Figure 2.

MTA3 Suppresses Metastasis and Stemness of ESCC Cells

(A) EC9706 cells transfected with shMTA3 or shCtrl were infected with lentiviruses carrying luciferase and then subjected to RNA extraction followed by qRT-PCR analysis of luciferase gene.

(B–G) The EC9706 cells with or without MTA3 depletion were infected with recombinant lentiviruses carrying luciferase and injected subcutaneously into the flanks of nude mice (n = 8). The inguinal lymph nodes of the animals were extracted and analyzed for the presence of metastatic cells by bioluminescence imaging (B), and proportion of inguinal lymph nodes metastasis in the nude mice (C). EC9706 cells transfected with shMTA3 or shCtrl were injected intravenously through the tail vein of nude mice (n = 6). Representative images of lung metastasis were shown (D, left panel), numbers of metastatic nodules per lung in the nude mice (D, right panel), and proportion of lung metastasis in the nude mice (E). (F) The inguinal lymph nodes were analyzed by H&E. Scale bars: left panels, 400 μm; right panels, 100 μm. (G) Lung architecture is shown by H&E. Scale bars: left panels, 400 μm; right panels, 100 μm.

(H) GSEA plots of enrichment of BOQOEST_STEM_CELL_UP signatures in MTA3^High tumors versus MTA3^Low tumors in the GSE23400 dataset.

(I) Representative images of spheres formed by EC9706 cells with MTA3 depleted (upper panel) or overexpressed (lower panel). Histograms showing the fold change in the number of spheres formed by EC9706 cells with MTA3 depleted (upper right panel) or overexpressed (lower right panel). Scale bars: 200 μm.

(J–L) (J) Flow cytometry analysis of the CD44⁺ population in EC9706 cells with MTA3 depleted (upper panel) or overexpressed (lower panel). Histograms showing the proportion of CD44⁺ cells in EC9706 cells with MTA3 depleted (upper right panel) or overexpressed (lower right panel). (K and L) Hoechst 33342 dye exclusion assay of the SP⁺ population in EC9706 cells with MTA3 depleted (K) or overexpressed (L). Histograms showing the proportion of SP⁺ cells in EC9706 cells with MTA3 depleted (K, right panel) or overexpressed (L, right panel).

(M) Representative images of immunofluorescence for CD44 proportion in EC9706 cells with MTA3 depleted. Scale bars: 40 μm.

Data are shown as the means of three independent experiments or representative data. Error bars indicate SEM. **p < 0.01, ***p < 0.001 by Student's t test or chi-square test, where appropriate. See also Figures S2–S5.