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. 2021 Jan 4;8(5):2002483. doi: 10.1002/advs.202002483

Figure 1.

Figure 1

Chemotherapy‐enriched THBS2‐deficient CD133+ liver cancer stem cells confer an enhanced ability to degrade the ECM by modulating MMP activity. A) Representative flow cytometry dot plots and quantification of CD133 expression in HCC cells with or without 5‐fluorouracil (5‐FU) treatment. DMSO was used as a control. B) Representative H&E and immunohistochemical staining for CD133 expression in orthotopic liver MHCC97L xenograft tumors with or without 5‐FU treatment. Bar graph shows quantitation of number of CD133+ cells. DMSO was used as a control. Scale bar: 50 µm. C) Representative images and quantification of gelatin degradation assays of sorted CD133+ cancer stem cell versus CD133− noncancer stem cell subpopulations in HCC cells. Red arrows point to the area of degraded gelatin. Scale bar: 10 µm. D) PCR array profiles of genes related to extracellular matrix and adhesion molecules. Venn diagrams (top) showing the number of downregulated (3) and upregulated genes (5) in CD133+ versus CD133− in 2 of 3 subpopulation sorted by HCC cell type (PLC8024, SNU182, and Huh7). Heatmap (bottom) of commonly dysregulated genes as analyzed by PCR array. Expression values were normalized to the expression of 5 housekeeping genes and presented as fold changes in CD133+ to CD133− subpopulations. E) Western blot validation of secreted THBS2 proteomic expression in sorted CD133 subpopulation of HCC cells. Coomassie blue was used as the loading control. F) Image showing the rationale used for THBS2 identification. G) Gelatin zymography for MMP2 and MMP9 activities and H) representative images and quantification of gelatin degradation assays in HCC cells with or without modulated THBS2 expression. Coomassie blue was used as the loading control. White arrows point to the area of degraded gelation. Scale bar: 10 µm. EV refers to the empty vector, OE refers to THBS2 overexpression, NTC refers to the nontarget control, and 1137 and 2606 refers to the two THBS2 shRNA knockdown clones. Data expressed as the mean ± SEM; * p < 0.05, ** p < 0.01, and *** p < 0.001 from A–C) Student's t‐test or H) one‐way ANOVA with Bonferroni's post‐test.