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

ABSTRACT: Stage 4S neuroblastoma, as defined by the International Neuroblastoma Staging System committee (INSS), is known to regress spontaneously and have a more favourable outcome compared with stage 4 tumours. Comparing the molecular differences between these two stages may provide insights into the progression of neuroblastoma. Our study aimed to explore the molecular differences in the tumour microenvironment (TME) between INSS stage 4S and stage 4 tumours to provide an insight into the mechanisms underlying the biological processes of neuroblastoma. We downloaded the datasets GSE120572 and GSE73517 from the GEO database and pre-processed them using the limma package. CIBERSORT deconvolution agorithm was applied to analyse the differences in 22 infiltrating immune leukocyte subsets between the two stages. We used gene ontology (GO) enrichment analysis to determine the biological process (BP) annotation of differentially expressed genes (DEGs) using the online WebGestalt tool. Hub genes were determined in the STRING database and Cytoscape, and the expression of these genes was verified in the Oncomine database. Then these critical genes were performed survival analysis in TARGET database. We further validated the hub genes using a transwell assay and wound healing assay to detect the function of the genes in the neuroblastoma cell line SK-N-BE(2). GO analysis revealed that the 216 DEGs between stage 4S and stage 4 were enriched in aggressive biological processes. Neuromedin U (NMU) and neurotensin (NTS), which were significantly associated with patients' overall survival rate, were verified to be elevated in stage 4, and to promote the proliferation and invasion of the SK-N-BE(2) cell. Tumour infiltrating leukocyte analysis showed a high infiltration of regulatory T cells and type 2 tumour-associated macrophages in stage 4 but not in stage 4S. Results of gene co-expression correlation, and the results of previous studies, suggest that NMU and NTS may play certain roles in modulating TME, thus facilitating the progression of neuroblastoma.

SUBMITTER: Yang D

PROVIDER: S-EPMC8176915 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundNeuromedin U (NMU) was identified as one of the hub genes closely related to colorectal cancer (CRC) progression and was recently shown to be a motility inducer in CRC cells. Its autocrine signalling through specific receptors increases cancer cell migration and invasiveness. Because of insufficient knowledge concerning NMU accessibility and action in the tumour microenvironment, its role in CRC remains poorly understood and its potential as a therapeutic target is still difficult to define.MethodsNMU expression in CRC tissue was detected by IHC. Data from The Cancer Genome Atlas were used to analyse gene expression in CRC. mRNA and protein expression was detected by real-time PCR, immunoblotting or immunofluorescence staining and analysed using confocal microscopy or flow cytometry. Proteome Profiler was used to detect changes in the profiles of cytokines released by cells constituting tumour microenvironment after NMU treatment. NMU receptor activity was monitored by detecting ERK1/2 activation. Transwell cell migration, wound healing assay and microtube formation assay were used to evaluate the effects of NMU on the migration of cancer cells, human macrophages and endothelial cells.ResultsOur current study showed increased NMU levels in human CRC when compared to normal adjacent tissue. We detected a correlation between high NMUR1 expression and shorter overall survival of patients with CRC. We identified NMUR1 expression on macrophages, endothelial cells, platelets, and NMUR1 presence in platelet microparticles. We confirmed ERK1/2 activation by treatment of macrophages and endothelial cells with NMU, which induced pro-metastatic phenotypes of analysed cells and changed their secretome. Finally, we showed that NMU-stimulated macrophages increased the migratory potential of CRC cells.ConclusionsWe propose that NMU is involved in the modulation and promotion of the pro-metastatic tumour microenvironment in CRC through the activation of cancer cells and other tumour niche cells, macrophages and endothelial cells. Video abstract.

Project description:Neuroblastoma is the most common extracranial solid tumour in children and is histologically classified by its Schwannian stromal cells. Although having fewer Schwannian stromal cells is generally associated with more aggressive phenotypes, the exact roles of other stromal cells (mainly macrophages and fibroblasts) are unclear. Here, we examined 41 cases of neuroblastoma using immunohistochemistry for the tumour-associated macrophage (TAM) markers CD68, CD163, and CD204, and a cancer-associated fibroblast (CAF) marker, alpha smooth muscle actin (?SMA). Each case was assigned to low/high groups on the basis of the number of TAMs or three groups on the basis of the ?SMA-staining area for CAFs. Both the number of TAMs and the area of CAFs were significantly correlated with clinical stage, MYCN amplification, bone marrow metastasis, histological classification, histological type, and risk classification. Furthermore, TAM settled in the vicinity of the CAF area, suggesting their close interaction within the tumour microenvironment. We next determined the effects of conditioned medium of a neuroblastoma cell line (NBCM) on bone marrow-derived mesenchymal stem cells (BM-MSCs) and peripheral blood mononuclear cell (PBMC)-derived macrophages in vitro. The TAM markers CD163 and CD204 were significantly up-regulated in PBMC-derived macrophages treated with NBCM. The expression of ?SMA by BM-MSCs was increased in NBCM-treated cells. Co-culturing with CAF-like BM-MSCs did not enhance the invasive ability but supported the proliferation of tumour cells, whereas tumour cells co-cultured with TAM-like macrophages had the opposite effect. Intriguingly, TAM-like macrophages enhanced not only the invasive abilities of tumour cells and BM-MSCs but also the proliferation of BM-MSCs. CXCL2 secreted from TAM-like macrophages plays an important role in tumour invasiveness. Taken together, these results indicate that PBMC-derived macrophages and BM-MSCs are recruited to a tumour site and activated into TAMs and CAFs, respectively, followed by the formation of favourable environments for neuroblastoma progression. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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

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