Date of Award

Summer 7-15-2022

Document Type

Thesis (Ph.D.)

Department or Program

Molecular and Systems Biology

First Advisor

Diwakar R. Pattabiraman

Second Advisor

Brock Christensen

Third Advisor

Michael Cole

Abstract

The Epithelial-to-Mesenchymal transition, a critical cellular process in development, is frequently co-opted by solid tumors to promote invasion and metastasis. In particular, the hybrid or intermediate EMT state, possessing both epithelial and mesenchymal characteristics, is associated with increased cancer stemness and plasticity. Similarly, intra-tumoral heterogeneity in solid tumors, in particular breast cancer, is associated with poor prognosis, tumor growth, proliferation, drug resistance, and metastasis. We sought to understand the link between the generation of intra-tumoral heterogeneity and the intermediate EMT state and their impact on tumor progression and patient prognosis. As part of my thesis work, I developed a model to study EMT by isolating single cell-derived clonal populations along the EMT spectrum of cell states using the SUM149PT basal-like breast cancer cell line and validated the impact of intermediate EMT states on tumor progression and metastasis in an orthotopic mouse model. Using this model, I developed and tested a novel multiplexed multi-round staining approach to identify and quantify E-M heterogeneity and EMT score in breast cancer. These metrics were further validated with high reproducibility on a large cohort of breast cancer patient samples, while developing the approach in a high throughput-adaptable way for broad use. This comprehensive analysis highlighted the impact of high E-M heterogeneity and overall intermediate EMT scores on decreased overall survival in these patients. Further, we used the SUM149PT model to understand the driving transcriptional networks of the intermediate EMT and identified CBFb as a regulator of metastasis in the intermediate state by promoting tumor heterogeneity and stabilizing the intermediate EMT state. Taken in its totality, this work provides the further support for understanding intermediate EMT states and their roles in cancer metastasis and heterogeneity, a framework for comprehensive identification of E-M tumor heterogeneity in patient samples, and therapeutic targets that can help to reduce the presence of this tumorigenic and metastasis-promoting phenotype in patients.

Original Citation

Chapter 1.4. Methods of quantifying EMT was adapted from the review article

Brown M. S.1,2,3,4, Muller K. E.4,5, & Pattabiraman D. R.1,2,5.“Quantifying the Epithelial-to-Mesenchymal Transition (EMT) from Bench to Bedside”. Cancers (Basel). 14, 1138 (2022).

Chapter 2 was adapted from the previously published method paper

Brown MS. 1,2,3,4, Abdollahi B1,4, Hassanpour S5, Pattabiraman DR. 1,2,5.“Quantifying epithelial mesenchymal heterogeneity and EMT scoring in patient tumor samples via Tyramide Signal Amplification (TSA)”. In press. Methods Cell Bio. (2022)

Chapter 3 was adapted from the previously published research article

Brown MS. 1,2,3,4, Abdollahi B1,4, Wilkins OM1,2,4, Lu H.3, Chakraborty P.4, Ognjenovic NB3, Muller KE4,5, Jolly MK4,5, ChristensenB.C5, Hassanpour S5, Pattabiraman DR1,2,5. “Phenotypic heterogeneity driven by plasticity of the intermediate EMT state governs disease progression and metastasis in breast cancer” In press. Science Advances. (2022) doi:10.1101/2021.03.17.434993

Author contributions: 1Wrote manuscript; 2Designed experiments; 3Performed experiments; 4Analyzed results; 5Advised on subject material and results

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