An overview of neuroblastoma cell lineage phenotypes and in vitro models

Abstract

This review was conducted to present the main neuroblastoma (NB) clinical characteristics and the most common genetic alterations present in these pediatric tumors, highlighting their impact in tumor cell aggressiveness behavior, including metastatic development and treatment resistance, and patients’ prognosis. The distinct three NB cell lineage phenotypes, S-type, N-type, and I-type, which are characterized by unique cell surface markers and gene expression patterns, are also reviewed. Finally, an overview of the most used NB cell lines currently available for in vitro studies and their unique cellular and molecular characteristics, which should be taken into account for the selection of the most appropriate model for NB pre-clinical studies, is presented. These valuable models can be complemented by the generation of NB reprogrammed tumor cells or organoids, derived directly from patients’ tumor specimens, in the direction toward personalized medicine.

Impact statement

This review provides an update on the mostly used cell line in vitro models for neuroblastoma (NB), a heterogeneous disease with high metastatic potential and resistance to treatment. The genetic and phenotypic profiles of the most used NB cell lines in the last 10 years are presented, considering the molecular markers that are involved in the distinct NB tumor phenotypes, including distinct core regulatory circuitries and non-coding RNAs. This gathered information can assist in the selection of the most appropriate NB in vitro model, based on the specific goals and objectives of each study.

Introduction

Neuroblastoma (NB) is an extracranial solid tumor in children and comprehends 8% to 10% of all pediatric cancers.¹ It is a heterogeneous disease that presents a broad spectrum of clinical behaviors; in children aged 18 months or older, it is often unresectable or metastatic, requires intensive multimodal therapy, and is associated with a survival rate lower than 50%.² On the other side of the spectrum, NB with spontaneous regression without chemotherapy is seen in low-risk subgroups.³

NB is derived from cells within the neural crest, likely sympathoadrenal progenitor cells that differentiate to sympathetic ganglion and adrenal catecholamine-secreting chromaffin cells.⁴ The presentation and symptoms at diagnosis reflect the tumor location, commonly in the adrenal medulla or anywhere along the sympathetic ganglia. Metastases are present in about 50% of patients at diagnosis, with bone marrow metastases corresponding to 80% of the cases. Metastases are also found in bones and regional lymph nodes, while the involvement of the central nervous system and lungs is rare.⁵

The International Committee of Pathology of Neuroblastoma classifies NB tumors into distinct subtypes, according to histological findings, which include the amount of Schwannian stroma present in the tumor and the mitosis-karyorrhexis index (MKI).⁶ Generally, the poorly differentiated or undifferentiated histology confers a worse prognosis to patients. Age is also an important prognostic indicator; in <18-month-old patients, poorly differentiated NB is still considered a favorable prognosis if the MKI is not high; however, in patients aged >18 months, poorly differentiated NB is invariably unfavorable.⁷

DNA or ploidy index, MYCN oncogene amplification status, and specific segmental chromosomal aberrations also influence NB prognosis,⁸ as it will be discussed later in this review. Furthermore, nerve growth factor (NGF) and other neurotrophins (NT) play important roles in the survival, differentiation, and activity of NB cells, mediated by tyrosine-kinase receptors (TrkA, TrkB, and TrkC).⁹ Favorable prognostic tumors usually express TrkA, the NGF receptor, while unfavorable prognostic tumors, with amplification of the MYCN oncogene, usually express TrkB and the ligand brain-derived neurotrophic factor (BDNF).^10,11 The TrkB-BDNF signaling pathway seems to function in an autocrine mode of action, increasing survival in NB cells and resistance to chemotherapy, via both the PI3K/Akt and the p44/42 MAPK signaling pathways, according to the chemotherapy drug used.¹²

NB most common genetic alterations

Although NB is the most common embryonic malignancy diagnosed during the first year of life,¹³ hereditary NB tumors occur in less than 1% of cases,¹⁴ where a few inherited mutations, such as the ones in ALK and PHOX2B genes, were reported to confer tumor predisposition.^15,16 In sporadic tumors, somatic mutations are not common¹⁷; however, chromosome aberrations are frequent and were shown to directly correlate with tumor aggressiveness and progression.¹⁸ These aberrations, which are critical risk factors previously identified in primary NB, can involve amplification of the MYCN oncogene, mutations of the ALK receptor, allelic deletions on the 1p and 11q chromosome regions, whole chromosome gains, and gains on the 17q chromosome region.^13,19–21

MYCN

Amplification of the proto-oncogene MYCN (MNA) is present in about 20%–25% of NB tumors, especially in high-risk patients.²² MNA was described in the early 1980s as a tumor aggressiveness factor,²³ and it is still considered the most widely used genetic marker for NB clinical prognosis.^22,24 MYCN is located on chromosome 2p24 and encodes a phosphoprotein of the MYC family of transcription factors, which binds to specific promoter sequences, favoring cell proliferation.²⁵ MYCN targets the proto-oncogene MDM2, a ubiquitin protein ligase which directly regulates the p53 protein²⁶; the TP53 tumor suppressor gene is rarely mutated in NB²⁷; however, the inhibition of the p53 pathway may occur through MYCN amplification, which can lead to the high expression of MDM2 protein. Overexpression of the ALK gene, also located on chromosome 2, in association with MNA, may also be found in sporadic NB driven by mutations, which are often identical to those identified in familial NB.^16,28

Chromosome 1p

Chromosome 1p deletions occur in 23% to 46% of NB tumors and are associated with poor prognosis and metastatic disease.^24,29,30 The commonly deleted region is on 1p36, which contains many genes associated to NB malignant behavior, including CHD5, TNFRSF25, CAMTA1, AJAP1, ERRFII, PIK3CD, RBP7, and TARDBP.^31–33 Among these, CHD5 was shown to be the most critical tumor suppressor gene in this chromosome region.³⁴ A high CHD5 expression is associated with a favorable prognosis; however, deletion or low expression of CHD5 is frequently observed in high-risk tumors.³⁵ CHD5 is a member of the chromatin remodeling gene family, encoding a protein with chromatin-remodeling, helicase, and DNA-binding motifs (CHD), with preferential expression in neural tissues, directly associated with nervous system development.³⁶ It controls cell proliferation, apoptosis, and senescence through p19 and p53 pathways regulation^35,37 and can be upregulated by the treatment with retinoic acid (RA), inducing cell differentiation via NGF/TrkA signaling.³⁸ RA is a well-known differentiating agent reported to reduce stemness characteristics³⁹ and treatments based on RA are currently used for high-risk NB.^14,40

Chromosome 11q

Deletions on chromosome 11q (del11q-q25) are present in 17% to 48% of NB tumors, depending of the cytogenetic method used for its detection.^{24,29,41–43} The main consequence of this aberration is the increase of genetic instability, leading to gene mutations and deregulation of the cell cycle.⁴⁴ Its presence is also related to a less favorable outcome and with an increased probability of disease recurrence, even in patients with localized tumors.⁴⁵ This poor outcome reinforces the relevance of the inclusion of the 11q chromosome status as one of the criteria for NB risk classification.

Although several molecular studies have concentrated on the identification of cancer driver genes on the 11q region, which includes CADM1, ATM, H2AFX, PHOXA2, SDHS, CCND1, NCAM, and CHK1 genes, the specific targets of this region that present key relevance to NB are still largely unknown.^43,46,47 The CADM1 gene (also known as TSLC1, NECL-2, IGSF4, and SynCAM1), located at 11q23.3, for example, is a suppressor gene altered in multiple types of cancer.⁴⁸ The CADM proteins family is reported to be involved in cell-cell adherence, potentially playing a role in epithelial–mesenchymal transition (EMT)-like processes and tumor progression.⁴⁹ In NB, the hypermethylation of CADM1 was correlated with reduced levels of mRNA and protein expression, in association with lower event-free and overall survival.⁵⁰ On the other hand, the overexpression of CADM1 in NB cells was shown to impair cell proliferation and viability as well as colony formation.⁵¹

Chromosome 17q

Gains on chromosome 17q are the most frequent chromosome aberrations in NB tumors,⁵² with a frequency that varies from 23% to 70%, often associated with age at diagnosis and advanced stages of the disease.²⁰ Although the accurate frequencies and boundaries of the chromosomal copy number variations on 17q and the molecular targets affected, have yet to be identified,²¹ gains extending from 17q23.1 to 17qter region have been described as promoting tumor progression.⁵² In addition, gains on 17q are positively correlated to MNA,^53,54 and combined with mutations in the PHOX2B gene, are considered to be critical for the acquisition of NB metastatic potential.⁵⁵

NB cell lineages phenotypes

In 1947, Murray and Stout described the common characteristics of neuroblasts derived from eight pairs of primary and metastatic tumors and their morphological changes in two-dimensional culture.⁵⁶ The cells, also referred to as sympathicoblasts, produced neurites of varying lengths, some of which begun to branch, acquiring a filamentous shape after four days in culture. It is now known that NB-derived cells are, in fact, heterogeneous and, when cultivated in vitro, represent populations with distinct morphological and tumorigenic properties, which reflect the biological and genetic specificities of original neuroblasts.^57,58 At least three different NB cellular phenotypes have been identified: neuroblastic (N-type), flat or substrate adherent (S-type), and intermediate (I-type), each of them presenting particularities that can be identified by specific molecular markers (Table 1).

Table 1.

Main phenotype markers for neuroblastoma cell lineages.

CELL MARKERS	N	S	I
S-type
Vimentin	−	+	−/+
CD44	−/+	+	−/+
Fibronectin	−/+	+	−/+
Dopamine beta-hydroxylase	+	−	−/+
N-type
Neurofilaments	+	−	−/+
β-tubulin	+	−	−/+
I-type
CD-133	−	−	+
c-KIT	−	−	+

Note: (+) High expression; (−/+) Moderate to low expression; (−) Rare expression.

The origin of these different phenotypes is still controversial, but there are two hypotheses. The first and most accepted hypothesis is known as transdifferentiation theory, which suggests that NB cells are capable of spontaneously interconverting from one phenotype to another.⁵⁹ According to this hypothesis, S-type and N-type cells originate from the undifferentiated I-type cells,^59–61 which could explain the appearance of S-type cells during the culture of N-type cell lines, or vice-versa, when I-type cells are still present. The clonal expansion theory, on the other hand, considers that both N and S-type cells could co-exist in culture, and that, under certain conditions, the less-aggressive S-type could dominate the cell population over the more aggressive N-type cells.^62,63

Regardless of the theory, it is clear that an NB cell lineage can contain a mixture of cell types, even if one is more predominant than the others. It is also important to consider that culture conditions can lead to new interactions between these cell types and contribute to distinct responses to a given tested stimuli or treatment. The activation of NOTCH pathway⁶¹ and hypoxia factors,⁶⁴ for example, have recently been described to be involved in the differentiation processes of these NB cells, although others may exist as well.

N-type cells

N-type cells are characterized as immature nerve cells, precursors to the neural crest sympathoadrenal lineage. In culture, they are small and rounded, with thin and long neurofilaments and a scant cytoplasm.^65–67 Although they present a high proliferation rate, form cell aggregates, and adhere weakly to the substrate,^68–70 their tumorigenicity is moderate.

β-tubulin heterodimer, a component of microtubules in neuronal cells, has been used as a specific marker for the highly proliferative N-type cells (Table 1).⁶⁷ The dynamic microtubule structures of this component are involved in cell movement and maintenance of cell shape, intracellular trafficking, and cell mobility during mitosis and meiosis.⁷¹ In the context of cancer, tubulin proteins are targets for tubulin-binding chemotherapeutics, which leads to mitotic arrest and cell death.^72,73 Changes in microtubule stability and expression of their isotypes have been correlated with a poor prognosis and chemotherapy resistance in several cancer types, including NB.^74,75

Neurofilaments represent the main structural component of the cytoskeleton in mature neurons, determining axon caliber and conductivity for proper neuronal function.⁷⁶ They are highly present in tumors of neuronal origin, and therefore, are largely used to characterize N-type cells,^65,66,77 even considering that they present a higher expression in benign or differentiated tumors than in malignant and less differentiated tumors of neuronal lineage.^77–81

Dopamine β-hydroxylase (Dβ-H) is another highly expressed marker in N-type cells with low expression in the S- or I-types.^66,82–84 Dβ-H is an enzyme that characterizes the neuronal differentiation of neuroblasts⁸³ and is involved in the conversion of dopamine to norepinephrine, released with other vesicular contents during synaptic transmission.⁸⁵

S-type cells

S-type cells are multipotent precursors to melanocytes, Schwann, and central nervous system glial cells that represent the neural crest non-neuronal components. They are characterized as non-tumorigenic and present a large and flat morphology with extensive cytoplasm, few and short neurofilaments, and a large cytoplasmic/nuclear ratio.^66,86 In culture, S-type cells are slow-growing, grow in a monolayer, and are specially characterized by their strong ability to adhere to the substrate.⁶⁹ The high expression of β-integrin, which provides a strong attachment to extracellular matrix (ECM) components, along with low levels of insulin-like growth factor receptor, may contribute to the less tumorigenic and less migratory phenotype of these cells.⁸⁷

CD44 is another cell surface receptor commonly used as marker of the NB S-type cells.⁸⁸ It binds to hyaluronic acid and many other ECM components and acts as a co-receptor for growth factors and cytokines.⁸⁹ Tumors of epithelial origin express CD44 in multiple isoforms, which has been associated with a role in regulating the EMT process and in conferring adaptive plasticity to cancer cells.^90,91 In NB, CD44 expression has been correlated with a favorable prognosis, being reported with low or absent expression in advanced stage tumors.⁹²

The high expression of ECM components also characterizes this cell phenotype.⁶⁶ Fibronectin is a secreted glycoprotein that is found in a soluble form circulating in the plasma, but also in an insoluble fibrillar form in the ECM, acting as a critical substrate for cell adhesion and migration.⁹³ In cancer cells, dysregulation of fibronectin cell adhesion and migration has been implicated in tumor development and metastasis, pointing out fibronectin as a cancer biomarker of EMT reversion and substrate-adherence cellular differentiation.^94,95

Vimentin, one of the cytoskeleton main intermediate filaments, is present in normal mesenchymal cells and is responsible for maintaining the cellular integrity, stress resistance, and the migration of cancer cells that have undergone EMT.^96,97 This protein is initially expressed by early neuronal precursors and is essential for neuritogenesis, as it is gradually replaced by neurofilaments.⁹⁸ Immature and motile glial cells also produce vimentin, whereas mature glial cells cease its expression and begin to express the glial fibrillar acidic protein (GFAP).⁹⁹ The overexpression of vimentin has been reported in many cancer types, in association with cell growth and invasion, reinforcing its role in cancer cells as a marker of the EMT process.¹⁰⁰ In NB, vimentin is mainly expressed in S-type cells, although it can also be expressed in I-type cells.^66,67 As the S-type cells do not express GFAP, it was suggested that they represent an embryonic neural crest precursor that may undergo terminal differentiation toward a glial, meningeal, or melanocytic phenotype.⁵⁹

I-type cells

I-type cells, firstly described as an intermediate phenotype, have been recently described as a progenitor of the N- and S-type cells, capable of self-renewal and bidirectional differentiation.⁶⁶ I-type cells present an intermediate morphology and mixed properties of N- and S-type cells, being flat-shaped with prominent nuclei and moderate amounts of cytoplasm, possibly presenting neurofilaments.^67,101 Differently than the S-type cells, which attach strongly to the underlying substrate and do not mound, or the N-type cells, which attach weakly to the substrate, the I-type cells adhere both to the substrate and to each other, mounding and forming multiple layers of cells.⁶⁹

Seven different antigens have been tested positively as specific markers to identify I-type cells: GPRC5C, LNGFR, TRKB, NOTCH1, PIGF2, CD133, and CD117.^65,66,102 CD133 is a transmembrane glycoprotein associated to progenitor cells and to tissue regeneration and cell differentiation processes in normal tissues. It was first described as a marker of primitive hematopoietic and neural stem cells,^103,104 and it has been used extensively as a marker of stem-like cells in NB.^105–107 CD133 is over-expressed in several NB cell lines, where it acts to repress neural differentiation and elongation, accelerating cell proliferation and colony formation.¹⁰⁸ The investigation of the clinical significance of CD133 expression in NB tumors has shown its association to a poor outcome¹⁰⁹ and chemoresistance.^110–112

CD117, also called c-KIT, is another marker mostly used to characterize I-type cells in NB. It is a tyrosine kinase receptor of the class III subfamily.¹¹³ The binding of its ligand, stem cell factor (SCF), and SCF/c-KIT signaling contributes to both tissue development and homeostasis, including the maintenance of the stemness status of precursor cells in several adult tissues.¹¹⁴ In NB, it was described as a marker of tumor-initiating cells, involved in cell growth and differentiation,¹⁰¹ clonogenic capacity, and drug resistance.¹¹⁵ c-KIT expression in NB cell lines was found to be induced under hypoxic conditions,¹¹⁶ which suggests that c-KIT+ cells represent an aggressive entity of NB, whose growth may be influenced by the tumor microenvironment.

Genetic regulators of NB phenotypes

Transcriptional plasticity in cancer cells can control cellular fates by regulation of transcriptional factors that can bind to promoters, enhancers, and super-enhancers, thus forming interconnected auto-regulatory loops that constitute the core regulatory circuitry (CRC).¹¹⁷ Distinct CRCs have also been shown to identify cell lineage phenotypes in NB. Boeva et al. identified three different identities in NB cell lineages¹¹⁸: the non-neuronal neural crest cells phenotype, driven by a CRC module containing the AP-1 transcription factor; the sympathetic noradrenergic phenotype, defined by a CReC module that includes the PHOX2B, HAND2, and GATA3 transcription factors, and a third phenotype consisting of a mix of both CRCs. Another profile was defined by Groningen et al.,¹¹⁹ which named two subsets of phenotypes in NB: the adrenergic lineage (ADRN), characterized by the CRC, including PHOX2a, PHOX2b, and DBH transcription factors; and the mesenchymal lineage (MES), characterized by the CRC that includes the SNAI2, VIM, and FN1 transcription factors. Later, the MES and ADRN populations were shown to be able to transdifferentiate into one another by a feedforward loop controlled by the NOTCH signaling pathway.⁶¹

MicroRNAs (miRNAs) can also differ in NB phenotypes. miRNAs are a class of non-coding endogenous RNA molecules that have been identified to play a critical role in cancer, regulating several target genes associated with aggressive tumor phenotypes, such as the ones that promote metastatic development and treatment resistance. In NB, the expression of miR-21, miR-221, and miR-335 was exclusively observed in non-neuronal cells, while the expression of miR-124 and miR-375 was only observed in neuroblastic cells.⁸⁶ The regulatory role of miR-335 in non-neuronal cells was described in the modulation of the expression levels of the neural differentiation modulators HAND1 and JAG1,⁸⁶ and in the suppression of cell invasion and metastasis by regulation of the TGF-β pathway.¹²⁰ Global miRNAs expression profiles were also shown to be distinct in NB tumors with diverse prognosis.^121,122

The most used NB cell lines as in vitro models

Cancer-derived cell lines have been widely used for research and proven to be useful models to understand the multiple and diverse processes in cancer, as well as for discovering and evaluating the efficacy of treatment; their unique molecular and phenotype characteristics should be taken into consideration when choosing the most appropriate one as an in vitro model.

During the preparation of this manuscript, the PubMed database was searched for human NB cell lines citations from the last 10 years. Sixteen cell lines were found (some of them sublines) that were cited at least five times on this defined period (Table 2). All of them were isolated from high-risk NB patients, classified as being in stage 4, according to the International Neuroblastoma Staging System,¹²³ except for cell line IMR-32, which was derived from a patient with an unknown stage. Most of the cell lines are derived from bone marrow metastases from patients that underwent several lines of treatment.

Table 2.

Clinical, biological and genetic features, cell lineage phenotypes and related cell lines of the most used NB cell lines.

Cell line	Gender	Age	INSS stage	Origin site	Treatment	Phenotype	Mycn status	Ch 1p status	Ch 11q status	Ch 17q status	Related cell lines	References
SK-N-SH	F	4 y	4	Bone marrow	CT/RT	I	Non-amplified	Normal	Normal	Gain	Sublines SH-SY5Y/SH-EP	68,130,132,133
BE(2)-C	M	25 mo	4	Bone marrow	CT/RT	I	Amplified	–	–		Parental SK-N-BE(2)	65,69,130,134,135
NB9	M	22 mo	4	Adrenal gland	None	I	Amplified	Loss	Normal	–	–	130,132,136
SH-SY-5Y	F	4 y	4	Bone marrow	CT/RT	N	Non-amplified	Normal	Normal	Gain	Parental SK-N-SH	65,67,126,133
SK-N-BE (1)	M	20 mo	4	Bone marrow	None	N	amplified	Loss	-	Normal	SK-N-BE (2)	65,128,130,134,135
SK-N-BE(2)	M	24 mo	4	Bone marrow	CT/RT	N	Amplified	Loss	Normal	Gain	Pre-therapy SK-N-BE(1)	128,132,135,137
BE(2)-M17	M	24 mo	4	Bone marrow	CT/RT	N	Amplified	Loss	Normal	Gain	Parental SK-N-BE(2)	65,66,130
SMS-KCN	M	11 mo	4	Adrenal gland	None	N	Amplified	Loss	–	Gain	Post-therapy SMS-KCNR	65,128,132
IMR-32	M	13 mo	_	Abdominal mass	None	N	Amplified	Loss	Normal	–	–	65,132,133,138
SK-N-FI	M	11 y	4	Bone marrow	CT	N	Non-amplified	Normal	Normal	Normal	–	34,139–141
SH-EP	F	4 y	4	Bone marrow	CT/RT	S	Non-amplified	Normal	Normal	Gain	Parental SK-N-SH	66,68,87
SK-N-AS	F	8 y	4	Bone marrow	-	S	Non-amplified	Loss		Gain	–	130,139,141,142
SK-N-MC	F	14 y	4	Supra-orbital lymph node	CT/RT	S	Amplified	Loss		Normal	Subline SK-N-MC-IXC	132,139,142
CHLA-20	F	24 mo	4	Bone marrow	CT	–	Non-amplified	–	–	Normal	Pre-therapy CHLA-15	128,131,133
NB1643	_	36 mo	4	Retroperitoneal mass	None	–	Amplified	Loss	–	Normal	–	140,143
NB19	F	12 mo	4	Bone marrow	CT	–	Amplified	Loss	–	–	–	130,132

Note: BM: bone marrow; AG: adrenal gland; INSS: International Neuroblastoma Staging System; AM: abdominal mass; RM: retroperitoneal mass; LN: lymph node; CT: chemotherapy; RT: radiotherapy; AMN: amplified MYCN; NAMN: non-amplified MYCN; (−): non determined.

The SH-SY5Y cell line and its parental line, SK-N-SH, represent the most frequently cited NB cell lines, found over 5000 times in the mentioned searched period. SK-N-SH represents an I-type cell line used to subclone a few different cell lines in 1978 by Biedler, including SH-SY5Y and SH-EP. In the above study, neuroblast-like cells from SK-N-SH were subcloned using metal cylinders isolation and single-cell culture. The first clone was called SH-SY, which was further subcloned into the SH-SY5, and finally into SH-SH5Y. Similarly, the epithelial-like cells from the SK-N-SH were also subcloned into SH-EP and SH-FE.¹²⁴ Therefore, the SH-SH5Y is an N-type cell line, although some studies demonstrate that it is also composed of S-type cells,⁶⁷ while the SH-EP represents a predominant S-type lineage. Both of these cell lines, however, present the same profile of genetic alterations, with no amplification of MYCN and no chromosome abnormalities on 1p and 11q, but with gains on 17q.^42,125,126 The SH-SY5Y and SK-N-SH are extensively used for the study of neuronal diseases,¹²⁷ as they can be differentiated into a more mature neuron-like phenotype with neuroblast-like morphology that is characterized by neuronal markers like tyrosine hydroxylase (TH) and DβH, characteristics of catecholaminergic neurons.⁶⁰

Contrary to the above described NB cell lines, others were collected at diagnosis, before any treatment. The IMR-32 and SMS-KCN represent the most frequently used ones; both are N-type cells with MYCN amplification and functional p53, along with the loss of chromosome 1p.^125,128,129 The SMS-KCN/SMS-KCNR, CHLA-15/CHLA-20, and SK-N-BE(1)/SK-N-BE(2) are paired cell lines, each pair obtained from the same patient before and after treatment (Childhood Cancer Repository – accessed at www.cccells.org/cellreqs-nbl), valuable for evaluating the effects of treatment agents at baseline and at different stages of treatment.

Almost 20 years ago, Thiele published a comprehensive review with an extensive data collection of over 100 NB cell lines.¹³⁰ Those are listed in tables based on the clinical features of patients from whom they have been derived, along with their chromosome and specific genetic alterations, biologic characteristics, and the effects of differentiation agents. In the same year, Keshelava et al. analyzed the drug resistance of 17 NB cell lines established from patients at different therapy stages.¹³¹

Future perspectives

Extraordinary advances were and are still obtained in basic and clinical cancer research with the use of “conventional” NB cell lines, established directly from patient cells immortalized in vitro. The use of these models provides the understanding of several phases of the tumorigenic process, including tumor progression and therapeutic resistance. Limitations, including the inability to reflect the natural biology of the patients’ tumor and the genetic intra- and interpatient heterogeneity, exist. These limitations can be overcome or reduced by the use of immortalization methods of specimens directly obtained from the cancer patients that reflect their in vivo tumor behavior and intrinsic molecular characteristics. One of these methods includes the conditional reprogrammed cells (CRCs) method, which allows the cells to be isolated directly from patients to surpass senescence when cultivated in the presence of irradiated mouse fibroblasts and the compound Y-27632, an inhibitor of the enzyme kinase Rho.^144,145 This technology has been used in the study of pancreas, prostate, lung, and bladder cancers.^146,147 A study conducted by Mahajan et al., in breast tumors, has shown that CRCs maintain their original cellular and genomic signatures when compared to their original cells.¹⁴⁸ More recently, CRCs were also established from murine NB cell lines from TH-MYCN mice, which were shown to preserve the cellular heterogeneity observed in in vivo.¹⁴⁹

Induced pluripotent human stem cells (hiPSCs) are based on the reprograming of differentiated adult cells of somatic origin that reach the stage of cell undifferentiation and pluripotency in vitro.^150,151 Due to the plasticity of the hiPSCs regarding the differentiation into various types of cells and tissues, they are widely used for the reconstruction of tissues or organs of patients with a variety of diseases. In cancer, as the CRC cells, they can be widely used to study several tumors phenotypes in a patient-specific model.^152,153

Patient-derived tumor xenografts, which are generated by transplanting fresh tumor tissues from patients into immunodeficient mice, can retain tumor heterogeneity and genomic stability, as well as reproduce complex cancer–stroma and cancer–matrix interactions, better predicting drug responses.^154,155 However, their application in medicine can be limited, due to its high cost and long processing time.^155,156 More recently, patient derived tumor organoids (PDTO) have been promising superior models, as they comprehend three-dimensional in vitro cellular structures derived from cancer stem cells that present self-renewal and self-organization capabilities, while also retaining the characteristics of their source tumor.^156,157 Beyond the lower cost and processing time, PDTOs enable easily accessible therapeutic effects in both health and tumoral organoids from the same patient, as well as CRISPR-Cas9 approaches for identification of key driver mutations.¹⁵⁸ Protocols to produce PDTOs from different pediatric solid tumors, including NBs, have already been developed and showed to be efficient for maintaining gene-expression profiles and epigenomes from tumors.¹⁵⁹ NB heterogeneities, such as phenotype markers and chromosomal aberrations, were also retained in PDTOs.¹⁶⁰

As these technologies of cancer organoids and tumor cells reprogramming evolve, enabling and facilitating their routine use in laboratories is necessary, as they will become essential tools for the generation of patient-derived cell lines that can subsequently be used for the discovery of novel predictive progression markers, and drug-screening toward personalized-medicine strategies.

Conclusions

One of the most critical and current challenge in NB is the treatment of high-risk patients, whose mortality rates have not decreased in the last four decades. The high rate of treatment failure is largely due to the high degree of cellular and genetic heterogeneity of these tumors, which leads to diverse mechanisms of treatment resistance. Although the current models of NB immortalized cell lines present limitations, they are still fundamental and valuable in vitro models for the understanding of such mechanisms. Their cell lineage diversity allows to address a variety of clinical processes, such as the response to differentiation-inducing agents and cytotoxic drugs, and the rapid progression from a primary tumor to metastasis. The availability and the choice of the most appropriate NB cell line, or NB cell line panel, based on its distinct cellular and genetic phenotypic profile is of utmost importance for clinical research, which may be complemented by the generation of NB reprogrammed tumor cells.

ORCID iDs

Fernanda de Almeida Brehm Pinhatti https://orcid.org/0000-0001-8137-5186

Selene Elifio-Esposito https://orcid.org/0000-0002-8676-134X