Functional Sphere Profiling Reveals the Complexity of Neuroblastoma Tumor-Initiating Cell Model

Abstract

Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically "profile" the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.

Neuroblastoma stem cells – mechanisms of chemoresistance and histonedeacetylase inhibitors

“…The sphere-forming cells, selected from the highly malignant NBL tumor samples are resistant to doxorubicin, cisplatin and etoposide compared to cells cultivated in medium with serum. Average CD133 expression was also increased in spheres but not in every analyzed sphere derived from NBL samples and cell lines [56]. This finding doubts the significance of CD133 as an universal marker of all CSCs in NBL and shows that is unreliable to detect CSCs using a single marker.…”

“…Hansfrod et al examined cells isolated from bone marrow metastases and those cells form spheres regardless of the risk group. While Coulon et al used isolated cells from primary tumors that had grown as spheres only when they were derived from HR NBL [56]. 16p13.3, 19p13.3, and 19q13.33 that are absent in CD133-cells.…”

“…However we are missing the basic information which is necessary for further research on CSCs and clinical application for their detection in diagnostics and CSC target therapy. It is necessary to: (i) define the CSC function in cancerogenesis, cancer development and their role in metastasis and chemoresistance; (ii) find a specific marker for CSCs in certain tumors; (iii) explain the role of different pathways that determine their behavior and (iv) resolve mechanisms of chemoresistance of CSCs [2,7,8,17,56]. …”

Reversal of cisplatin resistance in non-small cell lung cancer stem cells by Taxus chinensis var.

“…Therefore, several researchers are attempting to develop methods to reverse the chemoresistance of cancer stem cells. In this study, we developed a method to generate self-renewing and drug-resistant stem cells from the H460 cell line via sphere propagation, utilizing ultra-low-adherent substrates and growth factors such as EGF, bFGF, and B27, as described in previous studies (Coulon et al, 2011;Su et al, 2013). A number of studies have reported that cisplatin, widely used as a therapeutic agent against NSCLC, causes severe side effects (Karasawa and Steyger, 2015).…”

Novel Therapeutic Approaches for Neuroblastoma

“…Singh et al [37] have shown that human brain tumors contain CD133+ stem-like cells that are capable of growing tumors in immune-deficient mice. Cournoyer et al [40] have shown that CD133 high neuroblastoma (NB) cells have high tumor initiating cell properties, and Coulon et al [41] suggest that CD133, ABC transporter, Wnt and NOTCH genes are sphere markers in NB cells. Overall, 19-29 % of cells in glioblastomas and 6-21 % of cells of medulloblastomas are reported to be CD133+ and tumorigenic [33].…”