Background
Neuroblastoma (NB), one of the most common extracranial solid tumors of childhood, arises from embryonic neural crest cells. It may occur anywhere that sympathetic neural tissue is found, but most frequently occurs in the adrenal medulla. Other identifying characteristics of this tumor include the early age of onset, high metastatic disease frequency at diagnosis and the possibility of spontaneous regression [
1]. The most aggressive tumors display amplification of the
MYCN oncogene, which is usually associated with poor survival, even in localized disease.
Like most cancers, the effect of tumor microenvironment on disease progression is not to be underestimate, as highlighted by recent evidence [
2‐
7]. This influence is variable and it is determined by several factors, providing, in the early stages of tumor development, a physical barrier against tumorigenesis, with lymphocytes, macrophages and natural killer (NK) cells playing key roles in tumor suppression [
8‐
10]. During cancer progression, tumor cells may create a supportive milieu that promotes both tumor growth and metastasis by reprogramming the surrounding cells and molecules.
A role for mesenchymal stem cells (MSCs) in the promotion of tumor progression by interacting with tumor cells and other stroma cells in the complex network of the microenvironment has been proposed [
11]. It has been demonstrated that NB cells upon pre-incubation with MSCs developed a more invasive behavior towards the bone marrow, primary site of NB metastases. Interestingly, intratumorally injected BM-MSCs reduce NB tumor growth and prolong murine survival, while after systemic administration these cells fail to home and to reach the primary tumor sites [
12]; this observation suggests the need for contact between MSCs and NB cells. Moreover, MSCs have been shown to induce invasiveness of NB cells via stimulation of CXCR4 expression both by secretome production and enhanced SDF1/CXCR4 signaling. A crucial role of the CXCL12/CXCR4 axis in promoting the NB invasiveness and the cross-talk of NB cells with the microenvironment has been assessed [
13,
14].
The multiple properties of these cells such as self-renewal, differentiation plasticity and ability to modulate immune responses as well as their strong tropism for tumors make them crucial players in the development of a metastatic phenotype.
Little information is available regarding the biological and functional features of MSCs isolated from NB tissue (NB-MSCs) [
15]. Therefore, the purpose of this study was to characterize NB-MSCs in terms of morphology, phenotype, differentiation, immunological capacity, proliferative growth and gene expression profile to define their involvement in the tumor microenvironment and in NB progression.
Discussion
In spite of the different medical interventions available, the NB prognosis remains poor, accounting for over 15% of all pediatric cancer deaths. At diagnosis, about 50% of NB patients have metastases and even a higher percentage suffer from difficult to treat tumors [
22‐
25]. Among these patients only 40% survive long term despite treatment with different therapeutic approaches [
26‐
28]. The interaction of neuroblastoma cancer-initiating cells with their microenvironment has been reported to play an integral role in the maintenance of resistant disease and tumor relapse [
2].
The TME primarily consists of MSCs [
15]. They are multipotent cells, that can differentiate into several mesenchymal lineages, such as bone, adipose tissue, cartilage, tendon and muscle [
29‐
31]. Additionally, MSCs exert peculiar immunomodulatory effects on several cells involved in the immune response, both in vivo and in vitro, through mechanisms that are not yet completely elucidated [
4,
6,
7]. MSCs have been experimentally shown to be influenced by the tumor and subsequently regulate tumor functions. In fact extracellular matrix components have been shown to be influenced by tumor modified MSCs thereby promoting tumor proliferation and metastases [
3‐
7]. As reported by Nakata et al., the exposure of the MSCs to different types of tumor-derived exosomes, including NB, induce the production of pro-tumorigenic cytokines and chemokines, having a direct effect on tumor cell proliferation and survival, on angiogenesis and on the recruitment of other inflammatory cells that favour tumor progression [
32].
In the present study, NB-MSCs represent a population that is similar, but not identical, to healthy donor BM-MSCs. In detail, they exhibit different adipogenic differentiation and immunomodulatory capabilities. Moreover, NB-MSC gene expression profiling results suggest their potential role in promoting the invasiveness and metastatic traits of NB cancer cells.
Under standard conditions, we isolated and propagated MSCs derived from pediatric NB tissue at disease onset, and in two cases after administration of chemotherapy. MSCs isolated and propagated from pediatric NB tissue displayed the typical MSC morphology and phenotype [
33] and proliferative capacity superimposable to BM-MSCs. The similar expression levels of stemness markers, such as Nanog, OCT3/4 and SOX2, observed both in NB-MSCs and BM-MSCs, suggest that NB-MSCs, even when derived from the tissue/tumor microenvironment, maintain BM-MSC multipotentiality [
30,
31].
In the cell cycle analysis, NB-MSCs showed an increased number of cells in the G0-G1 phase compared to BM-MSCs. It has been reported that a high content of G1 phase-blocked cells in cancer might imply a still unclear situation defined as “dormancy” that poses fundamental questions, which extend beyond the cancer proliferation/suppressor balance in primary cancer [
34]. In fact, dormant tumor cells are characterized by G0/G1 phase arrest and chemotherapeutic drugs resistance [
34]. Our observation of NB-MSCs arrest in G0/G1 cell cycle phase could support the essential role of MSCs, in regulating cancer dormancy [
35,
36]. These data provide a valuable tool to understand the MSC role in tumorigenesis and therefore open new therapeutic avenues for the prevention of cancer recurrence.
Regarding in vitro differentiation capabilities, NB-MSCs revealed a similar osteogenic and chondrogenic differentiation potential compared with BM-MSCs; on the contrary, NB-MSCs did not differentiate into adipocytes. Even though, tumor-MSCs exhibit varying differentiation capabilities [
37], according to our results, only a small subset of MSCs isolated from pediatric neuroblastoma, teratoma, Ewing sarcoma, and rhabdomyosarcoma specimens may be induced to differentiate into adipocytes while they respond to osteogenic induction similarly to BM-MSCs [
34]. Nevertheless, the lack of evident adipocytic differentiation together with an enhanced EMT-related gene expression profile and a different immunological response, lead to the hypothesis that NB-MSCs may be already dysregulated. However, further studies are needed to determine whether these cells could be defined as “cancer stem cells”.
The immunomodulatory properties of MSCs have been demonstrated using different in vitro and in vivo study approaches. MSCs regulate immunity by interacting with innate immune cells (including macrophages, NK cells, and dendritic cells), and adaptive immune cells (including B and T cells) [
38‐
40]. In the present study, we report more evident NB-MSC anti-proliferative effects on immune cells compared with BM-MSCs, confirming the impact of the tumor microenvironment on MSC functions [
7]. It is well known that NK cells are potent anti-tumor cells [
41] and possess strong cytotoxic activity against NB, both in vitro [
42] and in vivo [
43]. Johann et al. [
15] have reported that NK cytotoxicity is significantly impaired after co-culture with tumor stromal cells. Galland et al. [
10] have demonstrated that squamous cell lung carcinoma derived-MSCs exert a more pronounced immunosuppressive effect on NK cell functions and phenotype compared with the non tumor derived-MSCs through different mechanisms. In our study, the NK cytotoxic capability was not influenced by co-culture either with BM-MSCs or with NB-MSCs. We believe that these contrasting data may be ascribed to the different culture conditions and the different starting population, purified NK or whole PBMCs.
Abnormal regulation of TP53 has a critical role in tumorigenesis. Velletri et al. [
44] reported that MSC immunomodulatory properties may be influenced by the TP53 mutational status, which leads to genome instability and subsequent functional alterations. About 50% of our NB patients resulted TP53 mutated, in disagreement with data reported in the literature, where 2% of patients are mutated. Even if these results may depend on the small number of patients, we believe that this mutational status could affect the properties of NB-MSCs and tumor microenvironment and dictate the conditions for cancer development and progression [
44]. Nevertheless, it is important to consider that although TP53 is rarely mutated in NB, it is functionally inactivated in the majority of both MYCN amplified and MYCN WT subsets NB [
45‐
47].
It has been reported that tumor-derived MSCs may promote cell invasiveness, migration and EMT of cancer cells in many tumors [
48,
49]. Recently, Rodriguez-Milla et al. identified about 500 genes differentially expressed in BM-MSCs from NB patients and BM-MSCs from normal donors. Of note, genes involved in many biological processes including neurological system process, apoptosis, cell adhesion, cell surface receptor and intracellular signaling were up-regulated while genes related to the immune response were down-regulated. These findings suggest a dysregulation of crucial cellular pathways in MSCs in response to NB tumor [
50].
Here, we found that the transcriptomic profiling of NB-MSCs isolated from tumor samples derived from NB patients was enriched in EMT-associated genes compared to BM-MSCs, in particular in CDH2 and MMP-9 genes. It is known that CDH2, also termed N-cadherin, favors transendothelial migration, and MMP-9, a key enzyme in extracellular matrix remodeling, promotes cell invasion and metastasis by degrading collagens and fibronectin. Moreover, high expression levels of CDH2 are associated with a poor prognosis in NB and MMP-9 is highly expressed in high-risk NB tumors [
51‐
53]. Furthermore, in our gene expression profiling data NB-MSCs express higher levels of CXCR4 while down-regulate CXCL12 expression levels compared with BM-MSCs, supporting the pivotal role of CXCL12/CXCR4 axis in promoting NB invasiveness.
All together, these data provide evidence supporting a role for NB-MSCs in facilitating the metastatic process of NB cells, although further in vivo studies are needed to demonstrate this hypothesis. We recognize that this study has some limitations. The sample size was small, but consistent with that of other studies investigating this specific population. Additionally, the follow-up of patients was too limited to be significant and provide correlations. Despite these limitations, our findings support a role for NB-MSCs as a crucial factor on the microenvironmental regulation of tumor progression and metastasis. Further studies analyzing a broader number of samples are mandatory to confirm these results.