Evaluation of α-tubulin, detyrosinated α-tubulin, and vimentin in CTCs: identification of the interaction between CTCs and blood cells through cytoskeletal elements

Abstract

Background: Circulating tumor cells (CTCs) are the major players in the metastatic process. A potential mechanism of cell migration and invasion is the formation of microtentacles in tumor cells. These structures are supported by α-tubulin (TUB), detyrosinated α-tubulin (GLU), and vimentin (VIM). In the current study, we evaluated the expression of those cytoskeletal proteins in CTCs.

Methods: Forty patients with breast cancer (BC) (16 early and 24 metastatic) were enrolled in the study. CTCs were isolated using the ISET platform and stained with the following combinations of antibodies: pancytokeratin (CK)/VIM/TUB and CK/VIM/GLU. Samples were analyzed with the ARIOL platform and confocal laser scanning microscopy.

Results: Fluorescence quantification revealed that the ratios CK/TUB, CK/VIM, and CK/GLU were statistically increased in MCF7 compared with more aggressive cell lines (SKBR3 and MDA-MB-231). In addition, all of these ratios were statistically increased in MCF7 cells compared with metastatic BC patients' CTCs (p = 0.0001, p = 0.0001, and p = 0.003, respectively). Interestingly, intercellular connections among CTCs and between CTCs and blood cells through cytoskeleton bridges were revealed, whereas microtentacles were increased in patients with CTC clusters. These intercellular connections were supported by TUB, VIM, and GLU. Quantification of the examined molecules revealed that the median intensity of TUB, GLU, and VIM was significantly increased in patients with metastatic BC compared with those with early disease (TUB, 62.27 vs 11.5, p = 0.0001; GLU, 6.99 vs 5.29, p = 0.029; and VIM, 8.24 vs 5.38, p = 0.0001, respectively).

Conclusions: CTCs from patients with BC aggregate to each other and to blood cells through cytoskeletal protrusions, supported by VIM, TUB, and GLU. Quantification of these molecules could potentially identify CTCs related to more aggressive disease.

Keywords: Breast cancer; CTCs; Cytoskeleton; Detyrosinated α-tubulin; Metastasis; Microtentacles; Vimentin; α-Tubulin.

Fig. 1

Expression of cytokeratin (CK), vimentin (VIM), and α-tubulin (TUB) in patients’ CTCs. Patients’ samples were stained with pancytokeratin (A45-B/B3) (green), vimentin (blue), α-tubulin antibodies (red), and 4′,6-diamidino-2-phenylindole (DAPI) (not shown). a–d Representative confocal laser scanning micrographs of patients’ CTCs (× 40). White arrow indicates the cytoskeleton bridges between CTCs supported by TUB, VIM, and CK. e–h Intercellular connections (white arrows) between a patient CTC and a blood cell (× 60). CTCs were positively stained for CK (green), TUB (red), and VIM (blue), whereas blood cells are positive for VIM and TUB

Fig. 2

Quantification of cytokeratin (CK), α-tubulin (TUB), detyrosinated α-tubulin (GLU), and vimentin (VIM) in patients with early and metastatic breast cancer. a Percentage of the corresponding circulating tumor cell (CTC) phenotypes in patients’ blood. Each patient was considered as positive for a distinct phenotype if she harvested at least on CTC in her blood with this phenotype. b Quantification of TUB, GLU, and VIM intensity in CTCs derived from patients with early and metastatic breast cancer. c Quantification of CK/TUB, CK/GLU, and CK/VIM ratios in CTCs derived from patients with early and metastatic breast cancer. d Patient CTCs stained with pancytokeratin (A45-B/B3, green) antibody and CD45 (hematopoietic cell marker, blue) antibody

Fig. 3

Expression of cytokeratin (CK), vimentin (VIM), and detyrosinated α-tubulin (GLU) in patients’ circulating tumor cells (CTCs). Patients’ samples were stained with pancytokeratin (A45-B/B3, green), detyrosinated tubulin (blue), and vimentin antibodies (red) and 4′,6-diamidino-2-phenylindole (DAPI, not shown). a–d Representative confocal laser scanning micrographs of patients’ CTCs (× 60) stained with pancytokeratin (A45-B/B3), vimentin, and GLU antibodies. Intercellular connections through cytoskeletal bridges (white arrows) were observed between CTCs. These microtentacles were supported by GLU, VIM, and CK. e–h CK, VIM, and GLU expression on a patient’s CTC (× 60), which is in contact with a peripheral blood mononuclear cell from a patient sample

Fig. 4

Progression-free survival (PFS) of patients with metastatic breast cancer. (I) Sequential samples from a patient with breast cancer. Quantification of α-tubulin (TUB) intensity (a), detyrosinated α-tubulin (GLU) intensity (b), vimentin (VIM) intensity (c), cytokeratin (CK)/TUB ratio (d), CK/GLU ratio (e), and CK/VIM ratio (f). (II) (a) PFS in patients with CK⁺GLU⁺VIM⁺-expressing CTCs (p = 0.004). (b) PFS in patients with CK⁺GLU⁻VIM⁺-expressing CTCs (p = 0.007)