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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

Chemotherapy alters heterogeneity of cells in small tumors. (A) Flow cytometric analysis on mouse neuroblastoma tumors of 5 mm diameter showed no remarkable difference in the expression of MAP2, β-catenin, and PDGFRβ in the two forms of tumor (grown from either AD or AI cells). (B) Doxorubicin treatment had no effect on the cellular heterogeneity on tumors of 5 mm diameter grown from AD cells; whereas the AI-tumors of similar size lost their heterogeneity, remained in their native form, and did not transition during doxorubicin treatment. (C) Combination of doxorubicin and metformin prevented the AD phenotype from establishing itself in the early stage (5 mm diameter) of tumor formation as defined by the absence of MAP2+ cells and the cells remained in the AI form. (D) Combined dose of Erlotinib (EGFR inhibitor) and PD173074 (FGFR inhibitor) prevented the AI phenotype from establishing itself in the early stage (5 mm diameter) of tumor formation as defined by the presence of MAP2+ cells and the cells remained in the AD form. (E) Combination of chemotherapy and growth factor receptor inhibitors resulted in the emergence of newer phenotypes as evident from the presence of unique/unfamiliar populations of MAP2+ and PDGFRβ+ cells. MFI, mean fluorescence intensity. AD, AI.
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Figure 10: Chemotherapy alters heterogeneity of cells in small tumors. (A) Flow cytometric analysis on mouse neuroblastoma tumors of 5 mm diameter showed no remarkable difference in the expression of MAP2, β-catenin, and PDGFRβ in the two forms of tumor (grown from either AD or AI cells). (B) Doxorubicin treatment had no effect on the cellular heterogeneity on tumors of 5 mm diameter grown from AD cells; whereas the AI-tumors of similar size lost their heterogeneity, remained in their native form, and did not transition during doxorubicin treatment. (C) Combination of doxorubicin and metformin prevented the AD phenotype from establishing itself in the early stage (5 mm diameter) of tumor formation as defined by the absence of MAP2+ cells and the cells remained in the AI form. (D) Combined dose of Erlotinib (EGFR inhibitor) and PD173074 (FGFR inhibitor) prevented the AI phenotype from establishing itself in the early stage (5 mm diameter) of tumor formation as defined by the presence of MAP2+ cells and the cells remained in the AD form. (E) Combination of chemotherapy and growth factor receptor inhibitors resulted in the emergence of newer phenotypes as evident from the presence of unique/unfamiliar populations of MAP2+ and PDGFRβ+ cells. MFI, mean fluorescence intensity. AD, AI.

Mentions: Since Neuro2A AI and AD phenotypes were differentially susceptible to doxorubicin exposure in vitro, we sought to determine the effects, if any, doxorubicin might exert on the Neuro2a phenotype of cells growing in vivo. Mice inoculated with AD and AI forms of Neuro2a cells were treated with doxorubicin. Initial tumor growth was found to be slower in the doxorubicin treated mice compared to the untreated mice (Figures 9A,B). At an early stage (5 mm in diameter), doxorubicin treated AD tumors maintained their heterogeneity similar to the untreated tumor (Figure 10A); whereas the AI-tumors remained in AI form and did not transition (Figure 10B). This observation shows that doxorubicin initially prevented transition to the more sensitive AD phenotype. Interestingly, as the tumors grew larger (10–15 mm in diameter), they became resistant to doxorubicin, displayed faster growth in vivo (Figure 9A), and both AD and AI forms were reconstituted (Figure 11A).


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

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

Chemotherapy alters heterogeneity of cells in small tumors. (A) Flow cytometric analysis on mouse neuroblastoma tumors of 5 mm diameter showed no remarkable difference in the expression of MAP2, β-catenin, and PDGFRβ in the two forms of tumor (grown from either AD or AI cells). (B) Doxorubicin treatment had no effect on the cellular heterogeneity on tumors of 5 mm diameter grown from AD cells; whereas the AI-tumors of similar size lost their heterogeneity, remained in their native form, and did not transition during doxorubicin treatment. (C) Combination of doxorubicin and metformin prevented the AD phenotype from establishing itself in the early stage (5 mm diameter) of tumor formation as defined by the absence of MAP2+ cells and the cells remained in the AI form. (D) Combined dose of Erlotinib (EGFR inhibitor) and PD173074 (FGFR inhibitor) prevented the AI phenotype from establishing itself in the early stage (5 mm diameter) of tumor formation as defined by the presence of MAP2+ cells and the cells remained in the AD form. (E) Combination of chemotherapy and growth factor receptor inhibitors resulted in the emergence of newer phenotypes as evident from the presence of unique/unfamiliar populations of MAP2+ and PDGFRβ+ cells. MFI, mean fluorescence intensity. AD, AI.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: Chemotherapy alters heterogeneity of cells in small tumors. (A) Flow cytometric analysis on mouse neuroblastoma tumors of 5 mm diameter showed no remarkable difference in the expression of MAP2, β-catenin, and PDGFRβ in the two forms of tumor (grown from either AD or AI cells). (B) Doxorubicin treatment had no effect on the cellular heterogeneity on tumors of 5 mm diameter grown from AD cells; whereas the AI-tumors of similar size lost their heterogeneity, remained in their native form, and did not transition during doxorubicin treatment. (C) Combination of doxorubicin and metformin prevented the AD phenotype from establishing itself in the early stage (5 mm diameter) of tumor formation as defined by the absence of MAP2+ cells and the cells remained in the AI form. (D) Combined dose of Erlotinib (EGFR inhibitor) and PD173074 (FGFR inhibitor) prevented the AI phenotype from establishing itself in the early stage (5 mm diameter) of tumor formation as defined by the presence of MAP2+ cells and the cells remained in the AD form. (E) Combination of chemotherapy and growth factor receptor inhibitors resulted in the emergence of newer phenotypes as evident from the presence of unique/unfamiliar populations of MAP2+ and PDGFRβ+ cells. MFI, mean fluorescence intensity. AD, AI.
Mentions: Since Neuro2A AI and AD phenotypes were differentially susceptible to doxorubicin exposure in vitro, we sought to determine the effects, if any, doxorubicin might exert on the Neuro2a phenotype of cells growing in vivo. Mice inoculated with AD and AI forms of Neuro2a cells were treated with doxorubicin. Initial tumor growth was found to be slower in the doxorubicin treated mice compared to the untreated mice (Figures 9A,B). At an early stage (5 mm in diameter), doxorubicin treated AD tumors maintained their heterogeneity similar to the untreated tumor (Figure 10A); whereas the AI-tumors remained in AI form and did not transition (Figure 10B). This observation shows that doxorubicin initially prevented transition to the more sensitive AD phenotype. Interestingly, as the tumors grew larger (10–15 mm in diameter), they became resistant to doxorubicin, displayed faster growth in vivo (Figure 9A), and both AD and AI forms were reconstituted (Figure 11A).

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