cancer. One of the major challenges in precision and personalized
medicine in cancer lies in the technical difficulties of ex-vivo cell culture and propagation
of the limited number of primary cells derived from patients. Therefore, our aims are to
1. Develop a biologically relevant platform for culturing cancer cells and characterize how
it influences the cell growth and phenotype compared to conventional 2-dimensional(2D)
cell culturing techniques, 2. Isolate secondary metabolites from endophytic fungi and screen
them on the platform for potential anticancer properties in a preliminary drug discovery
pipeline, 3. Design and develop biosensors for quantifying cell responses in real-time within
these systems.
Several biomaterial scaffolds with microscale architectures have been utilized for engineering
the tumor extracellular matrix, but very few studies have thoroughly characterized the
phenotypic changes in their cell models, which is critical for translational applications of biomaterial
systems. The overall objective of these studies is to engineer a biomimetic platform
for the culture of breast cancer cells in vitro and to quantify and profile their phenotypic
changes. In order to do this, we first evaluated a blank-slate matrix consisting of thiolated
collagen, hyaluronic acid and heparin, cross-linked chemically via Michael addition reaction
using diacrylate functionalized poly (ethylene glycol). The hydrogel network was used with
triple-negative breast cancer cells and showed significant changes in characteristics, with
cells self-assembling to form a 3D spheroid morphology, with higher viability, and exhibiting
significantly lower cell death upon chemotherapy treatment, as well as had a decrease in proliferation.
Furthemore, the transcriptomic changes quantified using RNA-Seq and Next-Gen
Sequencing showed the dramatic changes in some of the commonly targeted pathways in cancer
therapy. Furthermore, we were able to show the importance of our biomimetic platform
in the process of drug discovery using fungal endophytes and their secondary metabolites as
the source for potential anticancer molecules. Additionally, we developed gold nanoparticle
and antibody-based (ICAM1 and CD11b) sensors to quantify cell responses spatiotemporally
on our platform. We were able to show quenching of the green fluorescent fluorophores due
to the Förster Resonance Energy Transfer mechanism between the fluorophore and the gold
nanometal surface. We also observed antigen-dependent recovery of fluorescence and inhibition
of energy transfer upon the antibody binding to the cell-surface receptors. Future efforts
are directed towards incorporating the hydrogel system with antigen-dependent sensors in a
conceptually-designed microfluidic platform to spatiotemporally quantify the expression of
surface proteins in various cells of the tumor stroma. This includes the migration,infiltration,
and polarization of specific immune cells. This approach will provide further insight into the
heterogeneity of cells at the single-cell resolution in defined spaces within the 3D microfluidic
platform. / Doctor of Philosophy / Microsystems are a broad category of engineered technologies in the micro and nano scale
that have a diverse range of applications. They are emerging as a powerful tool in the field
of biomedical research, drug discovery, as well as clinical diagnostics and prognostics, especially
with regards to cancer. However, a major challenge in being able to offer personalized
medicine to cancer patients comes from the difficulty of growing cells from the patient's
tumor biopsy in a laboratory for further screening and analysis. There are also limited resources
available for real-time expression of proteins on cell-surfaces, that could be potential
biomarkers and targets for treatment.
Various natural and synthetic polymers are biocompatible and have been used widely in
engineering the tumor extracellular matrix. However, the effect of hydrogels derived from
these polymers on the specific tumor cells are not always well characterized. Our studies
explore the influence of a biohybrid hydrogel on breast cancer cells and our results show that
the microscale architecture of the hydrogel platform works as a suitable scaffold for recapitulating
the 3-dimensional(3D) breast tumor microenvironment, and can also be employed in
the drug discovery process. Additionally, we developed a nano-scale biosensor to enable the
quantification of specific cell-surface proteins in real-time. Ongoing and future efforts are focused
on designing and fabricating a microfluidic device with precise control over the design
of space and special chambers for cell culture. These will be used for studying interactions of
various cells in the tumor microenvironment that influence cancer progression. Integrating
these micro-scale systems, including sensors will allow researchers to quantify cell behavior
in response to the variable factors they are exposed to, as well as provide insight to answer
fundamental questions about cancer biology that are limited by the conventional 2D cell
culture systems.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/112667 |
| Date | 26 May 2021 |
| Creators | Menon, Nidhi |
| Contributors | Graduate School, Jones, Caroline N., Finkielstein, Carla V., Vaughn, Jennifer E., Lawrence, Christopher B., Johnson, Blake |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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