Contents
6.1 Introduction . . . 55
6.2 Neural Crest Differentiation . . . 55
6.3 Neuroblastoma Cellular Heterogeneity . . . 56
6.4 N-type Neuroblastic Cells . . . 56
6.5 S-type Non-Neural Cells . . . 57
6.6 I-type Stem Cells . . . 58
6.7 Transdifferentiation . . . 59
6.8 Conclusions . . . 60
References . . . 60
6.1 Introduction
Cellular heterogeneity, a hallmark of cancer, probably accounts for the variability of its clinical presentation and non-uniform response to treatment. For neuro- blastoma (NB), this heterogeneity results from the plasticity of the embryonic neural crest, from which this tumor originates (Biedler et al. 1997; Brodeur 2003). This chapter briefly reviews the lineages man- ifested by the developing neural crest and the biology of the distinct cell types in human NB.
6.2 Neural Crest Differentiation
The neural crest is a transient embryonic cell struc- ture generated from the neuroectodermal plate upon closure of the neural tube (Le Douarin and Ziller 1993). Migrating neural crest cells from the trunk re- gion of the embryo generate neuronal and glial cells of the peripheral nervous system, neuroendocrine and sensory ganglion cells, as well as non-neural pig- ment and smooth muscle-like cells. An important aspect of neural crest development germane to NB is that cell division continues along with the progres- sive restriction of differentiation potential and is even present in adrenal medullary cells postnatally (Mascorro and Yates 1989). Thus, two seemingly di- vergent cellular programs are operating simultane- ously: proliferation and differentiation.
Excellent studies have highlighted the amazing pluripotent nature of the neural crest anlage. Detailed studies using a chick/quail chimera showed that the local tissue microenvironment plays a pivotal role in effecting the differentiation lineages (Le Douarin and
Cellular Heterogeneity
Robert A. Ross
Ziller 1993). More recently, restrictive signaling fac- tors that promote commitment to particular cell fates in migratory and postmigratory neural crest precur- sor cells have been delineated (Lo et al. 2002; Hemmati et al. 2003; Luo et al 2003). For example, achaete–scute complex (e.g., HASH1) and atonal (ato) homologs (e.g., neurogenin) are required in vivo for develop- ment of autonomic and sensory neurons, respectively.
By contrast, melanocytes are generated by Wnt sig- naling, while TGF b promotes smooth muscle cell de- velopment. Finally, Notch and neuregulin promote satellite glial and Schwann cell differentiation.
6.3 Neuroblastoma Cellular Heterogeneity
Many of the cell phenotypes characteristic of the developing neural crest – neuroblasts, non-neuronal (Schwann, perineurial, or satellite) cells, and even melanocytes –are evident in the same NB (Shimada et al. 1999). Moreover, cellular heterogeneity and ex- tent of maturation (e.g., stroma-rich and stroma- poor tumors or high- and low-risk tumors based on histological grade) correlate with clinical behavior and are useful for prognostication of the disease (see Chap. 7; Shimada et al. 1999).
This same cellular heterogeneity is seen in NB cell lines. Three distinct cellular phenotypic variants have been described (Rettig et al. 1987; Biedler et al. 1988, 1997): sympathoadrenal (N-type) neuroblasts; large flattened, substrate-adherent (S-type) cells; and mor- phologically intermediate (I-type) cells (Fig. 6.1).
Studies over the past 25 years have shown that each phenotype represents a particular lineage within the neural crest. The availability of cell lines of the three cell types has led to an increased understanding of the differentiation and malignant potentials of each.
6.4 N-type Neuroblastic Cells
In vitro, the predominant neuroblastic (N) cells re- semble sympathoadrenoblasts – immature neural/
neuroendocrine precursors, with small rounded cell bodies and neuritic processes that vary widely in number and length. Cells adhere poorly to the under-
lying substrate but adhere well to each other to form cell clumps (pseudoganglia), achieving high satura- tion densities in culture (Rettig et al. 1987; Biedler et al. 1997; Spengler et al. 1997). Biochemically, they ex- press proteins for synthesis, binding, and degrada- tion of norepinephrine and acetylcholine (the two major neurotransmitters of the peripheral nervous system), as well as opioid and cholinergic receptors.
They express the neuroectodermal stem cell interme- diate filament nestin, as well as all three neurofila- ment proteins and chromogranin A (CgA) and secre- togranin II (SgII), depending on their degree of dif- ferentiation (Biedler et al. 1997; Ross et al. 2002;
Thomas 2003). In addition, they express dHAND and HASH-1, transcription factors that are markers of the early stages of neural crest development (Jögi et al.
2002).
Another transcription factor associated with NB and early neuronal development is MYCN (Chap. 4).
Expression of the oncoprotein is associated with in- creased mitosis and a dedifferentiated state in neu- roectodermal cells of the CNS. High-level expression requires a neuroblastic phenotype, as non-neuronal variants do not express the protein even in the cell lines with transcriptionally active, amplified MYCN genes (Spengler et al. 1997).
N-type cells are tumorigenic. They form colonies in soft agar and tumors in mice, with variable degrees of malignancy (Spengler et al. 1997); however, too few MYCN-nonamplified N-type cell lines have been test- ed to discern a relation between MYCN amplification status and malignant potential.
Experimental protocols can induce N-type cells to differentiate along either a neuronal or a neuro- endocrine pathway or de-differentiate to an imma- ture neural crest-like phenotype. Neuronal differenti- ation following addition of retinoids or cyclic AMP- elevating agents is characterized by decreases in cell division and amounts of CgA and MYCN protein and increases in SgII and neurofilament proteins and in the number and length of neurites (Ross et al. 2002).
Neuroendocrine differentiation induced by synthetic glucocorticoids results in cell flattening, increases in CgA and MYCN levels, and decreases in neurite for- mation, SgII, and neurofilaments (Ross et al. 2002).
Hypoxia has also been shown to affect neuroblastic
differentiation. Growth of N-type cells under hypox- ic conditions causes decreased expression of neu- ronal/ neuroendocrine-specific genes (e.g., CgA and neuropeptide Y) and increased expression of genes present in early neural crest development (c-kit, Notch-1, and HES-1) – indicators of de-differentia- tion (Jögi et al. 2002).
6.5 S-type Non-Neural Cells
In addition to neuroblasts, a second, clearly non-neu- ronal cell type is frequently observed in NB cell lines.
Termed S, for “substrate adherent”, it exhibits contact inhibition of growth, extensive migration on a sub- strate, and a limited lifespan in culture. Unlike the clearly defined neuronal lineage of N cells, the bio- chemical signature of S-type cells is more variable.
Studies have identified melanocytic properties (ty- rosinase, melanosomal glycoproteins, and melano- somes), Schwann or glial cell markers (chondroitin sulfate proteoglycans and large amounts of laminins and fibronectin), and/or smooth muscle cell features (alpha-smooth muscle actin and calponin) (Rettig et al. 1987; Tsokos et al. 1987; Jessen and Mirsky 1999;
Sugimoto et al. 2000). All of these lineages are consis- tent with a neural crest origin for the S cell, as devel- oping crest cells of the trunk give rise to non-neu- ronal Schwann, glial, melanocytic, and smooth mus- cle cell components in vivo. The presence of nestin in these cells is consistent with the S-cell phenotype as a neuroectodermal precursor of the non-neuronal lin- eages of the neural crest (Thomas 2003).
S cells differ from N cells in two other aspects.
Firstly, S cells display markers for HLA class-I anti- gens and b2-microglobulin, which are absent on N- type cells (Rettig et al. 1987). Secondly, unlike N cells, S-type cells will not grow in soft agar or form tumors in nude mice (Biedler et al. 1988; Spengler et al. 1997).
The NB tumors with abundant stroma (stroma- rich) generally have a better prognosis than stroma- poor tumors (Ambros and Ambros 1995; Shimada et al. 1999; Brodeur 2003). The discovery that, in vitro, N and S cells arise from a common precursor suggested that, in vivo, stromal cells could be of tumor origin.
One study, using paraffin nonisotopic in situ hy- bridization, concluded that stromal cells are of nontumor origin, presumably recruited by the neu- roblasts in the tumor (Ambros and Ambros 1995).
Subsequent studies, using short-term culture of
Figure 6.1
Phase-contrast photomicrographs of phenotypic cell variants derived from the LA-N-1 (N), SK-N-BE(2) (I), and SMS-KCN (S) neuroblastoma cell lines (magnification ×500)
bone marrow tumor cells or laser-capture microdis- section with bicolor fluorescence in situ hybridiza- tion, showed that both neuroblasts and Schwann cells had identical genetic markers – strong evidence that they arise from a neoplastic precursor (Valent et al.
1999; Mora et al. 2001). This topic is still under de- bate.
Equally important is the extent of the interaction between Schwann (or S-type) cells and neuroblastic (or N-type) cells in the survival/proliferation/differ- entiation of each phenotype. In early experiments, N cells co-cultured with S cells were much more differ- entiated and grew more slowly (B.A. Spengler and J.L.
Biedler, personal communication). Such results are consistent with studies of developing neurons and Schwann cells which show that reciprocal contact de- termines survival and differentiation (Jessen and Mirsky 1999). Also, conditioned medium from nor- mal Schwann cells in culture increases NB cell sur- vival and differentiation (Kwiatkowski et al. 1998) and contains a potent inhibitor of angiogenesis, thus providing a mechanistic basis for the benign behav- ior of stroma-rich tumors (Huang et al. 2000).
6.6 I-type Stem Cells
The I-type cell was initially identified in cultured cell lines because it appeared “intermediate” in morphol- ogy between N and S cells. It exhibits morphological features of both N-type cells (short neurite-like cell processes and growth to high saturation densities) and S-type cells (strong adhesion to and extensive migration over the substrate) (Biedler et al. 1997).
These cells also express proteins of both differen- tiation pathways – noradrenergic biosynthetic en- zymes, granins (CgA and SgII), and neurofilament proteins of neuroblasts as well as S cell proteins vimentin, EGF receptor, and CD44. Examples of I-type cells include the cell lines GOTO, NUB-7, BE(2)-C, SH-IN, and LA-N-2 (Biedler et al. 1988, 1997;
Ross et al. 1995, 2002).
Continuing research indicates that this cell repre- sents a unique cell type within the NB repertoire. Its ability to generate daughter cells with the same phe- notype (self-renewal) and to differentiate bidirection-
ally along either neuroblastic or Schwann/glial path- ways suggests that it is a neural crest cancer stem cell.
First demonstrated for BE(2)-C cell clones and subsequently for other I-type cell lines, I-type cells become neuroblastic when treated for 7–14 days with retinoic acid (RA), but differentiate into S-type cells following treatment with BUdR (Ross et al. 1995). Unlike N (or S) cells, I cells retain the ability to convert to two distinctly different cell line- ages.
The most provocative finding regarding the NB I- type stem cell is its malignant potential. As a group, these stem cells are more malignant than neuroblas- tic variants; they have four- to fivefold higher colony- forming efficiencies in soft agar than N cells and have an over sixfold greater capacity to form tumors in nude mice (Ross et al. 2003; Spengler et al. 1986;
Walton et al. 2004). Moreover, phenotype rather than MYCN amplification/overexpression determines ma- lignancy; e.g., NB I-type stem cells lacking MYCN amplification are more tumorigenic than N-type cells which contain >150-fold amplified genes. Thus, re- search to date on cell lines suggests that the I-like stem cell could be the truly tumorigenic cell component of NB tumors.
Malignant stem cells in tumors could exert a sig-
nificant negative effect on prognosis and long-term
survival; however, distinguishing putative stem cells
from those with a neuroblastic phenotype in tumor
sections by routine hematoxylin–eosin analysis is
difficult, if not impossible. To specifically search for
I-like cells, tumor sections immunostained conjoint-
ly with antibodies specific for N or S cells were
examined for the presence and frequency of double-
labeled cells (Ross et al. 2003). In preliminary an-
alyses, doubly labeled I-like cells were present in all
tumors (Fig. 6.2). When the tumors were grouped
as either good risk (typically local regional or
stage 4s) or poor risk (stages 3 or 4), the frequency
of I-like cells was significantly higher (~ fivefold)
in the latter group (B.A. Spengler, personal commu-
nication). The characterization of this previously
unnoticed NB cell type in cell lines and its potential
role in refractory high-risk tumors may have identi-
fied an important new target for experimental thera-
peutics.
6.7 Transdifferentiation
Transdifferentiation is the process whereby cells change from one unique phenotype into another unique phenotype without going through a develop- mentally less mature stage (Liu and Rao 2003). This process has been reported for cells from all three germ layers and, in particular, for cells of the hematopoietic and neural systems. However, distin- guishing de-differentiation/re-differentiation (a two- stage process involving reversion to a more immature stage before expression of the novel phenotype) from transdifferentiation is not easy, especially in hetero- geneous populations of cells or those where the inter- mediate cell type is not readily identifiable (Liu and Rao 2003). In studies with SH-SY5Y (N-type) and SH- EP (S-type), clones of the SK-N-SH cell line, cells with morphological and biochemical features of the other phenotype arose spontaneously and were subcloned (Ross et al. 1995). Of importance, transdifferentiated subclones each have a marker chromosome unique to the clone of origin; therefore, these lines did not arise by clonal selection of pre-existing variants, as has been suggested (Cohen et al. 2003), but represent the
conversion to a new cell phenotype. Similar pheno- typic conversions have been seen for the LA-N-1 and SK-N-BE(2) cell lines: N-type LA1–55n arose sponta- neously and was cloned from the S-type LA1–5s as were S-type LA1–19Bs cells from the N-type LA1–19n clonal cell line. Likewise, the twice-cloned BE(2)-M17 cell line gave rise to the BE(2)-M17F S-type clone. In all cases, the interconversion/transd- ifferentiation process occurred spontaneously and morphological, biochemical, and cytogenetic criteria were used to confirm the phenotype and cell of ori- gin. Transdifferentiation is very rare and it is the abil- ity to select for the different cell types in culture that has permitted its documentation. Whether the phe- nomenon observed in NB represents true transdiffer- entiation or a more complex process involving de- differentiation followed by differentiation along a second neural crest pathway has not been resolved.
Nevertheless, the interconversion of N- and S-type cells in culture would suggest that it may occur in vivo. The evolution of quiescent S-type cells into highly proliferative N or I cells mimics the clinical picture of a rapidly recurrent neuroblastoma follow- ing a period of clinical remission.
Figure 6.2
Neuroblastoma tumor section stained conjointly for expression of S100A6 (an S cell marker; red) and neurofilament 160 (an N- cell marker; gray). Examples of I-like cells expressing both pro- teins are indicated by filled arrows, whereas N cells express- ing only neurofilament protein are denoted by open arrows
6.8 Conclusions
Cellular heterogeneity is a common feature of human NB tumors and cell lines. Moreover, the different phe- notypes identified in fresh tumors are similar, if not identical, to those seen in cell lines; thus, cell lines may serve as useful surrogates in the investigation of the biochemical, differentiable, and tumorigenic het- erogeneity of human NBs. It is clear that the cell vari- ants differ markedly in growth potential, both in vit- ro and in vivo. A small amount of experimental data would also suggest that variants differ in the intrinsic sensitivities to commonly used chemotherapeutic agents. It is also clear that “cross-talk” may occur be- tween cell variants within tumors, further influenc- ing tumor viability, tumorigenicity, or response to therapy. The identification of putative stem cells within tumors and characterization in cell culture may offer new opportunities for developing strate- gies for more effective control of NB.
Acknowledgements.
Much of the research and thought represented in this chapter is the result of a highly productive collaboration between myself, J.L.
Biedler, and B.A. Spengler. Without their efforts and support, this work would not have been accom- plished.
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