A. MAITI1, A. MAITI1 1Roswell Park Cancer Institute,Breast Surgery,Buffalo, NY, USA
Introduction: Within the same tumor microenvironment phenotypic and functional heterogeneity arise due to plasticity of cancer cells as a consequence of environmental differences, genetic changes and reversible epigenetic changes. Individual tumor cells growing in culture also display heterogeneity in their intrinsic ability to progress and metastasize, however, molecular mechanism is still unknown. Tumor growth and metastasis are thought to be angiogenesis related processes. Recent reports suggested that cancer cells feed themselves by an angiogenesis independent pathway, known as Vacsulogenic mimicry (VM). We have examined the ability of matrigel to stimulate complex cell behavior by its heterogeneous composition.
Methods: Cells are mixed with matrigel and plated in low attachment plates. In order to understand differential gene expression pattern we stained MDA MB 231cells in formaldehyde fixed 3D matrigel matrix environment. In order to under the differential gene expression pattern, we isolated two phenotypically different groups of cells (Vessel and Tumorsphere forming cells) from the 3D matrigel culture by using microscopic suction procedure for gene expression analysis by qPCR. Epigenetic mechanisms mediated suppression of tumor suppressors or anti-angiogenesis marker genes are hall mark of VM formation and cancer progression, we sought to examine whether re-expression of those genes with Entinostat (MS-275), a selective inhibitor of class I histone deacetylase (HDAC) could abolish VM structures in 3D matrigel cell culture.
Results:When MDA-MB-231 cells are mixed with matrigel and cultured in low attachment plates, around 80% cells formed vessel like phenotype known as vascular mimicry (VM) and 20% cells form spheroids. Since CD44, a stem cell marker of enhances tumor cell plasticity, we examine CD44 expression in MDA-MB-231 cells grown in 3D matrigel matrix environment. We have observed that VM forming cells are stained CD44 positive while spheroid forming cells are negative in 3D matrigel culture. Both group of cells stained positive for VEGFc and HIF1α . Gene expression data suggested that VM forming cells have more expression of CD44 and HIF1α compared to spheroid forming cells. When we treated cells with MS-275, VM structure is totally abolished. QPCR data suggested that MS-275 treatment epigenetically re-express 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 .
Conclusion: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 genes could be a mechanism to control VM formation in triple-negative breast cancer cells.