A role for distinct cell types in determining malignancy in human neuroblastoma cell lines and tumors

Human neuroblastoma arises from the developing neural crest. Tumors are categorized clinically by their location, age at diagnosis, spread/metastasis, and degree of cellular maturation and heterogeneity. Our long-term studies have shown the presence in human neuroblastoma cell lines of three distinct cell types: I-type stem cells, N-type neuroblastic/neuroendocrine precursors, and S-type Schwannian/melanoblastic precursors. These distinct cell types can differentiate predictably along specific neural crest lineages in response to particular morphogens. As assessed by tumor formation in nude mice and anchorage-independent growth in soft agar, I-type stem cells are significantly more malignant than either N- or S-type cells. Recent research shows that three similar cell types are also present in human neuroblastoma tumors. Using immunocytochemical, laser-capture microdissection, or short-term culture methods to identify cell types in tumors of different stages and/or different outcomes, these studies have shown that (1) all tumors contain neuroblasts in various differentiation states; (2) presumptive I-type stem cells are present in tumors of all stages; and (3) stromal cells may be tumor-derived, i.e. S-type cells, as well as of normal origin. More importantly, there is a higher incidence of I-type cells in tumors that progress, consistent with the high malignant potential of this cell type in vitro. A better understanding of the cause and consequences of cellular heterogeneity of human neuroblastoma tumors is an important prerequisite to the development of more effective therapies for this often fatal disease.     

Expression of the smg p25A (a ras p21-like GTP-binding protein) gene in human neuroblastoma cell lines and tumor tissues

We have examined expression of the smg p25A (a ras p21-like GTP-binding protein) gene in neural crest-derived tumor cell lines and neuroblastoma tissues. The human neuroblastoma cell lines GOTO, IMR-32, NB-1, and SK-N-SH expressed the 1.6-kilobase smg-25A mRNA. SH-SY5Y and SH-IN, variant cell lines with a neuronal phenotype derived from SK-N-SH, expressed much more smg-25A mRNA than did SH-EP1, a variant line with an epithelium-like phenotype also derived from SK-N-SH. The primitive neuroectodermal tumor cell lines SK-N-MC and KU-SN and the Ewing's sarcoma cell lines RD-ES and SK-ES expressed the smg-25A mRNA to a much smaller extent than did neuroblastoma cell lines. Of 15 human neuroblastoma specimens tested, 13 expressed the smg-25A mRNA to various extents. When the relative ratio of the smg-25A mRNA level to the glyceraldehyde-3-phosphate dehydrogenase mRNA level was compared among neuroblastoma tumor tissues, the value was significantly higher in tumors histologically diagnosed as ganglioneuroblastoma. The smg-25A mRNA was not detected in the tissues of Hodgkin's lymphoma, Wilms' tumor, Ewing's sarcoma, or undifferentiated sarcoma of the liver. These results suggest that the smg-25A mRNA level is closely related to the neuronal differentiation state of tumors derived from the neural crest.     

Neuroblastoma cells isolated from bone marrow metastases contain a naturally enriched tumor-initiating cell

Neuroblastoma is a heterogeneous pediatric tumor thought to arise from the embryonic neural crest. Identification of the cell responsible for propagating neuroblastomas is essential to understanding this often recurrent, rapidly progressing disease. We have isolated and characterized putative tumor-initiating cells from 16 tumors and bone marrow metastases from patients in all neuroblastoma risk groups. Dissociated cells from tumors or bone marrow grew as spheres in conditions used to culture neural crest stem cells, were capable of self-renewal, and exhibited chromosomal aberrations typical of neuroblastoma. Primary spheres from all tumor risk groups differentiated under neurogenic conditions to form neurons. Tumor spheres from low-risk tumors frequently formed large neuronal networks, whereas those from high-risk tumors rarely did. As few as 10 passaged tumor sphere cells from aggressive neuroblastoma injected orthotopically into severe combined immunodeficient/Beige mice formed large neuroblastoma tumors that metastasized to liver, spleen, contralateral adrenal and kidney, and lung. Furthermore, highly tumorigenic tumor spheres were isolated from the bone marrow of patients in clinical remission, suggesting that this population of cells may predict clinical behavior and serve as a biomarker for minimal residual disease in high-risk patients. Our data indicate that high-risk neuroblastoma contains a cell with cancer stem cell properties that is enriched in tumor-initiating capacity. These cells may serve as a model system to identify the molecular determinants of neuroblastoma and to develop new therapeutic strategies for this tumor.     

Human neuroblastoma stem cells

Human neuroblastoma is an embryonic cancer of the neural crest. Cellular heterogeneity is a characteristic feature of both tumors and derived cell lines. Recent studies have revealed that both cell lines and tumors contain cancer stem cells. In culture, these cells are self-renewing, multipotent, and highly malignant; in tumors their frequency correlates with a worse prognosis. Their identification and characterization should now permit a targeted approach to more effective treatment of this often fatal childhood cancer.     

Restoration of promyelocytic leukemia protein-nuclear bodies in neuroblastoma cells enhances retinoic acid responsiveness

Neuroblastoma is the most common solid tumor of infancy and is believed to result from impaired differentiation of neuronal crest embryonal cells. The promyelocytic leukemia protein (PML)-nuclear body is a cellular structure that is disrupted during the pathogenesis of acute promyelocytic leukemia, a disease characterized by impaired myeloid cell differentiation. During the course of studies to examine the composition and function of PML-nuclear bodies, we observed that the human neuroblastoma cell line SH-SY5Y lacked these structures and that the absence of PML-nuclear bodies was a feature of N- and I-type, but not S-type, neuroblastoma cell lines. Induction of neuroblastoma cell differentiation with 5-bromo-2'deoxyuridine, all-trans-retinoic acid, or IFN-gamma induced PML-nuclear body formation. PML-nuclear bodies were not detected in tissue sections prepared from undifferentiated neuroblastomas but were present in neuroblasts in differentiating tumors. Expression of PML in neuroblastoma cells restored PML-nuclear bodies, enhanced responsiveness to all-trans-retinoic acid, and induced cellular differentiation. Pharmacological therapies that increase PML expression may prove to be important components of combined modalities for the treatment of neuroblastoma.     

ARID3B induces malignant transformation of mouse embryonic fibroblasts and is strongly associated with malignant neuroblastoma

ARID3B, a member of the AT-rich interaction domain (ARID) family of proteins, plays an essential role in the survival of neural crest during embryogenesis. Here, we report evidence that ARID3B is involved in the development of malignant neuroblastoma, a childhood tumor derived from neural crest. (a) ARID3B is expressed by all five cell lines derived from neuroblastoma tested by us. (b) Analysis of published DNA microarray data of fresh neuroblastoma tumors showed that ARID3B is expressed in 80% of stage IV tumors, whereas only in 9% of stage I-III+IVs tumors. (c) In vitro growth of several neuroblastoma cell lines is suppressed significantly by antisense as well as siRNA treatment. (d) An increase of the ARID3B expression level by transfection in the SY5Y neuroblastoma cell line enhances the malignancy in tumor growth assays in nu/nu mice. (e) ARID3B by itself can immortalize mouse embryonic fibroblasts (MEFs) in vitro and confers malignancy to MEF when transfected together with MYCN, the best characterized oncogene for neuroblastoma. Thus, ARID3B seems to play a key role in the malignant transformation of neuroblastoma and may serve not only as a marker of malignancy but also as a potential target for cancer therapy of stage IV neuroblastoma for which there is currently no effective treatment available.     

Importance of phenotypic and molecular characterization for identification of a neuroepithelioma tumor cell line, NUB-20

A neuroblastic-like cell line (NUB-20) was derived from a case of histopathologically diagnosed metastatic neuroblastoma. The metastatic tumor and nude mouse heterotransplant resembled neuroblastoma by histological criteria, in contrast to the primary tumor, which was differentially classified as Ewing's sarcoma. However, the cell line demonstrated a unique phenotype in culture with respect to morphology, immunohistochemical markers, and sensitivity to a battery of differentiation modulators. These characteristics, together with the presence of a chromosomal translocation (11;22),(q24;q12) and amplification with enhanced expression of the c-myc protooncogene rather than N-myc, established this tumor as neuroepithelioma. Neuroepithelioma is a tumor type distinct from, but related to, neuroblastoma in its development from the neural crest lineage. These results emphasize the growing importance of cytogenetic and molecular markers in the classification and characterization of human tumors.     

Comprehensive genomics linking between neural development and cancer: neuroblastoma as a model

Cancer cells are derived from their precursor cells, which normally develop to the matured cells to form individual organs. Neuroblastoma, one of the most common pediatric solid tumors, originates from possible cancer stem cells derived from the neural crest. During the development, neural crest cells segregate into several lineages such as sensory, enteric and sympathetic neurons. However, the genetic events to cause neuroblastoma occur only in the sympathetic precursor cells or cancer stem cells. Furthermore, spontaneous regression of a subset of neuroblastoma found in patients under one year of age mimics a developmentally programmed neuronal cell death that occurs in normal sympathetic neurons during the perinatal period. Thus, the genetic events to cause neuroblastoma may be programmed to occur in a lineage-specific as well as developmentally regulated manner. In this review, we discuss about the molecular link between neural development and the genesis of neuroblastoma based on our comprehensive genomics approach.     

131I-meta-iodobenzylguanidine in diagnosis and treatment of neuroblastoma

131I MIBG is taken up and stored by neural crest tumors, essentially pheochromocytoma and neuroblastoma. MIBG diagnosis in neuroblastoma has been attempted with the following results. 205 total body scintigrams were performed in 60 patients with neuroblastoma with doses of MIBG ranging from 0.5 to 1 mCi. 52 were positive, 6 in complete remission were negative, and 2 were false negatives in adults with tumors showing no secretion of metabolites. More than 90% of neuroblastoma are MIBG positive, and therefore MIBG imaging is now considered the most valuable means of diagnosis and staging of these tumors. 131I MIBG therapy has been attempted in 22 patients with neuroblastoma. They received multiple therapeutic doses of 41 to 2,090 mCi given IV at 3- to 6-week intervals. The results were 5 complete remissions, 10 partial remissions, 1 no change, 2 progressive disease and 1 lost to FU. Apart from bone marrow depression in patients with previous bone marrow involvement, the treatment was well tolerated. Six adults with other neural crest tumors were also treated. Pain relief in metastatic patients is a common and important result of MIBG therapy.     

Neuroblastoma as a Neurobiological Disease

While neuroscientists are often involved in the assessment and care of patients with central nervous system tumors, they are only rarely involved in the case of peripheral nervous system neoplasia. Neuroblastoma is a childhood tumor of the primitive sympathetic nervous system. It is at once one of the most common and one of the most deadly tumors of childhood. The prognosis for children with this tumor has not changed in the past two decades. Clearly, a fresh approach to neuroblastoma is needed. The neuroscientist has much to add to our understanding and treatment of neuroblastoma and its sequelae. Conversely, neuroblastoma has much to teach us regarding the normal development of the neural crest and the aberrant loss of neurons in this lineage. A neuroscientist's approach to neuroblastoma, its biology and clinical features, is presented herein.     

Studies on tyrosine hydroxylase in neuroblastoma in relation to urinary levels of catecholamine metabolites.

Tyrosine hydroxylase (TH) activity has been determined in 22 neuroblastoma tumors from 15 patients, in 1 pheochromocytoma, 20 adrenal glands, 10 other tumors and organs, and 4 specimens of sera. The enzyme activity was found only in the neural crest tumors and adrenal glands, but the levels were too low to be detected in the other tumors and in normal liver and kidney tissues. The average specific activity (mean +/- SE) of TH in 23 neural crest tumors was 0.559 +/- 0.101; in 20 adrenal glands was 0.418 +/- 0.124 nmol/mg protein/minute. In 13 patients with neuroblastoma and 1 patient with pheochromocytoma, both TH levels in the primary tumors and urinary excretion of vanillylmandelic acid (VMA) and homovanillic acid (HVA) were studied. The urinary excretion of VMA by 10 of 13 neuroblastoma patients and by the patient with pheochromocytoma was significantly to markedly elevated above normal levels; excretion of HVA by 12 of 13 neuroblastoma patients was similarly elevated. These results indicate that tyrosine hydroxylase, an enzyme specifically located in the adrenal medulla and monoaminergic neurons, is also present in neuroblastoma, a malignant tumor of similar embryologic origin, and in pheochromocytoma. Not only can TH activity in these tumors be demonstrated in vitro, but the elevated urinary excretion of VMA and/or HVA by the majority of patients with these tumors also indicates TH activity of the tumors in vivo.     

Absence of polysialylated NCAM is an unfavorable prognostic phenotype for advanced stage neuroblastoma

Background  

The expression of a neural crest stem cell marker, polysialic acid (polySia), and its main carrier, neural cell adhesion molecule (NCAM), have been detected in some malignant tumors with high metastatic activity and unfavorable prognosis, but the diagnostic and prognostic value of polySia-NCAM in neuroblastoma is unclear.     

The uptake of alpha-foetoprotein by C-1300 mouse neuroblastoma cells

Recent immunocytochemical and biochemical studies have shown the intracellular uptake of alpha-foetoprotein (AFP) by most neural crest and neural tube derivatives of developing mammals and birds. The neural crest origin of neuroblastomas has been known for a long time. While many mouse neuroblastoma cell lines can express several neuronal properties, other lines lack specialized neural functions and may re-express embryonal or foetal antigens, suggesting some reversion towards an earlier stage of differentiation. We have therefore tested the C-1300 Jackson mouse neuroblastoma cell line for its ability to incorporate AFP. The results obtained confirm the significant internalization of protein by these cells, both in vitro and in vivo. External photoscans of mice bearing tumours after injection with [131I]-AFP have proven the usefulness of the protein as a radiotracer for neuroblastoma localization.     

Characteristics of stem cells from human neuroblastoma cell lines and in tumors

Cellular heterogeneity is a hallmark of human neuroblastoma tumors and cell lines. Within a single neuroblastoma are cells from distinct neural crest lineages whose relative abundance is significant for prognosis. We postulate that a self-renewing multipotent tumor stem cell, which gives rise to diverse cell lineages, is the malignant progenitor of this cancer. To test this hypothesis, we have established 22 cloned, phenotypically homogeneous populations of the three major cell types from 17 neuroblastoma cell lines. In vitro, malignant neuroblastoma stem cells, termed I-type (intermediate type), have distinct morphologic, biochemical, differentiative, and tumorigenic properties. I-type cells express features of both neuroblastic (N) cells (scant cytoplasm, neuritic processes, neurofilaments, pseudoganglia, and granin and neurotransmitter enzyme expression) and substrate-adherent (S) cells (extensive cytoplasm and vimentin and CD44 expression). Moreover, they show bidirectional differentiation to either N or S cells when induced by specific agents. I-type cells are significantly more malignant than N- or S-type cells, with four- to five-fold greater plating efficiencies in soft agar and six-fold higher tumorigenicity in athymic mice. Differences in malignant potential are unrelated to N-myc amplification/overexpression or the ability to digest and migrate through the extracellular matrix. Immunocytochemical analyses of a small series of tumors reveal that frequency of cells coexpressing N and S cell markers correlates with poor prognosis. Thus, I-type stem cells may be instrumental in the genesis and growth of tumors in the patient. Their unique biology deserves attention and further investigation.