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Journal of Molecular and Clinical Medicine  2018, Vol. 1 Issue (4): 219-226    DOI: 10.31083/j.jmcm.2018.04.4221
Research article Previous articles | Next articles
Adrenergic to mesenchymal fate switching of neuroblastoma occurs spontaneously in vivo resulting in differential tumorigenic potential
Maria C. Lecca1, Marianne A. Jonker2, U. Kulsoom Abdul4, Asli Küçükosmanoglu4, Wessel van Wieringen2, 3, Bart A. Westerman1, 4, *()
1 Department of Oncogenomics, Amsterdam Medical Center (AMC), the Netherlands
2 Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam
3 Department of Mathematics, VU University, Amsterdam, De Boelelaan 1081a, 1081 HV Amsterdam
4 Department of Neurosurgery, Cancer Center Amsterdam, VU University Medical Center, Amsterdam
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Abstract  

Neuroblastoma is a pediatric tumor that originates from cells of the adrenergic lineage. Here we investigated the balance between differentiation and dedifferentiation in relation to tumor-engraftment potential in preclinical mouse models. We analyzed intratumoral heterogeneity through comparison of marker expression of normal adrenergic development versus tumor marker expression, which showed the presence of sympathoadrenal as well as mesenchymal subtypes of neuroblastoma cells. Subsequently, we evaluated long-term outgrowth capacity of these two (FACS-sorted) cell populations, which showed that adrenergic cells have a stronger long-term clonogenic potential. Engraftment of these sorted populations into mice revealed the occurrence of heterogeneous populations. Modelling of the interconversion rate indicated that cell fate transitions from the adrenergic to mesenchymal state were obtained gradually and stochastically as the tumors grew in mice. We found that adrenergic cells have an increased tumorigenic potential in mice without signs of beneficial cross talk between the two lineage populations. These findings indicate that neuroblastoma contains two rivalling differentiation states that exhibit differences in long term clonal/tumorigenic potential. We expect these states to be relevant for therapy resistance as a result of intratumoral heterogeneity.

Revised:  09 December 2018      Accepted:  15 December 2018      Published:  20 December 2018     
*Corresponding Author(s):  Bart A. Westerman     E-mail:  a.westerman@vumc.nl

Cite this article: 

Maria C. Lecca, Marianne A. Jonker, U. Kulsoom Abdul, Asli Küçükosmanoglu, Wessel van Wieringen, Bart A. Westerman. Adrenergic to mesenchymal fate switching of neuroblastoma occurs spontaneously in vivo resulting in differential tumorigenic potential. Journal of Molecular and Clinical Medicine, 2018, 1(4): 219-226.

URL: 

https://jmcm.imrpress.com/EN/10.31083/j.jmcm.2018.04.4221     OR     https://jmcm.imrpress.com/EN/Y2018/V1/I4/219

Fig. 1.  A number of neuroblastoma cell lines resemble tumors of mesenchymal origin. tSNE clustering of mRNA expression data of 87 pediatric tumor cell lines consisting of the neuronal tumors medulloblastoma (n=14), neuroblastoma (n=26), tumors of mesenchymal origin, Ewing Sarcoma (n=21), rhabdomyosarcoma (n=19) and osteosarcoma (n=9) and the blood derived tumor type ALL (n=7). The three main clusters observed are neuronal tumors (blue), mesenchymal tumors (green) and blood ALL (red). Note that some neuroblastoma and medulloblastoma cell lines cluster together with tumors of mesenchymal origin. Perplexity of the clustering was 28. Isogenic pair: SHEP2 and SY5Y.

Fig. S1.  K-means clustering shows that some neuroblastoma cell lines cluster together with tumors of a mesenchymal origin. (B) PCA plot showing that some neuroblastoma cell lines cluster together with tumors of a mesenchymal origin (purple arrows).

Fig. 2.  Neuroblastomas express markers of normal sympathoadrenal differentiation. (A) Schematic visualization of neural crest to sympathoadrenal differentiation. Neuroblastomas show expression of mesenchymal and adrenergic stages but not of premigratory neural crest stages (B) Immunostainings of primary cultures of neuroblastoma patients. Cells were stained with antibodies that were suited for IHC of paraffin embedded cells (upper panels) or immunofluorescence of cells grown onto glass slides (lower panels).

Fig. 3.  Sympathoadrenal neuroblastoma cells have a long term clonal expansion capacity (A) qPCR data showing that FACS sorted AMC700B cells for CD133 expression shows that MES (CD133+) cells have lower expression of ADRN genes. (B) Cell cycle profile based on click it FACS analysis showing that MES and sympathoadrenal cells have a similar cell cycle profile. (C) Light microscopy of FACS sorted mesenchymal and sympathoadrenal cells showing that sympathoadrenal cells have a viable morphology in contrast to the mesenchymal cells. (D) Schematic outline of the clonal expansion experiment. Cells were FACS sorted for CD133/CD24 staining and plated for clonal outgrowth into 384 wells plates. Each well was checked for the presence of maximal one sorted cell. Cells were serially expanded and assayed for CD133 expression. (E) Clonal outgrowth and serial expansion of CD133-700B cells (NE type) occurred more frequent than clonal outgrowth of CD133+ cells (MES type). NE cells could be serially expanded for multiple passages leading to long term cultures. (F) Histogram showing the relative amount of short term expanding cultures versus long term expanding cultures. CD133-NE type neuroblastoma cells show long term expanding cultures only. (ST) Short term clonal capacity (in percentage), (LT) long term clonal capacity are the chances to generate a culture from a single cell that lasts for less or more than 5 passages, respectively. (G) Titration of the number of cells per 6 wells results in enrichment of CD133-cells upon lower seeding densities that required clonal outgrowth after two week of expansion.

Fig. 4.  Sympathoadrenal neuroblastoma cell cultures are more tumorigenic (A) FACS plot of CD133/CD24 contained cells one day after sorting B) Average survival of mice when ADRN and MES type cells are compared from three isogenic pairs (200,000 cells [AMC700B]; 5x106 [AMC691] and 1x106 [SKNSH] were injected). (C) Macroscopic image of immunohistochemistry for vimentin showing a patchwork pattern of vimentin positive and negative domains. (D) Immunostainings of primary cultures of neuroblastoma patients. Cells were stained with antibodies that were suited for IHC of paraffin embedded cells (upper panels) of immunofluorescence of cells grown onto glass slides (lower panels). * p<0.05 log rank test.

Fig. 5.  Cell fate interconversion occurs stochastically in vivo (A) Injection of CD133low as well as CD133high cells gave rise to heterogeneous tumors as shown by expression of the MES identifier: VIM (shown in brown) with mutual exclusive absence of expression of SYN and CHGA in these clones. (B) Circos plots representative of the clonal evolution analysis where the moment of establishment of the intratumoral heterogeneity was estimated from clone size. When MES cells were injected, occurrence of MES negative clones was overlapping with the time of tumor initiation, consistent with the tumor outgrowth phenotype that was determined based on the previous in vitro and in vivo experiments. (C) Circos plots showing that many clones that were derived from sympathoadrenal cells converted to a mesenchymal fate. (D) These clones were formed significantly later than the time point of tumor injection of the tumor, i.e. the occurrence of mesenchymal population is uncoupled from the tumor initiation. Significant cases are shown by an Asterix.

Fig. S2.  Kaplan Meier plot showing that co-injection or separate injection of 691 mesenchymal and adrenal cells gives a similar survival of the mice.

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