Cellular and Molecular Characteristics of Established Neuroblastoma Cell Lines

1Shiga-ken Saiseikai Nursing School, Saiseikai Imperial Gift Foundation Inc, Ritto, Shiga, Japan 2Saiseikai Shiga Hospital, Saiseikai Imperial Gift Foundation Inc, Ritto, Shiga, Japan 3Department of Pediatrics, Kyoto Prefectual Univesity of Medicine, Kyoto, Japan 4Department of Pediatrics, University of Miyazaki, Miyazaki, Japan 5Department of Pediatrics, Kyoto City Hospital, Kyoto, Japan Review Open Access Journal of Cancer Research and Therapeutic Oncology


Introduction
The characteristics of childhood malignancy are different from those of adult malignancies, with respect to embryogenesis, histological findings, physiological attributes, and their incidence. In leukemia, lymphoma, brain tumor, neuroblastoma (NB), retinoblastoma, Wilms tumor, hepatoblastoma, and soft-tissue sarcomas including rhabdomyosarcoma and Ewing's sarcoma, the formation of embryonic tumors and sarcomas is prominent. In the nationwide record of childhood malignancies in Japan, hematopoietic malignancies (including leukemia and lymphoma) and solid tumor malignancies account for approximately 40% and 50% of total malignancies, respectively.
During the last 30 years in our pediatric departments in Kyoto, Miyazaki, and the second Kyoto, Japan, our basic policies in our departments emphasize on "Harmony and hard work through healthy competition among colleagues, " "Balancing clinical, educational, and research activities.
We previously attempted to establish cell lines derived from tumor tissue, bone marrow, or metastatic cells from peripheral blood and investigate their tumor-associated molecular characteristics, finally reporting their applicability in clinical studies.

Establishment of 16 NB cell lines from 12 patients
Tumor samples for cell culture were obtained from biopsy, operative, or autopsy specimens and finely minced with scalpels and cultured. Mononuclear cell fractions from bone marrow or peripheral metastatic cells were prepared via Ficoll-Hypaque density gradient centrifugation. Cells were cultured in RPMI 1640 medium containing penicillin (100 U/ml), streptomycin (100 µg/ml) and 15% heat-inactivated fetal calf serum at 37°C and 5% CO2. The media were changed every 3-4 d. Cell lines were considered to be successfully established on subculturing for more than 60 passages over a 2-year period [1]. The NB cell line is one of the most traditional human cell lines, and first NB cell lines NB-1 [2] and SK-N-SH [3] were established in 1973. NB-1 was established by Dr. Shinsaku Imashuku at our institute in the Departments of Pediatrics and Pathology and catecholamine metabolism was assessed in this NB cell line [2]. SK-N-SH [3] was established by Dr. June L Biedler at the Memorial Sloan-Kettering Cancer Center and its morphology, growth characteristics, tumorigenicity, and cytogenetics were assessed. Since then, more than 110 NB cell lines have been reported [4]; however, certain additional NB cell lines also exist. In this review, the cellular and molecular aspects of these established NB cell lines are discussed.

Monoclonal antibodies against NB cell surface antigens (1) Diagnosis of NB tumors with a panel of monoclonal antibodies
Many surface antigens are expressed on tumor cells, and monoclonal antibodies against tumor cell surface antigens have been developed; however, NB-specific antigens have not been reported. Although NB has not been diagnosed using a single monoclonal antibody, a panel of monoclonal antibodies enabled us to detect NB cells.     however, HLA-DQ-mediated induction has not been reported, demonstrating the biological independent among HLA-DR, -DP, and -DQ antigens [14].

Differentiation of NB cells into schwannian cells is
based on pathological evidence that S100-rich tumors of NB patients have a better prognosis. Upon treatment with bromodeoxyuridine (BrdU), flat epithelial cells increased and 2 ' , 3'-cyclinc nuclotide-3'-phosphodiesterase activity was significantly elevated, indicating schwannian differentiation upon BrdU treatment [15].             These results provide the initial evidence that CNTF signaling is conserved in some of the NB cell lines, suggesting that not only the Jak-STAT pathway but also the MAPK pathway are activated by CNTF through gp130 in NB cell lines [29].    in tumor recurrence in the patient from these two cell lines [31].

NB and apoptosis
NB at stage 4S is limited to infants aged less than 1 year and is a localized primary tumor (as in stage 1, 2A, or 2B) with metastasis limited to the skin, liver, and/or bone marrow.
Stage 4S is characterized by distant metastasis; however, spontaneous regression with a favorable prognosis has been clinically observed in the early 1970s. In addition, recent advancements in studies on the cellular and molecular characteristics of these cells revealed that NB cells have a similar morphology; however, they comprise heterogeneous cell groups. The mechanism underlying spontaneous regression is uncertain; however, cell differentiation and cellular apoptosis are considered to be related. Prognosis of advanced NB is still poor, and novel therapeutic trials such as molecular targeted therapies are urgently required; therefore, the association between "NB and apoptosis" is discussed herein. Thus, our study was the first to report that FR induces sustained activation of JNK and p38 MAPK in a ROS-dependent manner in FR-sensitive NB cells ( Figure. 25), but not in FR-resistant NB cells. Furthermore, our study was the first to show that the suppression of intracellular ROS production and the downstream JNK/p38 MAPK pathways are associated with FR resistance in NB cells [32]. At present, a phase I clinical trial to eradicate residual tumor cells in refractory NB patients is expected to assess this FR-induced therapy and hopefully cure this difficult-to-treat disease.  This was the first study to report that EGFR protein is expressed on the cell surface in NB tissue and NB cell lines and that molecular-targeted therapies may effective for NB. Our results indicate the feasibility of targeting EGFR as a novel treatment strategy for NB.