Neuromedin U and neurotensin may promote the development of the tumour microenvironment in neuroblastoma

Introduction

Neuroblastoma arises during foetal or early post-natal life from the sympathoadrenal lineage of the neural crest and presents a broad clinical spectrum characterized by enormous biological and genetic heterogeneity. Neuroblastoma can spontaneously regress, mature, or demonstrate an aggressive and malignant phenotype that is poorly responsive to current multimodal therapy (Castleberry, 1997; Luksch et al., 2016). In what the International Neuroblastoma Staging System Committee (INSS) defines as stage 4, tumour cells metastasize to distant tissues via blood and lymphatic vessels, with obviously increased lymphangiogenesis. INSS stage 4S is a unique category of metastatic disease in infants with dissemination localizedin the liver and skin, with minimal marrow involvement (i.e., <10% of total nucleated cells identified as malignant by bone biopsy or by bone marrow aspirate) (PDQ Pediatric Treatment Editorial Board, 2002). Considering that stage 4S can regress spontaneously and has a favourable outcome compared with stage 4, we may be able to obtain insight into the progression of neuroblastoma by comparing the molecular differences between these two stages.

The tumour microenvironment (TME) is the cellular and matrix environment that includes tumour cells, immune cells, stroma cells, and the extracellular matrix (McCarthy, 2014). The imbalance between the effector and regulatory cell interactions in the TME are known to contribute to the major immune evasion mechanisms of tumours.Tumour-infiltrating lymphocytes (TILs) play a pivotal role in regulating tumour growth and anti-tumour progression. Researchers have confirmed that the infiltration of CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, memory T lymphocytes, and natural killer (NK) cells is linked to a favourable outcome in several cancers including breast cancer, colorectal cancer, and neuroblastoma (Galon, Fridman & Pages, 2007; Mina et al., 2015). Conversely, a high level of immunosuppressive immune cells including regulatory T lymphocytes (Tregs), myeloid-derived suppressor cells (MDSCs), and tumour-associated macrophages type 2 (TAM-M2) may be involved in weakening the efficacy of anti-neoplasm immune responses (Carlson & Kogner, 2013) leading to aggressive tumour growth and an unfavourable outcome.

The molecular and cellular mechanisms underlying the interaction between immune cells and neuroblastoma have been studied. MYCN amplification has been determined to be independent high risk factor for stratification in neuroblastoma study (Luksch et al., 2016), however, the existence of key genes and the modulation of tumour microenvironment by these remain to be elucidated. In this study, we attempted to research the overall gene alteration between stage 4S and stage 4, and to explore the impact of key genes on TME processes to explain the mechanisms of neuroblastoma development.

Materials and Methods

Results

Gene ontology analysis revealed that genes were enriched in aggressive biological progression

We obtained 309 and 1,103 DEGs from the GSE120572 and GSE73517 datasets, respectively, when comparing INSS stage 4S and stage 4. Of these, 216 intersected DEGs were further screened from the two datasets (Fig. 1A).

Consequently, GO analysis revealed that BP terms of the 216 DEGs were enriched in positive regulation of the development process, cell adhesion, signal transduction, and negative regulation of cell death, suggesting that these genes are involved in aggressive tumour development (Fig. 1B, Table S3).

NMU and NTS were identified as elevated hub genes in stage 4

We constructed protein-protein interactions (PPI) networks in the STRING database to learn more about the function of the identified genes. Their networks were subsequently visualized by Cytoscape (Fig. 2A, Fig. S1). The top 25 hub genes were screened out with the Degree method using CytoHubba (Fig. 2B, Table S4).

We then extracted expression values of the 25 genes in stage 4S and stage 4 from the Oncomine database, respectively, and compared the differences between the two stages. Results showed NMU and NTS were significantly elevated in stage 4, without obvious differences being found between the two stages for the remaining 23 genes (Figs. 2C, 2D).

Combining data from TARGET database, we found that patients’ overall survival rate in high NMU expression group was significantly lower than that in low expression group (P = 2.25e−06). NTS presented similar action (P = 0.019) (Figs. 2E, 2F).

Significant tumour-associated lymphocytes infiltration occurred in stage 4

By integrating the 22 immune cells basic gene expression matrix, which as a background gene expression file, to GSE120572 and GSE73517, we found there was no significant (P > 0.05) immune cell infiltration in INSS stage 4S for any of the two datasets. Conversely, obvious infiltration of tumour-associated macrophages type 2 (TAM-M2) and regulatory T cells (Tregs) was determined in stage 4, for both datasets (Figs. 3A–3D).

NMU and NTS promote cell proliferation and invasion of SK-N-BE(2)

The results of the Western blot assay showed obvious interference of the target genes (Figs. 4A, 4B). The results of the transwell assay showed that the invaded cell number in si-NMU and si-NTS group were significantly less than the corresponding negative control (P < 0.0001, t = 12.81; P < 0.0001, t = 8.714), respectively (Figs. 4C–4E, 4F–4H). The results of the wound-healing assay revealed the interference of NMU and NTS led to a significant migration distance less than the negative control (P = 0.0003, t = 11.69; P = 0.0002, t = 13.04), respectively (Figs. 4I–4J, 4K–4L, 4M). Our results suggested that NMU and NTS could facilitate the invasion and migration of SK-N-BE(2).

NMU and NTS were significantly correlated with immune-related genes

Results of genes co-expression analysis showed that expression of NMU was positively associated with CD4, CD25, FOXP3, NFAT, IL-4 and IL-13 (Figs. 5A–5F, 5M–5R). NTS presented positive relationships with CD163, CD206, IL-10, TGF-β, STAT3 and STAT4, both in GSE120572 and GSE73517 datasets (Figs. 5G–5L, 5S–5X).

Discussion

The tumour microenvironment is the cellular environment comprising tumour cells, immune cells, stromal cells, and the extracellular matrix. The TME plays three important roles in stimulating the development of tumours by providing a growing environment for tumours, disabling the efficacy of anti-tumour drugs, and assisting tumour cell evasion from immune surveillance (Kerkar & Restifo, 2012; Patel et al., 2018; Potter, 2007). The mechanisms underlying the interaction between immune cells and neuroblastoma cells have been studied, but many questions remain unanswered, including the identification of key genes and how they to modulate TME. INSS stage 4S neuroblastoma is a special category of metastatic disease that may regress spontaneously. It has been shown to have a favourable outcome when compared to INSS stage 4 (Nickerson et al., 2000; Schleiermacher et al., 2003); thus, comparing the molecular differences between the two stages may provide some explanation for tumour progression. We demonstrated the significant infiltration of Tregs and TAM-M2 in stage 4 cases from two datasets, without obvious immune cell infiltration in stage 4S.

Tregs are immune cells characterized by immunosuppression and hypoergia (Campbell, 2015) and can restrain the function of effector cells and attenuate the body’s immune response, accelerating tumour angiogenesis and development (Sakaguchi et al., 2020). With single-cell sequencing analysis, Zheng et al. (2017) proved that non-functional CD8⁺ T cells and Tregs were enriched in liver cancer tissues, suggesting that Tregs may inhibit CD8⁺ T cell activity to promote liver cancer progression. Further, tumour-associated macrophages (TAMs) participate in the process to facilitate tumour immune escape through polarization (Hao et al., 2012). Studies have shown that tumour-derived IL-4, IL-5, IL-6, IL-10, and IL-13 can stimulate TAM polarization towards TAM-M2 (Martinez et al., 2008). Further, cytokines including IL-4, TGF-β, IL-17, VEGF, PDGF, M-CSF, and MMPs secreted by TAM-M2 (Coffelt, Hughes & Lewis, 2009) are involved in tumour invasion, metastasis, angiogenesis, and lymphangiogenesis. IL-10 derived from TAM-M2 has been implicated in the suppression of NK cells and cytotoxic T lymphocytes (Benner et al., 2019), thereby reducing the tumour cells to be killed. The accumulation of Tregs and TAM-M2 is a significant event in tumour development.

It is difficult to determine the underlying causes of the changes in TME between INSS stage 4S and stage 4. We sought to determine the molecular alterations influencing TME development and found that neuromedin U (NMU) and neurotensin (NTS) are hub genes elevated in stage 4 compared to stage 4S. In addition, we compared the gene expression differences between MYCN amplified and MYCN non-amplified groups in these stages, however, NMU and NTS were not found in the differentially expressed genes. This suggested that NMU and NTS may not be driven by MYCN amplification (The results were not presented). Studies that have linked NMU to cancer indicate that NMU is significantly upregulated in most malignancies, including neuroblastoma (Martinez et al., 2017; Przygodzka et al., 2019). NMU upregulation in the tissues and cell lines of malignancies correlated with increased invasiveness and metastatic potential of tumour cells and advanced stages, such as head and neck carcinoma, pancreatic tumour and lung cancer (Tokumaru et al., 2004; Wang et al., 2016; Yang et al., 2018). Lebedev et al. (2019) demonstrated that the increased expression of NMU was associated with poor prognosis in neuroblastoma. Similarly, the oncogenic action of NTS has been described in different cancers with effects at each step of progression (Ouyang et al., 2017). For example, NTS promotes glioma cell invasiveness through NTSR1 in vitro and in vivo (Ouyang et al., 2015). NMU and NTS are known to exert functions by binding to their relevant receptors and activating the corresponding signalling pathways to directly or indirectly accelerate tumours into malignant characteristics.

The influence of NMU and NTS on the tumour microenvironment has not been reported. NMU and NTS receptors, which belong to G-protein-coupled receptor (GPCR) families, were found to be expressed on the membrane of immune cells including T cells, B cells, macrophages, dendritic cells, mast cells, and on tumour cells (Hedrick et al., 2000; Kim et al., 2006; Magazin et al., 2004; Saada et al., 2012). Johnson et al. (2004) confirmed that the binding of NMU to NMUR increased the synthesis and secretion of IL-4, 5, 6, 10, and 13 in CD4⁺ T cells, and may be mediated as follows: activated phospholipase C (PLC) induced by G protein hydrolysed phosphoinositide 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5 Tris-phosphate (IP3); IP3 subsequently elicited Ca²⁺ release from the endoplasmic reticulum, which resulted in calmodulin binding, stimulation of calcineurin, and dephosphorylation of NFAT family members, thereby facilitating the production of ILs (Johnson et al., 2004; Park et al., 2012; Smith-Garvin, Koretzky & Jordan, 2009). The binding of IL-4 and IL-13 to their receptors is linked to macrophage polarization to the M2 type via activation of the JAK-STAT pathways (Martinez et al., 2008; Mosser & Edwards, 2008). Kim et al. (2006) suggested that NTS immediately mediated the stimulation of the JAK-STAT1 pathway in macrophages. The JAK-STAT pathway is reported to be pivotal for TAM polarization to the M2 type (Wilson, 2014). IL-10 and TGF-β are powerful stimulants secreted by TAM-M2 and can transform CD4⁺ T cells into Tregs (Liu et al., 2013; Shen et al., 2015; Zhou et al., 2017).

In this study, we found NMU was significantly correlated with the expression of CD4, CD25, FOXP3, NFAT, IL-4 and IL-13, suggesting NMU may perform positive action in Tregs activation and engage in TAM-M2 polarization related cytokines release from CD4⁺ T cells. Meanwhile, NTS presented positive relationships with CD163 and CD206, which are the molecular biomarkers of TAM-M2, reminding us that NTS may involve in polarization of tumour infiltrating macrophages to M2 subtype. Co-expression of IL-10, TGF-β, STAT3 and STAT4 partially demonstrated NTS may facilitate M2 cells to release cytokines, thereby in return to strengthen CD4⁺ T cells activation. Moreover, results showed NMU and NTS were inversely correlated with neuroblastoma patients’ overall survival rate. These further provided us an evidence that NMU and NTS may influence neuroblastoma development by interfering its microenvironment.

Our data, and the results of previous studies, suggest that NMU and NTS may mediate the interaction of cancer cells and immune cells and may influence the progressionof neuroblastoma (Fig. 6), however further in vitro and in vivo experiments should be undertaken to confirm this speculation.

Conclusions

Our results indicate that NMU and NTS may play roles in the deterioration of the neuroblastoma microenvironment and may provide evidence for future treatments.

Supplemental Information