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Reversible adaptive plasticity: a mechanism for neuroblastoma cell heterogeneity and chemo-resistance.

Chakrabarti L, Abou-Antoun T, Vukmanovic S, Sandler AD - Front Oncol (2012)

Bottom Line: The AI tumorspheres were found to be more resistant to chemotherapy and proliferated slower in vitro compared to the AD cells.Our results demonstrate that neuroblastoma cells are plastic, dynamic, and may optimize their ability to survive by changing their phenotype.Phenotypic switching appears to be an adaptive mechanism to unfavorable selection pressure and could explain the phenotypic and functional heterogeneity of neuroblastoma.

View Article: PubMed Central - PubMed

Affiliation: The Joseph E. Robert Center for Surgical Care, Children's National Medical Center Washington, DC, USA.

ABSTRACT
We describe a novel form of tumor cell plasticity characterized by reversible adaptive plasticity in murine and human neuroblastoma. Two cellular phenotypes were defined by their ability to exhibit adhered, anchorage dependent (AD) or sphere forming, anchorage independent (AI) growth. The tumor cells could transition back and forth between the two phenotypes and the transition was dependent on the culture conditions. Both cell phenotypes exhibited stem-like features such as expression of nestin, self-renewal capacity, and mesenchymal differentiation potential. The AI tumorspheres were found to be more resistant to chemotherapy and proliferated slower in vitro compared to the AD cells. Identification of specific molecular markers like MAP2, β-catenin, and PDGFRβ enabled us to characterize and observe both phenotypes in established mouse tumors. Irrespective of the phenotype originally implanted in mice, tumors grown in vivo show phenotypic heterogeneity in molecular marker signatures and are indistinguishable in growth or histologic appearance. Similar molecular marker heterogeneity was demonstrated in primary human tumor specimens. Chemotherapy or growth factor receptor inhibition slowed tumor growth in mice and promoted initial loss of AD or AI heterogeneity, respectively. Simultaneous targeting of both phenotypes led to further tumor growth delay with emergence of new unique phenotypes. Our results demonstrate that neuroblastoma cells are plastic, dynamic, and may optimize their ability to survive by changing their phenotype. Phenotypic switching appears to be an adaptive mechanism to unfavorable selection pressure and could explain the phenotypic and functional heterogeneity of neuroblastoma.

No MeSH data available.


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Stem cell-like properties of AD and AI phenotypes of mouse neuroblastoma cell line. (A) Dissociated AI or AD phenotypes of Neuro2a cells reseeded at limited dilution in NC or D10 media exhibited self-renewal capability over time by reforming tumorspheres or growing as adhered cells, respectively. (B) Graphical representation of the percent self-renewal capacity of the AD and AI cells as measured by the percent of wells (seeded at 1 cell/well) growing adherent cells or tumorspheres, respectively. (C) Neuro2a cell-derived AI tumorspheres and AD adhered cells were allowed to differentiate followed by immunostaining for stem cells (nestin), neurons (Tuj1), oligodendrocytes (O4), and GFAP (astrocytes). (D) Graphical representation of the percent immunopositive cells revealed multipotency and ubiquitous expression of nestin for both phenotypes. Data points expressed as mean ± S.D. (n = 3). (E) Western blot analysis supports the abundant expression of nestin by both AD and AI phenotypes of Neuro2a cells. (F) Flow cytometric analysis supports that nestin is a ubiquitous marker of both tumor cell phenotypes. MFI, mean fluorescence intensity. (G) Western blot analysis showing complete absence of E-cadherin and abundance of vimentin and SNAIL proteins in both AD and AI phenotypes of neuro2a cells. Scale bar, 100 μm.
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Figure 4: Stem cell-like properties of AD and AI phenotypes of mouse neuroblastoma cell line. (A) Dissociated AI or AD phenotypes of Neuro2a cells reseeded at limited dilution in NC or D10 media exhibited self-renewal capability over time by reforming tumorspheres or growing as adhered cells, respectively. (B) Graphical representation of the percent self-renewal capacity of the AD and AI cells as measured by the percent of wells (seeded at 1 cell/well) growing adherent cells or tumorspheres, respectively. (C) Neuro2a cell-derived AI tumorspheres and AD adhered cells were allowed to differentiate followed by immunostaining for stem cells (nestin), neurons (Tuj1), oligodendrocytes (O4), and GFAP (astrocytes). (D) Graphical representation of the percent immunopositive cells revealed multipotency and ubiquitous expression of nestin for both phenotypes. Data points expressed as mean ± S.D. (n = 3). (E) Western blot analysis supports the abundant expression of nestin by both AD and AI phenotypes of Neuro2a cells. (F) Flow cytometric analysis supports that nestin is a ubiquitous marker of both tumor cell phenotypes. MFI, mean fluorescence intensity. (G) Western blot analysis showing complete absence of E-cadherin and abundance of vimentin and SNAIL proteins in both AD and AI phenotypes of neuro2a cells. Scale bar, 100 μm.

Mentions: Cancer stem cell properties include self-renewal capacity, potent in vivo tumorigenic activity, and an undifferentiated state with the ability to differentiate (Al-Hajj et al., 2003; Singh et al., 2003; Dean et al., 2005; Bao et al., 2006). The phenotypic transition could be a property of a rare cell subpopulation within the cell lines (such as cancer stem cells) or a general cell property. To evaluate these possibilities we performed a self-renewal assay that is considered a stem-like quality. AI and AD forms of Neuro2a cells were dissociated and reseeded at a limiting dilution. Cells cultured in NC media reformed tumorspheres, while those cultured in D10 formed monolayers of adhered cells (Figure 4A). The efficiency of clonal growth of cells seeded at 1 cell/well was similar in AI and AD phenotypes, suggesting self-renewal potential in both phenotypes (Figure 4B).


Reversible adaptive plasticity: a mechanism for neuroblastoma cell heterogeneity and chemo-resistance.

Chakrabarti L, Abou-Antoun T, Vukmanovic S, Sandler AD - Front Oncol (2012)

Stem cell-like properties of AD and AI phenotypes of mouse neuroblastoma cell line. (A) Dissociated AI or AD phenotypes of Neuro2a cells reseeded at limited dilution in NC or D10 media exhibited self-renewal capability over time by reforming tumorspheres or growing as adhered cells, respectively. (B) Graphical representation of the percent self-renewal capacity of the AD and AI cells as measured by the percent of wells (seeded at 1 cell/well) growing adherent cells or tumorspheres, respectively. (C) Neuro2a cell-derived AI tumorspheres and AD adhered cells were allowed to differentiate followed by immunostaining for stem cells (nestin), neurons (Tuj1), oligodendrocytes (O4), and GFAP (astrocytes). (D) Graphical representation of the percent immunopositive cells revealed multipotency and ubiquitous expression of nestin for both phenotypes. Data points expressed as mean ± S.D. (n = 3). (E) Western blot analysis supports the abundant expression of nestin by both AD and AI phenotypes of Neuro2a cells. (F) Flow cytometric analysis supports that nestin is a ubiquitous marker of both tumor cell phenotypes. MFI, mean fluorescence intensity. (G) Western blot analysis showing complete absence of E-cadherin and abundance of vimentin and SNAIL proteins in both AD and AI phenotypes of neuro2a cells. Scale bar, 100 μm.
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Related In: Results  -  Collection

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Figure 4: Stem cell-like properties of AD and AI phenotypes of mouse neuroblastoma cell line. (A) Dissociated AI or AD phenotypes of Neuro2a cells reseeded at limited dilution in NC or D10 media exhibited self-renewal capability over time by reforming tumorspheres or growing as adhered cells, respectively. (B) Graphical representation of the percent self-renewal capacity of the AD and AI cells as measured by the percent of wells (seeded at 1 cell/well) growing adherent cells or tumorspheres, respectively. (C) Neuro2a cell-derived AI tumorspheres and AD adhered cells were allowed to differentiate followed by immunostaining for stem cells (nestin), neurons (Tuj1), oligodendrocytes (O4), and GFAP (astrocytes). (D) Graphical representation of the percent immunopositive cells revealed multipotency and ubiquitous expression of nestin for both phenotypes. Data points expressed as mean ± S.D. (n = 3). (E) Western blot analysis supports the abundant expression of nestin by both AD and AI phenotypes of Neuro2a cells. (F) Flow cytometric analysis supports that nestin is a ubiquitous marker of both tumor cell phenotypes. MFI, mean fluorescence intensity. (G) Western blot analysis showing complete absence of E-cadherin and abundance of vimentin and SNAIL proteins in both AD and AI phenotypes of neuro2a cells. Scale bar, 100 μm.
Mentions: Cancer stem cell properties include self-renewal capacity, potent in vivo tumorigenic activity, and an undifferentiated state with the ability to differentiate (Al-Hajj et al., 2003; Singh et al., 2003; Dean et al., 2005; Bao et al., 2006). The phenotypic transition could be a property of a rare cell subpopulation within the cell lines (such as cancer stem cells) or a general cell property. To evaluate these possibilities we performed a self-renewal assay that is considered a stem-like quality. AI and AD forms of Neuro2a cells were dissociated and reseeded at a limiting dilution. Cells cultured in NC media reformed tumorspheres, while those cultured in D10 formed monolayers of adhered cells (Figure 4A). The efficiency of clonal growth of cells seeded at 1 cell/well was similar in AI and AD phenotypes, suggesting self-renewal potential in both phenotypes (Figure 4B).

Bottom Line: The AI tumorspheres were found to be more resistant to chemotherapy and proliferated slower in vitro compared to the AD cells.Our results demonstrate that neuroblastoma cells are plastic, dynamic, and may optimize their ability to survive by changing their phenotype.Phenotypic switching appears to be an adaptive mechanism to unfavorable selection pressure and could explain the phenotypic and functional heterogeneity of neuroblastoma.

View Article: PubMed Central - PubMed

Affiliation: The Joseph E. Robert Center for Surgical Care, Children's National Medical Center Washington, DC, USA.

ABSTRACT
We describe a novel form of tumor cell plasticity characterized by reversible adaptive plasticity in murine and human neuroblastoma. Two cellular phenotypes were defined by their ability to exhibit adhered, anchorage dependent (AD) or sphere forming, anchorage independent (AI) growth. The tumor cells could transition back and forth between the two phenotypes and the transition was dependent on the culture conditions. Both cell phenotypes exhibited stem-like features such as expression of nestin, self-renewal capacity, and mesenchymal differentiation potential. The AI tumorspheres were found to be more resistant to chemotherapy and proliferated slower in vitro compared to the AD cells. Identification of specific molecular markers like MAP2, β-catenin, and PDGFRβ enabled us to characterize and observe both phenotypes in established mouse tumors. Irrespective of the phenotype originally implanted in mice, tumors grown in vivo show phenotypic heterogeneity in molecular marker signatures and are indistinguishable in growth or histologic appearance. Similar molecular marker heterogeneity was demonstrated in primary human tumor specimens. Chemotherapy or growth factor receptor inhibition slowed tumor growth in mice and promoted initial loss of AD or AI heterogeneity, respectively. Simultaneous targeting of both phenotypes led to further tumor growth delay with emergence of new unique phenotypes. Our results demonstrate that neuroblastoma cells are plastic, dynamic, and may optimize their ability to survive by changing their phenotype. Phenotypic switching appears to be an adaptive mechanism to unfavorable selection pressure and could explain the phenotypic and functional heterogeneity of neuroblastoma.

No MeSH data available.


Related in: MedlinePlus