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


METHODS
Specimens 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.


RESULTS
NB-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.


CONCLUSIONS
We 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.
['Pelizzo|Gloria|G|http://orcid.org/0000-0002-5253-8828', 'Veschi|Veronica|V|', 'Mantelli|Melissa|M|', 'Croce|Stefania|S|', 'Di Benedetto|Vincenzo|V|', "D'Angelo|Paolo|P|", 'Maltese|Alice|A|', 'Catenacci|Laura|L|', 'Apuzzo|Tiziana|T|', 'Scavo|Emanuela|E|', 'Moretta|Antonia|A|', 'Todaro|Matilde|M|', 'Stassi|Giorgio|G|', 'Avanzini|Maria Antonietta|MA|', 'Calcaterra|Valeria|V|']
D014408:Biomarkers, Tumor D001854:Bone Marrow Cells D000072645:Cancer-Associated Fibroblasts D002453:Cell Cycle D002454:Cell Differentiation D002469:Cell Separation D002478:Cells, Cultured D002675:Child, Preschool D018920:Coculture Techniques D005260:Female D020869:Gene Expression Profiling D006801:Humans D007150:Immunohistochemistry D016130:Immunophenotyping D007223:Infant D008297:Male D059630:Mesenchymal Stem Cells D009154:Mutation D009447:Neuroblastoma D011159:Population Surveillance D012042:Registries D015398:Signal Transduction D059016:Tumor Microenvironment
Microenvironment Neuroblastoma Mesenchymal stromal cells Stemness EMT Children