Abstract 1553: Evaluation of a tubulin, detyrosinated tubulin and vimentin expression in CTCs; identification of the interaction between CTCs and blood cells through cytoskeletal elements
Authors: Kallergi Galaktea , Martin Stuart S. , Katsarlinos Panagiotis , Georgoulias Vassilis 
Published: July 2016

Abstract
Background: Circulating tumor cells are the major player in metastatic procedure (CTCs). A potential mechanism of cell migration and invasion is the formation of microtentacles in tumor cells. These structures are supported by alpha-Tubulin, detyrosinated, alpha Tubulin (GLU) and Vimentin.
The goal of the current study is to identify the expression of the above cytoskeletal proteins on CTCs.
Methods: We used three different representative breast cancer cell lines MCF7, SKBR3 and MDA-MB 231 spiked in of normal donor's blood as controls. Furthermore 15 metastatic breast cancer patients were enrolled in this study. Tumor cell were isolated using the ISET platform. Spots were then stained with two different combinations of antibodies; pancytokeratin/Vimentin/a-Tubulin and pancytokeratin/Vimentin (VIM)/GLU. Cells were then analyzed with both confocal laser scanning microscopy and the ARIOL system.
Results: We observed that Vimentin was highly expressed in MDA-MB231 cells whereas lower expression was observed in SKBR3 and MCF7 cells. Fluorescence intensity quantification, using the ARIOL system, revealed that the ratio CK/VIM was 41.11, 29.03 and 4.58 in MCF7, SKBR3 and MDA-MB 231 respectively. CK/Tubulin ratio was also higher in MCF7 compared to other cell lines 6.24, 2.29 and 4.18 respectively. In accordance with this, CK/GLU was 20.13 in MCF7 cells, 11.87 in SKBR3 and 11.68 in MDA-MB 231. Interestingly, in SKBR3 and MDA-MB 231 cell lines, alpha-tubulin and vimentin filaments were observed to mediate tumor to blood cell interaction through filamentous bridges between cells.
Consequently, we analyzed 15 breast cancer patients. Eight (53%) of them harvested CTCs in their blood. One of these patients had clusters of CTCs in her blood. In this patient as well as in other CK(+) patients, CTC-to-CTC communication through cystoskeletal bridges could be revealed. These cell connections show localization of alpha-tubulin, Vimentin and GLU; moreover, the same proteins form cytoskeletal structures which allow connections between CTCs and blood cells.
In all breast cancer patients the CK/Vimentin and CK/Tubulin ratio was lower than that observed in MCF7 cells. Conversely, there was a heterogeneity of the CK/GLU ratio since in four patients was lower than that of MCF7 cells and in three others higher.
Conclusions: The importance of understanding the mechanisms that underlie CTC aggregation have been highlighted by recent reports showing that clusters of CTCs have up to 50-fold higher metastatic potential. Our study shows that CTCs can aggregate with each other and with blood cells through cytoskeletal structures supported by. Vimentin, a-Tubulin and Glu-tubulin. Quantification of these cytoskeletal elements revealed that CTCs present a phenotype similar to the most aggressive cell lines.
Citation Format: Galaktea Kallergi, Stuart S. Martin, Panagiotis Katsarlinos, Vassilis Georgoulias. Evaluation of a tubulin, detyrosinated tubulin and vimentin expression in CTCs; identification of the interaction between CTCs and blood cells through cytoskeletal elements. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1553.