Abstract LB-200: Alpha-catulin as a marker of a specific population of invasive breast cancer cells
Authors: Gielata Mateusz , Karpińska Kamila , Kobielak Agnieszka 
Published: September 2020

Abstract
Breast cancer is the most commonly diagnosed cancer in women worldwide. The epithelial-to-mesenchymal transition (EMT) has been associated with breast cancer progression. However transient, reversible nature of EMT and the fact that only a small minority of carcinoma cells may undergo an EMT in primary tumors makes observing an EMT in vivo a great challenge. In our previous studies, we observed that the upregulation of alpha-catulin expression correlates with the transition of tumor cells from epithelial to mesenchymal morphology. Therefore we developed a reporter system, Catulin-GFP that allows us for the first time to isolate and characterize in vivo a small population of invasive triple negative breast cancer cells from the xenograft model and test their cancer initiating potential. It is a reporter driving GFP expression from catulin gene promoter. In vitro 3D invasion assay in matrigel confirmed specific localization of the GFP positive reporter cells in invading population. Whereas depletion of catulin results in the decreased invasive potential of the tumor cells. Furthermore localization of the GFP positive reporter cells overlaps with the expression of known EMT markers like Vimentin, N-cadherin and many more. In addition, flow cytometry analysis of those reporter cell lines showed that GFP signal partially overlaps with known breast cancer stem cell markers CD44+ and CD24- and that catulin depletion results in the change of CD44 expression pattern. Moreover spheres serial replating assay showed decreased stemness potential of catulin depleted spheres. MDA-231 - triple negative breast cancer cell line with catulin reporter was injected into a mammary fat pad of Nod.Scid mice. FACS analysis was performed on the tumors that formed after 8 weeks. GFP+/CD49f+ and GFP-/CD49f+ cells were collected and isolated RNA has been recently subjected to RNAseq analysis and pathway analysis has been performed. 1620 genes have been found to be downregulated and 1670 to be upregulated affecting many cancer invasion related pathways. Part of the tumors were cut and stained with antibodies for known EMT markers (N-cadherin, Vimentin, CD44, and others) to determine the localization of specific populations within the tumor. Moreover MDA-231 catulin knockdown cells with decreased invasive potential and control cells expressing TurboGFP were injected into a mammary fat pad of Nod.Scid mice. RNA from tumors formed after 8 weeks has been also subjected to RNAseq analysis in order to establish transcriptional profile of catulin deficient tumors. Our data show that the Catulin-GFP reporter system marks invasive cells that localize highly outermost in spheres as well as in whole tumors and allows us to perform transcriptional profiling of those cells for characterization of early invasion markers and potential therapeutic targets for breast cancer.
Citation Format: Mateusz Gielata, Kamila Karpińska, Agnieszka Kobielak. Alpha-catulin as a marker of a specific population of invasive breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-200.