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- All Subjects: Breast Cancer
- Creators: Andersen, Karen
- Creators: Department of Chemistry and Biochemistry
- Member of: Theses and Dissertations
- Status: Published
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
The synergistic effects between Vorinostat and Tamoxifen observed through a phase II study on breast cancer patients resistant to hormone therapy may involve more than the modulation of ER-alpha to reverse Tamoxifen resistance in ERBC cells. RT-qPCR of genes expressed in Tamoxifen resistant cells, trefoil factor 1(TFF1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC), were evaluated along with ESR1 and Diablo as a control. MYC was observed to have increased expression in the treated cells, whereas the other genes had a decrease in their expression levels after the cells were treated for 3 days with Vorinostat IC30 of 1 µM. As for targeting the AR, MCF7 Tamoxifen sensitive and resistant cells were not affected by the AR antagonists to determine an IC50. The cell viability for all MCF7 sub-clones only decreased for high concentrations of 5.56 µM - 50 µM in Bicalutamide and 16.67 µM – 50 µM of MDV1300. Furthermore, hormone depletion of MCF7 G11 Tamoxifen resistant sub-clones did not show a great response to DHT stimulation or the AR antagonists. In the RT-qPCR, the MCF7 G11 cells showed an increase in mRNA expression for ER, AR, and PR after 4 hours of treatment with estradiol. As for the DHT treatment, ER, AR, PR, and PSA had a minimal increase in the fold change, but the fold change in AR was less than in the estradiol treatment. The Mayo Clinic will investigate the possible usage of AR as a biomarker through immunohistochemistry.
40,000 fatalities annually. The severe impact of breast cancer can be attributed to a poor
understanding of the mechanisms underlying cancer metastasis. A primary aspect of cancer
metastasis includes the invasion and intravasation that results in cancer cells disseminating from
the primary tumor and colonizing distant organs. However, the integrated study of invasion and
intravasation has proven to be challenging due to the difficulties in establishing a combined tumor
and vascular microenvironments. Compared to traditional in vitro assays, microfluidic models
enable spatial organization of 3D cell-laden and/or acellular matrices to better mimic human
physiology. Thus, microfluidics can be leveraged to model complex steps of metastasis. The
fundamental aim of this thesis was to develop a three-dimensional microfluidic model to study the
mechanism through which breast cancer cells invade the surrounding stroma and intravasate into
outerlying blood vessels, with a primary focus on evaluating cancer cell motility and vascular
function in response to biochemical cues.
A novel concentric three-layer microfluidic device was developed, which allowed for
simultaneous observation of tumor formation, vascular network maturation, and cancer cell
invasion/intravasation. Initially, MDA-MB-231 disseminated from the primary tumor and invaded
the acellular collagen present in the adjacent second layer. The presence of an endothelial network
in the third layer of the device drastically increased cancer cell invasion. Furthermore, by day 6 of
culture, cancer cells could be visually observed intravasating into the vascular network.
Additionally, the effect of tumor cells on the formation of the surrounding microvascular network
within the vascular layer was evaluated. Results indicated that the presence of the tumor
significantly reduced vessel diameter and increased permeability, which correlates with prior in vivo
data. The novel three-layer platform mimicked the in vivo spatial organization of the tumor and its
surrounding vasculature, which enabled investigations of cell-cell interactions during cancer
invasion and intravasation. This approach will provide insight into the cascade of events leading up
to intravasation, which could provide a basis for developing more effective therapeutics.
Globally, breast cancer is the most common cancer in women. The disease and treatment cause many unfavorable symptoms such as fatigue, pain, and psychological stress. Research suggests that stress may negatively impact survival and recurrence outcomes of breast cancer patients. Cognitive-behavioral stress management (CBSM) programs have been found to improve these outcomes. In this thesis, I propose using virtual reality as a tool for CBSM to improve symptoms and outcomes for non-metastatic breast cancer patients.
Globally, breast cancer is the most common cancer in women. The disease and treatment cause many unfavorable symptoms such as fatigue, pain, and psychological stress. Research suggests that stress may negatively impact survival and recurrence outcomes of breast cancer patients. Cognitive-behavioral stress management (CBSM) programs have been found to improve these outcomes. In this thesis, I propose using virtual reality as a tool for CBSM to improve symptoms and outcomes for non-metastatic breast cancer patients.