Abstract P5-09-03: Intrinsic heterogeneity of triple-negative breast cancer cells triggers vascular mimicry in 3D matrigel matrix environment
Authors: Maiti A , Takabe K , Hait NC 
Published: February 2018

Abstract
Within the same tumor microenvironment phenotypic and functional heterogeneity arise among cancer cells as a consequence of genetic change, environmental differences, and reversible epigenetic changes in cellular properties. However, it is thought that cancer stem cells are drivers of drug resistance and metastasis. Individual tumor cells growing in culture also display heterogeneity in their intrinsic ability to progress and metastasize. It remains unclear whether intrinsic and extrinsic heterogeneity contribute to the emergence of distinct progressive phenotypes that contribute more to cancer stem cells to disseminate. To this study, we have examined the ability of matrigel to stimulate complex cell behavior that is a consequence of its heterogeneous composition. We have observed that mixing matrigel with metastatic triple negative breast cancer MDA-MB-231 cells, which mimic in vivo tumor microenvironment, around 80-90% cells created network like structures resembling a clinical phenotype known as vascular mimicry (VM) and around 10-20% cells form spheroids. BT549 another triple-negative breast cancer cells also responded similarly, forming cellular networks and spheroids when mixing with matrigel. Since CD44, a marker of epithelial-to-mesenchymal transition has shown enhances tumor aggressiveness by promoting cell plasticity, we decided to examine CD44 expression in MDA-MB-231 cells grown in 3D matrigel matrix environment. We have observed that VM forming cells are showing CD44 positive staining compared to spheroid forming cells which showed negative staining in formaldehyde-fixed 3D matrigel culture of MDA-MB-231 cells, while both group of cells stained positive for VEGFC. Next, we sought to isolate two phenotypically different groups of cells (VM and Tumorsphere forming cells) from the 3D matrigel culture by using microscopic suction procedure for gene expression analysis by qPCR. Our gene expression data suggested that VM forming cells have more expression of VM inducer genes such as CD44 and HIF1α compared to spheroid forming cells isolated from the same 3D matrigel culture of MDA-MB-231 cells. Spheroid forming cells express significant level of endothelial cell adhesion marker, CD31 compared to VM forming cells. Epigenetic mechanisms mediated suppression of tumor suppressors or anti-angiogenesis marker genes are hall mark of VM formation and cancer progression, we examine whether re-expression of those genes with Entinostat (MS-275), a selective inhibitor of class I histone deacetylase (HDAC) can abolish VM structures in 3D matrigel cell culture. Data suggested that MS-275 treatment in 3D culture drastically reduced VM structure by epigenetically re-expression of anti-angiogenic genes; SERPINF1, THBS1 and THBS2 and tumor suppressor genes; APC, PTEN and p21. While MS-275 treatment also downregulated Vimentin, VEGF-A and CD44. Our results suggest that the VM phenotype arises in a subpopulation of cells from a conserved transcriptional response in 3D matrigel environment. Epigenetically re-expression of anti-angiogenic gene expression could be a mechanism to control VM formation in triple-negative breast cancer cells.
Citation Format: Maiti A, Takabe K, Hait NC. Intrinsic heterogeneity of triple-negative breast cancer cells triggers vascular mimicry in 3D matrigel matrix environment [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-09-03.