Abstract 940: Altering intratumoral heterogeneity in HER2+ breast cancers with PTEN inactivation and trastuzumab therapy
Authors: Burnett Joseph P. , Fouladdel Shamileh , Truchan Nathan , Azizi Ebrahim , Wicha Max , Sun Duxin 
Published: July 2017

Abstract
Within the context of HER2+ breast cancer, PTEN mutation has been associated with poor patient survival, trastuzumab resistance, and induction of the epithelial to mesenchymal transition. Accumulating evidence supports the theory that breast cancers are organized in a hierarchical manner with the presence of cancerous stem, lineage restricted progenitor, and differentiated-like cells. Recent literature has also indicated that CSCs may exist in distinct epithelial and mesenchymal states which retain unique properties. In order to understand the influence of PTEN inactivation within the context of a hierarchical model of cancer, and examine which populations themselves are targeted by trastuzumab, we have performed analysis of traditional CSC markers and single cell lineage tracing experiments. Here, a lentiviral vector containing shPTEN and dsRed reporter are introduced into HER2+ cell line BT474. Following bulk sorting of dsRed+ cells, long term culture of parent and shPTEN cells with and without trastuzumab for 4 weeks reveal no obvious changes in traditional EMT-CSC markers CD44+/CD24- and only modest changes in Aldefluor + cells. In order to take a CSC-marker agnostic approach we performed a single cell colony formation assay and identify that trastuzumab enriches for rare cells with colony forming potential. The majority of single cell derived colonies exhibited distinct morphologies stable with long term culture which are similar regardless of PTEN knockdown or trastuzumab therapy. However, quantification reveals PTEN knockdown results in a higher frequency of colonies which exhibit distinctly mesenchymal morphology and less of those with epithelial morphology. Interestingly rare colony forming cells could recapitulate all morphologies present in the parental cell line, suggesting they were derived from a true CSC. Single cell multiplex RT-qPCR of 96 target genes was performed on mixed and distinct morphology colonies using Fluidigm’s C1 and BioMark HD instruments and TaqMan gene expression assays. Principal component and t-SNE analysis was capable of separating morphologically distinct colonies and revealed multiple cell clusters within each colony. Using hierarchical clustering it is evident that a minority of cells which appear as unique clusters in PCA analysis are those which are proliferative and express distinct lineage markers reflective of the colony itself. Taken together these results suggest that PTEN knockdown shifts the equilibrium from primarily epithelial lineage differentiation to a more mesenchymal differentiation while trastuzumab enriches for stem and progenitor cells.
Citation Format: Joseph P. Burnett, Shamileh Fouladdel, Nathan Truchan, Ebrahim Azizi, Max Wicha, Duxin Sun. Altering intratumoral heterogeneity in HER2+ breast cancers with PTEN inactivation and trastuzumab therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 940. doi:10.1158/1538-7445.AM2017-940