Dissertation Defense: Raneen Rahhal

Candidate: Raneen Rahhal

Major: Tumor Biology

Advisor: Grady Pearson, Ph.D.

Title: Intratumor Heterogeneity Drives the Evolution of Multiple Populations with Metastatic Initiating Capabilities

Heterogeneity within primary tumors allows cancer cells to acquire traits advantageous to their survival and growth, but a better understanding of how this heterogeneity influences metastasis is required. Using a genetically engineered mouse model (GEMM) of triple negative breast cancer (TNBC), we investigated the evolution of tumor heterogeneity during disease progression, and its contribution on metastasis. Here, we show that late malignant tumors (LM) have an enhanced ability to invade and form pulmonary metastases compared to early malignant (EM) tumors. To understand the changes taking place during progression to promote this enhanced metastatic capability, we employed scRNA-seq and bulk RNA-seq. RNA-seq revealed that LM tumors are enriched in programs that contribute to invasion and metastasis such as epithelial-mesenchymal-transition (EMT), hypoxia, and ECM-receptor interactions. Even though the contribution of EMT on metastasis is unclear, the significant upregulation of EMT genes in LM tumors led us to assess experimental lung metastases for two canonical EMT markers, EpCAM and Vimentin. We found that lung metastases formed from LM tumors were phenotypically heterogeneous with respect to EpCAM and Vimentin expression, but there was not a clear association between the phenotype of the primary tumor and that of the resulting experimental lung metastases. scRNA-seq revealed the transcriptional heterogeneity that exists between and within LM and EM tumors, showing subpopulations with unique molecular signatures. During tumor progression, LM tumors experience a loss of mammary epithelial lineage as observed by the upregulation in luminal genes such as Krt8, Wfdc18, Ptn, Kit, Barx2, and Prom1. This was not accompanied by a gain in basal features as observed with the constant or reduced expression of Trp63, Col17a1, and Krt14. To better understand the contribution of tumor heterogeneity on metastasis, we identified three subpopulations that were unique to LM tumors including EpCAM low, EpCAM high Itga2 low, and EpCAM high Itga2 high tumor cells. While EpCAM low subpopulations exhibited an enhanced ability to invade in vitro, EpCAM high subpopulations displayed a greater ability to form lung metastases. Importantly, we found that multiple subpopulations are comprised of metastasis-initiating cells (MICs), and this metastatic capability was independent of their EMT status. Additionally, our findings suggest that mesenchymal-epithelial-transition (MET) is not required for tumor cells to survive and proliferate after the colonization of a secondary site. Collectively, our findings reveal the existence of MICs in multiple tumor subpopulations, and provides insight to the contribution of heterogeneity and EMT on metastasis.