Investigation of Intratumoral Heterogeneity in Sarcoma Progression and Metastasis: From Tumor Propagating Cell Dynamics to Clonal Evolution

Sarcoma is composed of heterogeneous cancer cells characterized by molecular and phenotypic differences. It has been hypothesized that intratumoral heterogeneity can derive from a subpopulation of tumor cells with stem-cell like properties known as tumor propagating cells (TPCs), and/or from different tumor clones progressing in response to evolutionary forces. However, the functional consequences of cellular heterogeneity in sarcoma and the molecular alterations associated with the different sarcoma cell phenotypes is poorly understood. To identify a cell surface marker of sarcoma TPCs, I showed that CD146 is enriched on the surface of side population (SP) cells and are enhanced for tumorigenicity and self-renewal, suggesting they are enriched for TPCs. Furthermore, they represent a distinct but overlapping population of TPCs from SP cells. Gene expression profiling identified activation of Notch and TGF-β pathways are common to both SP and CD146+ TPCs. Inhibition of Notch signaling inhibited tumor self-renewal. To test the stability of TPCs, I performed a series of lineage tracing experiments by labeling different tumor cells in an autochthonous mouse model of undifferentiated pleomorphic sarcoma driven by KrasG12D and p53 mutations. I showed that Non-SP cells that are not enriched for TPCs can give rise to SP cells enriched for TPCs de novo in transplanted tumors. In vivo lineage tracing of tumor clones by multiple fluorescent reporters and DNA barcoding demonstrated that competition and selection induce substantial changes in tumor architecture over time. Specifically, primary tumor growth is driven by a loss of clonal heterogeneity, and local recurrence following therapy is driven by multiple clones. In contrast, a single metastatic clone (MC) is responsible for advanced metastasis and arise through biological selection. Gene expression profiling of MCs compared to Non-MCs found suppression of functional genes in the metastatic cells of the primary tumor. The suppression of these putative metastasis suppressor genes may represent early changes that contribute to metastatic capacity. In summary, findings described in this thesis revealed that the heterogeneous sarcoma cells consist of CD146+ TPCs, a potentially dynamic TPC state; and as the tumor progresses, the clonal architecture of the tumor alters in response to biological competition and selection.Ph.D