Investigating the Role of Heterogeneity in Breast Cancer and Experimental Design

Abstract

Breast cancer is predicted to be the most frequent newly diagnosed cancer and second leading cause of cancer-associated death among women in the United States in 2018. Intratumoral heterogeneity plays an important role in disease progression and therapeutic resistance in many types of cancer, including breast cancer. However, the forces that drive heterogeneity, particularly functional heterogeneity, remain largely unknown. During my doctoral studies, I developed a sequencing based molecular barcoding detection system to track the clonal dynamics of heterogeneous, single-cell derived populations of human and murine breast cancer cells in pre-clinical models. Using these tools, I demonstrated that the host systemic environment influences the degree of heterogeneity of human breast cancer xenografts and that particular systemic changes (such as those induced by the presence of a distant TNBC tumor that is capable of perturbing the immune system) can trigger the outgrowth of tumor cell subpopulations that would otherwise remain dormant. Furthermore, the systemically driven differential patterns of clonal selection had functional consequences, including variable chemotherapeutic responsiveness. Additionally, I worked with my colleagues to use heterogeneous, single-cell derived clonal populations of murine breast cancer models to demonstrate the importance of considering functional heterogeneity during experimental design. We demonstrated that a heterogeneous parental cell line could be an inappropriate control for gene-edited cell lines that underwent a selection or subcloning step. Accordingly, heterogeneity must be considered to prevent the generation of false-positive or false-negative results and inaccurate interpretations of data. We employed a modified gene-editing protocol to perform proof-of-concept and discovery studies that provide a model for generating appropriately matched edited and control cell lines when working with functionally heterogeneous populations. These studies demonstrate for the first time that modulations to the host systemic environment can direct the selection of particular subclonal populations with functional relevance, highlighting the need for an increased understanding of heterogeneity and the processes the guide its formation in experimental disease models and in clinical settings. With an increased understanding of the forces that generate and maintain tumor heterogeneity, it may become possible to interdict the process, potentially slowing disease progression and improving therapeutic outcomes.