Isolation of cancer stem cells from three human glioblastoma cell lines

Characterization of two selected clones

Fortunata Iacopino, Cristiana Angelucci, Roberto Piacentini, Filippo Biamonte, Annunziato Mangiola, Giulio Maira, Claudio Grassi, Gigliola Sica

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Cancer stem cells (CSC) were isolated via a non-adherent neurosphere assay from three glioma cell lines: LI, U87, and U373. Using a clonal assay, two clones (D2 and F11) were selected from spheres derived from LI cells and were characterized for the: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2); proliferation; differentiation capability (determined by the expression of GalC, βIII-Tubulin and GFAP); Ca2+ signaling and tumorigenicity in nude mice. Both D2 and F11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowly than LI cells with a two-fold increase in duplication time. Markers of differentiation (βIII-Tubulin and GFAP) were expressed at high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, and βIII-Tubulin was down-regulated in D2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplication time of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependent Ca2+- channels but they exhibited increased intracellular Ca2+ levels in response to ATP. These Ca2+ signals were larger in LI cells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATP treatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injected in athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones have characteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiation markers and Ca2+-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 might represent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches.

Original languageEnglish
Article numbere105166
JournalPLoS One
Volume9
Issue number8
DOIs
Publication statusPublished - Aug 14 2014

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human cell lines
Neoplastic Stem Cells
Glioblastoma
Stem cells
stem cells
Clone Cells
Cells
clones
Cell Line
Tubulin
Nestin
calcium
Differentiation Antigens
tubulin
Nude Mice
cells
Assays
Adenosine Triphosphate
Stem Cells
Serum-Free Culture Media

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)
  • Agricultural and Biological Sciences(all)

Cite this

Isolation of cancer stem cells from three human glioblastoma cell lines : Characterization of two selected clones. / Iacopino, Fortunata; Angelucci, Cristiana; Piacentini, Roberto; Biamonte, Filippo; Mangiola, Annunziato; Maira, Giulio; Grassi, Claudio; Sica, Gigliola.

In: PLoS One, Vol. 9, No. 8, e105166, 14.08.2014.

Research output: Contribution to journalArticle

Iacopino, Fortunata ; Angelucci, Cristiana ; Piacentini, Roberto ; Biamonte, Filippo ; Mangiola, Annunziato ; Maira, Giulio ; Grassi, Claudio ; Sica, Gigliola. / Isolation of cancer stem cells from three human glioblastoma cell lines : Characterization of two selected clones. In: PLoS One. 2014 ; Vol. 9, No. 8.
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