Cell-surface Vimentin: A mislocalized protein for isolating csVimentin(+) CD133(-) novel stem-like hepatocellular carcinoma cells expressing EMT markers

Abstract

Recent advances in cancer stem cell biology have shown that cancer stem-like cells with epithelial-mesenchymal transition (EMT) phenotypes are more aggressive and cause relapse; however, absence of a specific marker to isolate these EMT stem-like cells hampers research in this direction. Cell surface markers have been identified for isolating cancer stem-like cells, but none has been identified for isolating cancer stem-like cells with EMT phenotype. Recently, we discovered that Vimentin, an intracellular EMT tumor cell marker, is present on the surface of colon metastatic tumor nodules in the liver. In our study, we examined the potential of targeting cell surface Vimentin (CSV) to isolate stem-like cancer cells with EMT phenotype, by using a specific CSV-binding antibody, 84-1. Using this antibody, we purified the CSV-positive, CD133-negative (csVim(+) CD133(-) ) cell population from primary liver tumor cell suspensions and characterized for stem cell properties. The results of sphere assays and staining for the stem cell markers Sox2 and Oct4A demonstrated that csVim(+) CD133(-) cells have stem-like properties similar to csVim(-) CD133(+) population. Our investigation further revealed that the csVim(+) CD133(-) cells had EMT phenotypes, as evidenced by the presence of Twist and Slug in the nucleus, the absence of EpCAM on the cell surface and basal level of expression of epithelial marker E-cadherin. The csVimentin-negative CD133-positive stem cells do not have any EMT phenotypes. csVim(+) CD133(-) cells exhibited more aggressively metastatic in livers than csVim(-) CD133(+) cells. Our findings indicate that csVim(+) CD133(-) cells are promising targets for treatment and prevention of metastatic hepatocellular carcinoma.

Keywords: EMT; HCC; liver cancer stem cells; metastasis.

Figure 1. A novel csVim⁺CD133- population isolated from primary HCC exhibited stem-like properties

(a) Images of spheres formed in Matrigel by csVim⁺CD133⁻ and csVim⁻CD133⁺ populations on days 3 and 7. (b) Stem cell-associated transcription factors Sox2 and Oct4a were detected using fluorescence microscopy in Matrigel culture. (c) Cells from each population were laid on Matrigel at densities of 5, 10, 25, and 50 cells/well, with 15 replicate samples per density. The numbers of spheres formed at different seeding densities in 15 wells were calculated. (d) Percentages of csVim⁺CD133⁻ and csVim⁻CD133⁺ populations in sub-G1, G1, S, and G2 phases, determined by flow cytometry. Representative data were shown as mean ± SEM.

Figure 2. A novel csVim⁺CD133⁻ subpopulation enriched from primary HCC showed differentiation

(a) csVim⁺CD133⁻ cells under normal conditions (nontreated) and in hepatocyte-specific medium. (b) Immunofluorescent confocal microscopy was used to detect albumin expression in csVim⁺CD133⁻ cells in both nontreated and hepatocyte-specific media. (c) Glycogen storage content of csVim⁺CD133⁻ cells in nontreated and hepatocyte-specific media, determined by periodic acid-Schiff staining. (d) csVim⁺CD133⁻ cells formed lumen- and asterisk-shaped structures in cholangiocyte-specific culture conditions.

Figure 3. csVim⁺CD133⁻ cells exhibit EMT like phenotypes and metastasize at a higher rate than do csVim⁻CD133⁺ and csVim⁻CD133⁻ cells

(a) Freshly sorted csVim⁺CD133⁻, csVim⁻CD133⁺ and csVim⁻CD133⁻ cells were stained for EpCAM, Twist and Slug. (b) Fifty thousand csVim⁺CD133⁻ (four mice) or csVim⁻CD133⁺ cells (five mice) or csVim⁻CD133⁻ cells (three mice) were injected intraosseously into the right legs of NSG mice. Two weeks postinoculation, the right legs were amputated. The images are representative of liver metastases observed at week 4 postinoculation. (c) Numbers of gross metastatic liver nodules. P value is 0.0486 (d) Hematoxylin-eosin-stained sections of metastatic liver tumor tissues (original magnification ×400). One-way Anova analyses: *P < .05; **P < .01; ***P< .001.