chheda, urmi
(2019)
ANALYSIS OF GENE SIGNATURES OF MIGRATORY PHENOTYPES USING
THREE-DIMENSIONAL BREAST MICROTUMOR MODELS.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
Breast cancer is the second most common cause of cancer death in women. Yet, many challenges remain to be addressed such as treatment of local recurrence and metastasis; ability to predict the patients at risk of progression from pre-invasive to invasive disease; and overtreatment of patients that are not at risk. Recent studies show that the tumor microenvironment promotes acquisition of migratory phenotype by the tumor cells, which is one of the essential steps for pre-invasive to invasive transition. However, lack of experimental models with controlled tumor microenvironments to capture this transition is impeding progress in the field. To overcome the limitations of the current in vitro models, our lab has previously developed uniform-sized, three-dimensional (3D) in vitro microtumor models with controlled microenvironments using polyethylene glycol dimethacrylate (PEGDMA) hydrogel microwells. Without any external stimulus, the precise control over the size of T47D microtumors exhibits three key hallmarks of pre-invasive to invasive disease transition observed in vivo: increasing tumor size drives hypoxia and metabolic stress; heterogeneous tumor cells spontaneously emerge; and cells begin to migrate from the parent tumor. Thus, our lab has previously developed three controlled model systems: 1) non-hypoxic, non-migratory small microtumors (150μm/Day 6); 2) hypoxic, migratory large microtumors (600μm/Day 6); and 3) non-hypoxic, yet migratory small microtumors treated with tumor-secreted factors (secretome) from the conditioned media (CM) of large microtumors (150/CM Day 6).
The objective of this thesis was to analyze the gene signatures of migratory phenotypes using microarray data generated on these controlled breast microtumors (T47D). More specifically, the major goal was to delineate the role of hypoxic microenvironment, time of progression and tumor-secreted factors (CM) in inducing migratory phenotypes in non-migratory parental T47D cells. To understand the mechanisms of spontaneous emergence of migratory phenotypes in large hypoxic migratory microtumors (600μm/Day 6) and small non-hypoxic yet migratory microtumors (150/CM Day 6), we performed quantitative analysis of RNA expression using Affymetrix gene chip cDNA microarrays. We utilized non-hypoxic small microtumors (150μm/Day 6) and early stage large microtumors (600μm/Day 1) as controls of non-migratory phenotypes. The raw data were analyzed using Transcriptome Analysis Console (TAC) and Ingenuity Pathway Analysis (IPA). Our preliminary analyses show different genes affected by hypoxia in 600μm/Day 6 microtumors when compared to 600μm/Day 1 or 150μm/Day 6 tumors while no hypoxia-regulated genes were seen in 150/CM Day 6 microtumors when compared with 150μm/Day 6 microtumors. We discovered different sets of genes affecting migration function in large hypoxic microtumors (600μm/Day 6) and small CM-treated non-hypoxic microtumors (150/CM Day 6). Interestingly, experimental observations from our lab have revealed differences in the migratory pattern of these two microtumor models. Specifically, large hypoxic microtumors exhibited directional migration while CM-treated non-hypoxic small microtumors exhibited non-directional migration. Further studies are required to correlate the differences in the gene signatures identified by microarray analysis with the differences in these migration patterns. Thus, our controlled microtumors models may provide insights into different possible mechanisms implicated in the transition from non-migratory to migratory phenotypes. Future studies are underway to identify specific tumor secreted factors as potential biomarkers or therapeutic targets to halt primary tumor progression.
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| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
15 April 2019 |
| Date Type: |
Publication |
| Defense Date: |
4 April 2019 |
| Approval Date: |
15 April 2019 |
| Submission Date: |
12 April 2019 |
| Access Restriction: |
5 year -- Restrict access to University of Pittsburgh for a period of 5 years. |
| Number of Pages: |
98 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Pharmacy > Pharmaceutical Sciences |
| Degree: |
MS - Master of Science |
| Thesis Type: |
Master's Thesis |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Ductal carcinoma in situ
Invasive Ductal carcinoma |
| Date Deposited: |
15 Apr 2019 18:32 |
| Last Modified: |
15 Apr 2024 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/36486 |
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