Fig. 2 In vivo tumorigenicity of 4T1, CTC1, and DTC1 cells and EMT traits of DTC1-derived CTC lines. In vivo tumorigenicity of 4T1, CTC1, and DTC1 cells.

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Fig. 2 In vivo tumorigenicity of 4T1, CTC1, and DTC1 cells and EMT traits of DTC1-derived CTC lines. In vivo tumorigenicity of 4T1, CTC1, and DTC1 cells and EMT traits of DTC1-derived CTC lines. (A) 4T1, CTC1, and DTC1 (1.25 × 105 cells) were transplanted subcutaneously into BALB/c mice. Dot plot shows individual and mean tumor weights for each group at the end of the experiment including numbers of transplanted mice. One-way ANOVA with post hoc multiple testing and Bonferroni correction, *P < 0.05 and ***P < 0.001. (B) 4T1 (1.25 × 105 cells; n = 5), CTC1 (5 × 105, 1 × 106, and 2 × 106 cells; each n = 10), and DTC1 (1.25 × 105 cells; n = 5) were transplanted subcutaneously into BALB/c mice. Shown are pictures of primary tumors. Dot plot shows individual and mean tumor weights for each group at the end of the experiment. One-way ANOVA with post hoc multiple testing and Bonferroni correction, *P < 0.05 and ***P < 0.001. (C) Metastatic index per cell was calculated as numbers of lung metastatic colony divided by initially injected cell numbers. Dot plot shows metastatic index per cell and frequencies of lung metastasis per mouse. One-way ANOVA with post hoc multiple testing and Bonferroni correction, ***P < 0.001. (D) Schematic representation of the establishment of CTC sublines from DTC1-transplanted mice. Shown are representative pictures of CTC1-, 4T1-, and DTC1-derived CTCs displaying various degrees of EMT. (E) Dot plot shows mean EMT score grouped by mouse from n = 3 independent scoring results. 4T1 (epithelial, score 0) and CTC1 (mesenchymal, score 400) are included as controls. (F) IHC staining of EpCAM and E-cadherin in E/m-type (CTC6-6, CTC6-11, and CTC8-12) and M/e-type (CTC8-6, CTC8-5, and CTC8-1) CTCs derived from DTC1 transplantations. Shown are representative pictures from n = 3 independent staining. (G) The cell metabolism of E/m-type (CTC6-6, CTC6-11, and CTC8-12) and M/e-type (CTC8-6, CTC8-5, and CTC8-1) CTCs was assessed by MTT assay (initial cell number, 1000 cells). Shown are means with SD from n ≥ 3 independent experiments performed in triplicates. t test of E/m type versus M/e type cells is indicated. (H) Adhesion assay to bEnd.3 endothelial cells was performed with E/m-type (CTC6-6, CTC6-11, and CTC8-12) and M/e-type (CTC8-6, CTC8-5, and CTC8-1) CTCs. Shown are mean adhesion rates with SD from n ≥ 3 independent experiments performed in triplicates. t test of E/m versus M/e cells is indicated, ***P < 0.001. (I) The invasion capacity of E/m-type (CTC6-6, CTC6-11, and CTC8-12) and M/e-type (CTC8-6, CTC8-5, and CTC8-1) CTCs was detected by Transwell invasion assay. Shown are mean OD at 590 nm quantifications of invaded cells as boxplot whiskers graph with SD from n ≥ 4 independent experiments performed in duplicates. t test of E/m versus M/e cells is indicated. Xiao Liu et al. Sci Adv 2019;5:eaav4275 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).