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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

Neuroblastoma tumor cell heterogeneity in mouse model. (A) Flow cytometric phenotyping analysis on mouse neuroblastoma tumors of 10 mm diameter showed no remarkable difference in the expression of MAP2, β-catenin, and PDGFRβ in the two forms of tumor. MFI, mean fluorescence intensity. (B) Western blot analysis on mouse neuroblastoma tumors (5, 10, and 15 mm in diameters) revealed the heterogeneity of MAP2, PDGFRβ, and β-catenin expression in either forms of tumor suggesting that phenotypic transitions occur in vivo. (C) Immunofluorescence staining on frozen sections from mouse neuroblastoma tumors (grown from either AD or AI cells) of 10 mm diameter supports the flow cytometric and Western blot analysis of tumor cell heterogeneity. (D) H&E staining of tumors reveals that both AD and AI forms of Neuro2a cells gave rise to histopathologically similar tumors in mice. (E)In vivo tumorigenic potential of the AD and AI phenotypes of Neuro2a cells were compared by inoculating the mice with either form of cell phenotype and measuring the tumor over time. Both cell types gave rise to very large tumors and the growth rates were indistinguishable. Data points expressed as mean ± S.D. (n = 12). Scale bar, 50 μm.
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Figure 7: Neuroblastoma tumor cell heterogeneity in mouse model. (A) Flow cytometric phenotyping analysis on mouse neuroblastoma tumors of 10 mm diameter showed no remarkable difference in the expression of MAP2, β-catenin, and PDGFRβ in the two forms of tumor. MFI, mean fluorescence intensity. (B) Western blot analysis on mouse neuroblastoma tumors (5, 10, and 15 mm in diameters) revealed the heterogeneity of MAP2, PDGFRβ, and β-catenin expression in either forms of tumor suggesting that phenotypic transitions occur in vivo. (C) Immunofluorescence staining on frozen sections from mouse neuroblastoma tumors (grown from either AD or AI cells) of 10 mm diameter supports the flow cytometric and Western blot analysis of tumor cell heterogeneity. (D) H&E staining of tumors reveals that both AD and AI forms of Neuro2a cells gave rise to histopathologically similar tumors in mice. (E)In vivo tumorigenic potential of the AD and AI phenotypes of Neuro2a cells were compared by inoculating the mice with either form of cell phenotype and measuring the tumor over time. Both cell types gave rise to very large tumors and the growth rates were indistinguishable. Data points expressed as mean ± S.D. (n = 12). Scale bar, 50 μm.

Mentions: To determine the in vivo representation and stability of the in vitro phenotypes, mice were inoculated separately with Neuro2a cells of AD or AI phenotypes. Nestin, MAP2, β-catenin, and PDGFRβ expression were analyzed in tumors reaching 5, 10, and 15 mm in diameter. Flow cytometry and Western blot analyses revealed similar abundance of each protein in tumors of corresponding sizes irrespective of the phenotype injected (Figures 7A,B). This finding suggests that either both AI and AD phenotypes were present in vivo, or that a third phenotype containing both AI and AD markers was predominant in vivo. To distinguish between these possibilities we stained frozen tumor sections with nestin, MAP2, PDGFRβ, and β-catenin specific antibodies. This analysis showed heterogeneity of cells in situ (Figure 7C). In light of ubiquitous nestin expression, scattered areas with differential MAP2, PDGFRβ, and β-catenin staining represent Neuro2a cells of AD (MAP2) or AI (PDGFRβ and β-catenin) phenotype. This conclusion is also supported by co-staining of nestin and MAP2 showing both Nestin + MAP2+ and Nestin + MAP2− cells in tumors irrespective of their phenotype prior to inoculation (Figure 7C). The pockets of cells with distinct phenotypes were not localized to a specific region in the tumor mass, but seemed randomly distributed (data not shown). Not surprisingly, the histopathologic appearance (Figure 7D) and in vivo growth (Figure 7E) of the tumors were also indistinguishable. Therefore, Neuro2a cells driven to AI or AD phenotype in vitro display a tendency to transition, establish, and maintain an approximate 1:1 ratio of the two phenotypes in vivo.


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

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

Neuroblastoma tumor cell heterogeneity in mouse model. (A) Flow cytometric phenotyping analysis on mouse neuroblastoma tumors of 10 mm diameter showed no remarkable difference in the expression of MAP2, β-catenin, and PDGFRβ in the two forms of tumor. MFI, mean fluorescence intensity. (B) Western blot analysis on mouse neuroblastoma tumors (5, 10, and 15 mm in diameters) revealed the heterogeneity of MAP2, PDGFRβ, and β-catenin expression in either forms of tumor suggesting that phenotypic transitions occur in vivo. (C) Immunofluorescence staining on frozen sections from mouse neuroblastoma tumors (grown from either AD or AI cells) of 10 mm diameter supports the flow cytometric and Western blot analysis of tumor cell heterogeneity. (D) H&E staining of tumors reveals that both AD and AI forms of Neuro2a cells gave rise to histopathologically similar tumors in mice. (E)In vivo tumorigenic potential of the AD and AI phenotypes of Neuro2a cells were compared by inoculating the mice with either form of cell phenotype and measuring the tumor over time. Both cell types gave rise to very large tumors and the growth rates were indistinguishable. Data points expressed as mean ± S.D. (n = 12). Scale bar, 50 μm.
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Figure 7: Neuroblastoma tumor cell heterogeneity in mouse model. (A) Flow cytometric phenotyping analysis on mouse neuroblastoma tumors of 10 mm diameter showed no remarkable difference in the expression of MAP2, β-catenin, and PDGFRβ in the two forms of tumor. MFI, mean fluorescence intensity. (B) Western blot analysis on mouse neuroblastoma tumors (5, 10, and 15 mm in diameters) revealed the heterogeneity of MAP2, PDGFRβ, and β-catenin expression in either forms of tumor suggesting that phenotypic transitions occur in vivo. (C) Immunofluorescence staining on frozen sections from mouse neuroblastoma tumors (grown from either AD or AI cells) of 10 mm diameter supports the flow cytometric and Western blot analysis of tumor cell heterogeneity. (D) H&E staining of tumors reveals that both AD and AI forms of Neuro2a cells gave rise to histopathologically similar tumors in mice. (E)In vivo tumorigenic potential of the AD and AI phenotypes of Neuro2a cells were compared by inoculating the mice with either form of cell phenotype and measuring the tumor over time. Both cell types gave rise to very large tumors and the growth rates were indistinguishable. Data points expressed as mean ± S.D. (n = 12). Scale bar, 50 μm.
Mentions: To determine the in vivo representation and stability of the in vitro phenotypes, mice were inoculated separately with Neuro2a cells of AD or AI phenotypes. Nestin, MAP2, β-catenin, and PDGFRβ expression were analyzed in tumors reaching 5, 10, and 15 mm in diameter. Flow cytometry and Western blot analyses revealed similar abundance of each protein in tumors of corresponding sizes irrespective of the phenotype injected (Figures 7A,B). This finding suggests that either both AI and AD phenotypes were present in vivo, or that a third phenotype containing both AI and AD markers was predominant in vivo. To distinguish between these possibilities we stained frozen tumor sections with nestin, MAP2, PDGFRβ, and β-catenin specific antibodies. This analysis showed heterogeneity of cells in situ (Figure 7C). In light of ubiquitous nestin expression, scattered areas with differential MAP2, PDGFRβ, and β-catenin staining represent Neuro2a cells of AD (MAP2) or AI (PDGFRβ and β-catenin) phenotype. This conclusion is also supported by co-staining of nestin and MAP2 showing both Nestin + MAP2+ and Nestin + MAP2− cells in tumors irrespective of their phenotype prior to inoculation (Figure 7C). The pockets of cells with distinct phenotypes were not localized to a specific region in the tumor mass, but seemed randomly distributed (data not shown). Not surprisingly, the histopathologic appearance (Figure 7D) and in vivo growth (Figure 7E) of the tumors were also indistinguishable. Therefore, Neuro2a cells driven to AI or AD phenotype in vitro display a tendency to transition, establish, and maintain an approximate 1:1 ratio of the two phenotypes in vivo.

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