Transient treatment with epigenetic modifiers yields stable neuroblastoma stem cells resembling aggressive large-cell neuroblastomas

Abstract

Cancer stem cells (CSCs) are plastic in nature, a characteristic that hampers cancer therapeutics. Neuroblastoma (NB) is a pediatric tumor of neural crest origin, and half of the cases are highly aggressive. By treating NB cell lines [SKNAS, SKNBE(2)C, CHP134, and SY5Y] with epigenetic modifiers for a short time, followed by sphere-forming culture conditions, we have established stem cell-like NB cells that are phenotypically stable for more than a year. These cells are characterized by their high expression of stemness factors, stem cell markers, and open chromatin structure. We referred to these cells as induced CSCs (iCSCs). SKNAS iCSC and SKNBE(2)C iCSC clones (as few as 100 cells) injected s.c. into SCID/Beige mice formed tumors, and in one case, SKNBE(2)C iCSCs metastasized to the adrenal gland, suggesting their increased metastatic potential. SKNAS iCSC xenografts showed the histologic appearance of totally undifferentiated large-cell NBs (LCNs), the most aggressive and deadly form of NB in humans. Immunohistochemical analyses showed that SKNAS iCSC xenografts expressed high levels of the stem cell marker CXCR4, whereas the SKNAS monolayer cell xenografts did not. The patterns of CXCR4 and MYC expression in SKNAS iCSC xenografts resembled those in the LCNs. The xenografts established from the NB iCSCs shared two common features: the LCN phenotype and high-level MYC/MYCN expression. These observations suggest both that NB cells with large and vesicular nuclei, representing their open chromatin structure, are indicative of stem cell-like tumor cells and that epigenetic changes may have contributed to the development of these most malignant NB cells.tumor initiating cells | prominent nucleoli N euroblastoma (NB) is the most common extracranial pediatric malignancy. Although some NBs are easily treatable, nearly 50% of the tumors exhibit aggressive behavior. The International Neuroblastoma Risk Group Classification is used to classify NB at diagnosis (1). High-risk NBs are associated with older age, advanced stages, unfavorable histologic features, widespread tumor dissemination, and poor long-term survival (1). Current treatment for these NBs includes high-dose chemotherapy or myeloablative cytotoxic therapy with autologous hematopoietic stem cell transplantation (2). However, late relapse is often seen despite achieving complete clinical remission. A subset of high-risk NBs, which is refractory to current frontline therapy designed for high-risk NB, is termed ultra-high-risk NB (3, 4).NB is histopathologically defined as neuroblastic tumors with less than 50% Schwannian stromal components. On the basis of the degree of neuronal differentiation, NB is further divided into undifferentiated (UD), poorly differentiated, and differentiating subtypes (5, 6). A unique histological subset of NBs within the UD and poorly differentiated subtypes has also been identified (7,8). These tumors are uniformly composed of large cells with sharply outlined nuclear membranes and one to four prominent nucleoli and are referred...

Downregulation of c-Myc is involved in TLR3-mediated tumor death of neuroblastoma xenografts

“…8,12,13 Through triggering epigenetic changes, MYCN-non-amplified NB cells could be transformed to aggressive, stem cell-like cells. 14 In up to 70% of human cancers, c-Myc protein expression is deregulated and considered as an attractive target for cancer therapy. 15 Its transcriptional targets may promote cell cycle progression, tumor cell growth, and cell differentiation, and regulate cellular metabolism and cell adhesion as well as apoptosis.…”

Clinical Implications of Neuroblastoma Stem Cells

“…The iCSC xenografts were thus classified as totally undifferentiated "large-cell" NB, according to the International Neuroblastoma Pathology Classification [2,3,23,25]. In fact, as reported in our study, all of the other iCSC xenografts from SKNBE(2)C, CHP134, and SY5Y have the LCN phenotype [10]. Fig.…”

“…Our in vivo studies show that the NB iCSCs possess a high tumor-initiating ability and a high metastatic potential. SKNAS iCSC and SKNBE(2)C iCSC clones (as few as 100 cells) injected subcutaneously into SCID/Beige mice formed tumors, and in one case, SKNBE(2)C iCSC metastasized to the adrenal gland, suggesting their increased metastatic potential [10]. Important histopathological observations were also made on the NB iCSC xenografts, and highlights of these findings are described in below.…”

“…We referred to these cells as induced CSC (iCSC) [10]. Our in vivo studies show that the NB iCSCs possess a high tumor-initiating ability and a high metastatic potential.…”

“…We have recently reported the establishment of phenotypically stabilized stem cell-like NB cells (refer to as iCSC, see below) by short-term treatments of conventional monolayer NB cell lines with epigenetic modifiers [10]. The study addresses a fundamental problem that has affected a complete success in treating patients with cancers.…”