Microenvironment in neuroblastoma: isolation and characterization of tumor-derived mesenchymal stromal cells

Abstract

BackgroundIt has been proposed that mesenchymal stromal cells (MSCs) promote tumor progression by interacting with tumor cells and other stroma cells in the complex network of the tumor microenvironment. We characterized MSCs isolated and expanded from tumor tissues of pediatric patients diagnosed with neuroblastomas (NB-MSCs) to define interactions with the tumor microenvironment.MethodsSpecimens were obtained from 7 pediatric patients diagnosed with neuroblastoma (NB). Morphology, immunophenotype, differentiation capacity, proliferative growth, expression of stemness and neural differentiation markers were evaluated. Moreover, the ability of cells to modulate the immune response, i.e. inhibition of phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cytotoxic function, was examined. Gene expression profiles, known to be related to tumor cell stemness, Wnt pathway activation, epithelial-mesenchymal transition (EMT) and tumor metastasis were also evaluated. Healthy donor bone marrow-derived MSCs (BM-MSC) were employed as controls.ResultsNB-MSCs presented the typical MSC morphology and phenotype. They showed a proliferative capacity superimposable to BM-MSCs. Stemness marker expression (Sox2, Nanog, Oct3/4) was comparable to BM-MSCs. NB-MSC in vitro osteogenic and chondrogenic differentiation was similar to BM-MSCs, but NB-MSCs lacked adipogenic differentiation capacity. NB-MSCs reached senescence phases at a median passage of P7 (range, P5-P13). NB-MSCs exhibited greater immunosuppressive capacity on activated T lymphocytes at a 1:2 (MSC: PBMC) ratio compared with BM-MSCs (p = 0.018). NK cytotoxic activity was not influenced by co-culture, either with BM-MSCs or NB-MSCs. Flow-cytometry cell cycle analysis showed that NB-MSCs had an increased number of cells in the G0-G1 phase compared to BM-MSCs. Transcriptomic profiling results indicated that NB-MSCs were enriched with EMT genes compared to BM-MSCs.ConclusionsWe characterized the biological features, the immunomodulatory capacity and the gene expression profile of NB-MSCs. The NB-MSC gene expression profile and their functional properties suggest a potential role in promoting tumor escape, invasiveness and metastatic traits of NB cancer cells. A better understanding of the complex mechanisms underlying the interactions between NB cells and NB-derived MSCs should shed new light on potential novel therapeutic approaches.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-5082-2) contains supplementary material, which is available to authorized users.

Peer Review #3 of "Cellular components in tumor microenvironment of neuroblastoma and the prognostic value (v0.1)"

“…Moreover, some cells, including osteoblasts and MSCs, may enhance bone and bone marrow metastases (Ma et al, 2011;Timaner et al, 2019), which are common in stage 4 NB patients. Pelizzo G. (Pelizzo et al, 2018) had isolated and characterized the tumor-derived MSCs and proposed the potential role of MSCs in promoting tumor invasion and metastasis. Additionally, some cells are tissue-specific, such as epithelial cells clustered in bladder urothelial carcinoma, cervical carcinoma, head and neck squamous cell carcinoma, and other epithelial-derived cancers.…”

Targeting the Tumor Microenvironment in Neuroblastoma: Recent Advances and Future Directions

“…TAMs also contribute to the stimulation of hypoxic microenvironment in NB by inducing transcription of hypoxia inducible factor (HIF 2α) [29]. Besides macrophages, the presence of cancer associated fibroblasts (CAFs), mesenchymal stromal cells (MSCs), endothelial cells and inflammatory immune cells including regulatory T cells (T reg ), myeloid derived suppressor cells (MDSCs); also contributes to highly vascular, angiogenic, hypoxic and immunosuppressive microenvironment of NB [7,[30][31][32]. Immunosuppressive microenvironment in NB is generated due to various reasons, which include (1) infiltrating immunosuppressive immune cells including macrophages, regulatory T cells and myeloid derived suppressor cells, (2) soluble factors secreted in neuroblastoma microenvironment which mediates immunosuppression like TGF beta, IL10, and galectin-1, (3) defects in antigen-presenting machinery (APM) and low levels of MHC class I molecule displayed by NB cells.…”

“…These are multipotent cells that can be differentiated into different lineages including bone, skeletal muscles, tendon and cartilage [147]. The role of MSCs in neuroblastoma tumor progression and metastasis is well documented [32,148,149]. These cells interact with the tumor cells and other stromal cells to mediate tumor progression.…”

“…These cells interact with the tumor cells and other stromal cells to mediate tumor progression. Several reports suggested that these cells can modulate TME by affecting immune cell responses [32,146]. A study by Ma et al has shown that neuroblastoma cells express both receptors for stromal cell derived factor 1 (SDF 1), i.e., CXCR4 and CXCR7.…”

“…The expression of CXCR4 correlated with the bone marrow metastasis in primary neuroblastoma tumors [153]. Pelizzo et al isolated and characterized MSC from tumor tissues of seven pediatric neuroblastoma patients [32]. The gene expression profiling and the functional properties revealed that these stromal cells contribute to tumor immune escape and metastatic traits of neuroblastoma.…”