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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.


Related in: MedlinePlus

Molecular markers differentiate AD and AI phenotypes of neuroblastoma in vitro. (A) Western blot analysis of proteins that play vital role in neuroblastoma homeostasis revealed differences in expression of PDGFRβ, MAP2, Dcx, NCAM, survivin, and β-catenin between the AD and AI phenotypes. (B) The table represents relative protein expression of AD and AI cell types as determined by Western blot analysis. (C) Immunofluoresence analysis in vitro demonstrated that MAP2 is exclusively expressed by the AD cells whereas β-catenin and PDGFR-β is overexpressed in AI cells compared to the AD cells. (D) Flow cytometric analysis confirmed that MAP2 is expressed by the AD cells only, whereas β-catenin and PDGFRβ are overexpressed in AI cells. Scale bar, 50 μm. MFI, mean fluorescence intensity.
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Figure 6: Molecular markers differentiate AD and AI phenotypes of neuroblastoma in vitro. (A) Western blot analysis of proteins that play vital role in neuroblastoma homeostasis revealed differences in expression of PDGFRβ, MAP2, Dcx, NCAM, survivin, and β-catenin between the AD and AI phenotypes. (B) The table represents relative protein expression of AD and AI cell types as determined by Western blot analysis. (C) Immunofluoresence analysis in vitro demonstrated that MAP2 is exclusively expressed by the AD cells whereas β-catenin and PDGFR-β is overexpressed in AI cells compared to the AD cells. (D) Flow cytometric analysis confirmed that MAP2 is expressed by the AD cells only, whereas β-catenin and PDGFRβ are overexpressed in AI cells. Scale bar, 50 μm. MFI, mean fluorescence intensity.

Mentions: To determine whether the AI and AD phenotypes of Neuro2a cells have distinct molecular markers, we analyzed expression of eleven proteins that play a vital role in neuroblastoma homeostasis. These included proteins regulating proliferation (β-catenin/FZD1, SHH/Gli1/PTCH1, TrkB/BDNF, PDGFR), apoptosis (FZD1, Gli1), chemo-resistance (FZD1, Telomerase), angiogenesis (VEGF/VEGFR, PDGFRβ, SHH), metastasis (SHH, MAP2, TrkB), adhesion and migration (NCAM, TrkB, Dcx), and differentiation and neurite extension (MAP2, Dcx) (Eggert et al., 2000; Oltra et al., 2005; Nakamura et al., 2006; Flahaut et al., 2009; Korja et al., 2009; Krishnan et al., 2009; Bahnassy et al., 2010; Oue et al., 2010; Souzaki et al., 2010; Wesbuer et al., 2010; Zhang et al., 2010; Schiapparelli et al., 2011). Western blot analysis revealed differences in expression of PDGFRβ, MAP2, Dcx, NCAM, survivin, and β-catenin (Figures 6A,B), thereby identifying these molecules as potential unique markers of AI or AD phenotypes. To test the value of these molecules as markers of in vivo cellular commitment to AD or AI phenotypes we performed immunofluorescence microscopy. This analysis confirmed the findings for MAP2, β-catenin, and PDGFRβ, while staining for Dcx, NCAM, and survivin did not discriminate (Figure 6C). Flow cytometric analysis confirmed differences in the expression of MAP2, β-catenin, and PDGFRβ while all neuroblastoma cells were nestin positive (Figure 6D). These results showed that Nestin + MAP2+ cells are indicative of the AD phenotype while Nestin + MAP2− are markers of the AI phenotype.


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

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

Molecular markers differentiate AD and AI phenotypes of neuroblastoma in vitro. (A) Western blot analysis of proteins that play vital role in neuroblastoma homeostasis revealed differences in expression of PDGFRβ, MAP2, Dcx, NCAM, survivin, and β-catenin between the AD and AI phenotypes. (B) The table represents relative protein expression of AD and AI cell types as determined by Western blot analysis. (C) Immunofluoresence analysis in vitro demonstrated that MAP2 is exclusively expressed by the AD cells whereas β-catenin and PDGFR-β is overexpressed in AI cells compared to the AD cells. (D) Flow cytometric analysis confirmed that MAP2 is expressed by the AD cells only, whereas β-catenin and PDGFRβ are overexpressed in AI cells. Scale bar, 50 μm. MFI, mean fluorescence intensity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3412992&req=5

Figure 6: Molecular markers differentiate AD and AI phenotypes of neuroblastoma in vitro. (A) Western blot analysis of proteins that play vital role in neuroblastoma homeostasis revealed differences in expression of PDGFRβ, MAP2, Dcx, NCAM, survivin, and β-catenin between the AD and AI phenotypes. (B) The table represents relative protein expression of AD and AI cell types as determined by Western blot analysis. (C) Immunofluoresence analysis in vitro demonstrated that MAP2 is exclusively expressed by the AD cells whereas β-catenin and PDGFR-β is overexpressed in AI cells compared to the AD cells. (D) Flow cytometric analysis confirmed that MAP2 is expressed by the AD cells only, whereas β-catenin and PDGFRβ are overexpressed in AI cells. Scale bar, 50 μm. MFI, mean fluorescence intensity.
Mentions: To determine whether the AI and AD phenotypes of Neuro2a cells have distinct molecular markers, we analyzed expression of eleven proteins that play a vital role in neuroblastoma homeostasis. These included proteins regulating proliferation (β-catenin/FZD1, SHH/Gli1/PTCH1, TrkB/BDNF, PDGFR), apoptosis (FZD1, Gli1), chemo-resistance (FZD1, Telomerase), angiogenesis (VEGF/VEGFR, PDGFRβ, SHH), metastasis (SHH, MAP2, TrkB), adhesion and migration (NCAM, TrkB, Dcx), and differentiation and neurite extension (MAP2, Dcx) (Eggert et al., 2000; Oltra et al., 2005; Nakamura et al., 2006; Flahaut et al., 2009; Korja et al., 2009; Krishnan et al., 2009; Bahnassy et al., 2010; Oue et al., 2010; Souzaki et al., 2010; Wesbuer et al., 2010; Zhang et al., 2010; Schiapparelli et al., 2011). Western blot analysis revealed differences in expression of PDGFRβ, MAP2, Dcx, NCAM, survivin, and β-catenin (Figures 6A,B), thereby identifying these molecules as potential unique markers of AI or AD phenotypes. To test the value of these molecules as markers of in vivo cellular commitment to AD or AI phenotypes we performed immunofluorescence microscopy. This analysis confirmed the findings for MAP2, β-catenin, and PDGFRβ, while staining for Dcx, NCAM, and survivin did not discriminate (Figure 6C). Flow cytometric analysis confirmed differences in the expression of MAP2, β-catenin, and PDGFRβ while all neuroblastoma cells were nestin positive (Figure 6D). These results showed that Nestin + MAP2+ cells are indicative of the AD phenotype while Nestin + MAP2− are markers of the AI phenotype.

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