Cellular and Molecular Characteristics of Established Childhood Soft-Tissue Sarcoma Cell Lines

From 1984 to 2013, we established 29 childhood tumor cell lines, including 13 soft-tissue-sarcoma cell lines. Here, we provide an overview of these soft-tissue sarcoma cell lines, their origins, characteristics, and highlight their potential as valuable research tools for fundamental research and development of new treatments. The cell lines were established from three patients with rhabdomyosarcoma (RMS), five patients with Ewing sarcoma family tumors, four patients with rhabdoid tumors, and one patient with clear cell sarcoma of soft parts. In particular, we demonstrate the potential of the MRT cell lines in preclinical in vivo and in vitro studies to evaluate molecular target therapy using gefitinib and trastuzumab activated antibody-dependent cellular cytotoxicity. Moreover, the clear cell sarcoma cell line, MP-CCS-SY, was established from a metastatic tumor in the left Achilles tendon of a 17-year-old girl, which represents a rare cell line for this cancer, and will help to gain a better understanding of the molecular biology of this malignancy and serve as a useful tool for developing boron neutron capture therapy.


Introduction
Soft-tissue sarcomas originate from immature embryonal mesoderm or mesenchymal tissues, and can therefore potentially develop from any anatomical site. Recent advances in progressive therapy have led to a high cure rate for soft-tissue sarcomas with combinations of chemotherapy, surgery, radiation therapy, and supportive therapy. In addition, chromosomal analysis and more recent advanced molecular biological techniques that can identify chromosomal aberrations have improved both the diagnosis and prognosis of the disease, along with insight into the underlying mechanism of tumor biology.

No
Cell Tumor samples from patients for cell culture were obtained by biopsy, operation, or autopsy and were finely minced with a scalpel and cultured. Mononuclear cell fractions from the bone marrow or peripheral metastatic cells were prepared by Ficoll-Hypaque centrifugation. The cells were cultured in RPMI 1640 medium containing penicillin (100 U/ml), streptomycin (100 µg/ml), and 15% heated-inactivated fetal calf serum at 37℃ in a 5% CO 2 atmosphere. The medium was replaced every 3-4 days. Cell lines were considered to be established when they were cultured for more than 60 passages over a 2-year period [1].

Incidence and pathological findings in RMS
RMS is derived from the immature embryological mesoderm or mesenchymal tissues during the formation of skeletal muscle cells, and is the most common soft-tissue sarcoma (50%), accounting for 5% of all childhood cancers. RMS can be classified into five groups: embryonal, botryoid, spindle, alveolar, and pleomorphic, as per the 1992 World Health Organization Classification of Soft Tissue Tumors [2]. Antibodies against skeletal muscle-specific proteins (e.g., desmin, alpha-and gamma-muscle actin, alpha-sarcomeric actin) are effective for the immunofluorescence-based diagnosis of RMS [3,4]

Expression of MyoD1 in RMS cell lines
Myoblast determination protein 1 (MyoD1) expression has been found to be limited to RMS, and is considered to be responsible for the lack of differentiation to mature skeletal muscle cells [5][6][7].  and SCMC-RM2). Thus, using recent molecular biology techniques, we were able to confirm the presence of the PAX3/FKHR chimera gene to identify and diagnose alveolar RMS more specifically [8,9].  According to Barr et al. [8], the PAX3/FKHR chimera product does not only indicate "an embryonal RMS cell line" as outlined in our previous study [11] but can also refer to alveolar RMS, as highlighted in the example described above ( Figure 2).

Moreover, amplification of the N-Myc proto-oncogene
MYCN has generally been considered to be related to neuro-   However, we did not find a clear association between the type of chimeric mRNA and clinical features such as sex, age, primary site, and histopathology of the patients. All of the chimeric mRNAs were generated from in-frame junctions and are considered to encode fusion proteins that may be involved in the molecular mechanism underlying the progression of ESFT [17].  To date, there are less than 10 MRT cell lines available.

EWS/FLI1 chimeric gene in established EW sarcoma cell lines
Four of these were established in our departments, and thus the cellular and genetic characterization of these cell lines will be useful for new diagnosis methods and novel therapeutic development.

KP-MRT-NS cell line
The

KP-MRT-RY cell line
The KP-MRT-RY cell line was established from a 1-month-old infant who presented with abdominal distension (Table 1, Figure 10). The left renal tumor was resected, and the KP-MRT-RY cell line was cultured and characterized as described previously [20].

MP-MRT-AN cell line
The MP-MRT-AN cell line was cultured from liver biopsy specimens ( Figure 11). Immunohistochemical assays detected the expression of vimentin and cytokeratin. RT-PCR assays revealed that this cell line did not express smooth muscle myosin heavy chain isoforms or MyoD1 [21].   Figure   15). 8 Figure. 14 Effect of gefitinib on two MRT cell lines Figure. 15 Inhibition of tumor growth by gefitinib in MRT cell lines Overall, these results demonstrated that gefitinib has antitumor effects in MRT cells in vitro and in vivo, and thus has promise as a novel and therapeutic strategy for MRT [24].
Preclinical studies on the activation of antibody-dependent cellular cytotoxicity by trastuzumab against MRT cells Trastuzumab, a humanized monoclonal antibody against human epidermal growth factor receptor-2 (HER-2), has been shown to be effective against breast cancer and other cancers. Therefore, we also examined the expression of HER-2 in our four MRT cell lines by indirect immunofluorescence, flow cytometry, and immunohistochemistry [20].

Establishment of the MP-CCS-SY cell line
To date, there are only 10 established cell lines of CCS [26,27]. We established the MP-CCS-SY cell line from metastasis of the left femoral bone tumor in a 17-year-old SY girl ( Figure   18).
The tumor cells grew as an adherent monolayer. A small number of melanosomes were detected in the cytoplasm by electron microscopy (Figure 19), which immunoreacted with two melanoma-associated antibodies, HMB45, and Melan-A ( Figure   20A, B). Western blot analysis further demonstrated the existence of a Melan-A antigen in this cell line ( Figure 20C) [26].  Figure 21B) indicating a potential role in the malignant progression of CCS [26]. The availability of this MP-CCS-SY cell line will help to improve understanding of the molecular biology of this malignancy and should serve as a useful tool for developing boron neutron capture therapy [28].