Establishment and characterization of NCC‑ssRMS2‑C1: a novel patient‑derived cell line of spindle cell/sclerosing rhabdomyosarcoma

ImageRyuto Tsuchiya1,2 · Yuki Yoshimatsu1 · Rei Noguchi1 · Yooksil Sin1 · Takuya Ono1 · Akane Sei1 · Fumitaka Takeshita3 · Jun Sugaya4 · Fumihiko Nakatani4 · Akihiko Yoshida5 · Seiji Ohtori2 · Akira Kawai4 · Tadashi Kondo1

Received: 9 May 2021 / Accepted: 15 June 2021 / Published online: 23 June 2021
© Japan Human Cell Society 2021

Spindle cell/sclerosing rhabdomyosarcoma (ssRMS) is a rare subtype of rhabdomyosarcoma (RMS) that has fascicular spindle cell and/or sclerosing morphology. SsRMS has a diverse molecular background and is categorized into three groups: congenital/infantile ssRMS with a gene fusion involving the NCOA2 and VGLL2, ssRMS with the MYOD1 mutation, and ssRMS with no recurrent identifiable genetic alterations. Because ssRMS is a newly defined disease concept of RMS, the optimal treatment methods have not been determined. This results in unfavorable prognosis and consequently signals the urgent need for continuous research. Patient-derived cell lines are essential tools in basic and translational research. However, only two ssRMS cell lines with the MYOD1 mutation have been reported to date. Thus, we established a novel ssRMS cell line named NCC-ssRMS2-C1 using a surgically resected tumor tissue from an adult ssRMS patient. NCC-ssRMS2-C1 cells retained the copy number alterations corresponding to the original tumor and are categorized into the group with no recur- rent identifiable genetic alterations. NCC-ssRMS2-C1 cells demonstrated constant proliferation, spheroid formation, and capability for invasion in vitro, reflecting the malignant features of the original tumor tissue. In a drug screening test, ssRMS demonstrated remarkable sensitivity to romidepsin, trabectedin, actinomycin D, and bortezomib. Hence, we conclude that the NCC-ssRMS2-C1 cell line is the first ssRMS cell line which belongs to the group with no recurrent identifiable genetic alterations, and it will be a useful resource in both basic and translational studies for ssRMS.

Keywords Sarcoma · Spindle cell/sclerosing rhabdomyosarcoma · Patient-derived cancer model · Patient-derived cell line

Spindle cell/sclerosing rhabdomyosarcoma (ssRMS) is a* Tadashi Kondo [email protected]
1 Division of Rare Cancer Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
2 Department of Orthopaedic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
3 Department of Translational Oncology, Fundamental Innovative Oncology Core Center, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
4 Department of Musculoskeletal Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
5 Department of Diagnostic Pathology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
subtype of rhabdomyosarcoma (RMS) that has fascicu- lar spindle cell and/or sclerosing morphology [1]. While ssRMS was once classified under embryonal RMS, as a subtype of RMS, it has since been defined as an inde- pendent subtype since the WHO classification of 2013 [2]. SsRMS accounts for 3–10% of all RMS cases, and it affects infants, children, and adults [1, 3]. The typical site of origin is the head and neck region, followed by the extremities [4]. Genetically, ssRMS is categorized into three groups according to the latest WHO classification [1]. The first group, congenital/infantile ssRMS, exhibits a gene fusion involving the NCOA2 and VGLL2 [5, 6]. The second group comprises ssRMS with the recurrent gene mutation of MYOD1 p.Leu122Arg.
The mutation was identified in 40–60% of ssRMS in adolescents and adults and associated with poor clinical outcomes [5, 7, 8].The third group of ssRMS shows no recurrent identifiable genetic alterations [1]. To date, there has been no consen- sus for the optimal treatment strategy for ssRMS [9]. It has been reported that ssRMS, as well as other RMS subtypes, initially exhibited a favorable response to the vincristine, actinomycin D, and cyclophosphamide (VAC) regimen but tumor recurrence or progression was observed in more than 50% of the cases [10]. Therefore, novel therapeutic methods are urgently needed to improve the prognosis of ssRMS.

Patient-derived cell lines are essential tools in basic and translational research. Although long-term cultur- ing of cell lines results in genetic alterations, and further, changes in drug sensitivity, short-term cultured cell lines can maintain the characteristics of the original tumor [11–14]. Increasing utilization of cell lines, genomic pro- filing, and drug screening tests have been conducted in recent years for cancer drug research [15–19]. However, because ssRMS is a new pathological concept, only two ssRMS cell lines with the MYOD1 mutation have been reported [20, 21], and ssRMS cell lines of other genetic groups have not been established to date according to Cellosaurus [22]. The lack of available ssRMS cell lines means that large-scale genomic profiling or drug screen- ing tests have not been possible in ssRMS. Thus, it is
necessary to establish novel ssRMS cell lines to promote and enable research.
Here, we report the first known ssRMS cell line which belongs to the group with no recurrent identifiable genetic alterations, NCC-ssRMS2-C1, established from a surgically resected specimen from a patient with ssRMS. We charac- terized the NCC-ssRMS2-C1 cells and conducted a drug screening test.

Materials and methods
Patient history

The donor patient was a 40-year-old man with ssRMS. The patient presented in the previous hospital with a chief com- plaint of a rapidly growing mass on his back, confirmed to be a large tumor by magnetic resonance imaging (Fig. 1A, B). The tumor was suspected to be malignant, prompting referral to the National Cancer Center Hospital (Tokyo, Japan). Open biopsy was then performed, and the tumor was diagnosed as ssRMS. Considering its histological type and the large size of the tumor, neoadjuvant chemotherapy (VAC: vincristine, actinomycin D, cyclophosphamide) was provided prior to surgery. After four courses of neoadjuvant Clinical and pathologi- cal data. Magnetic resonance imaging showing a large mass with mixed intensity of high and low in A T2-weighted image and B STIR. Yellow arrows indicate the tumor. C H&E staining showing long fascicles of atypical spindle cells within hyalinized stroma. D The tumor showing strong diffuse expres- sion of MyoD1chemotherapy, wide resection was performed. Pathologi- cally, the tumor showed long fascicles of atypical spindle cells within hyalinized stroma (Fig. 1C) and was diffusely positive for MyoD1 on immunohistochemistry (Fig. 1D). A part of the resected tumor was used to establish the cell line described in this study. The ethical committee of the National Cancer Center approved the use of clinical materi- als for this study and written informed consent was obtained from the donor patient.
Histological analysis

Histology examination was performed on 4-μm-thick sec- tions from a representative paraffinized tumor sample. The sections were deparaffinized and stained with hematoxylin and eosin (H&E).

Immunohistochemistry was also performed using the depar- affinized tumor sample. The sections were exposed to 3% hydrogen peroxide for 15 min to block any endogenous per- oxidase activity and were then washed in deionized water for 2–3 min. The preparations were subjected to heat-induced epitope retrieval. Primary antibody against MyoD1 (EP212, 1:200, Cell Marque, Rocklin, CA, USA) was used. The slides were incubated for 1 h at room temperature, and sub- sequently labeled with peroxidase (EnVision system, Dako, Santa Clara, CA, USA).

Cell culture
The surgically resected tumor tissue was used to establish the cell line as previously described [23]. The cells were maintained in Dulbecco’s Modified Eagle Medium/Nutri- ent Mixture F-12 (Gibco, Grand Island, NY, USA) supple- mented with 5% heat-inactivated fetal bovine serum (Gibco), 100 μg/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque, Kyoto, Japan), 0.4 µg/mL hydrocortisone (Sigma- Aldrich, St. Louis, MO, USA), 5 ng/mL EGF (Sigma- Aldrich), 10 ng/mL bFGF (Sigma-Aldrich), 5 µg/mL insulin (Sigma-Aldrich), and 10 µM Y-27632 (Selleck Chemicals, Houston, TX, USA: Rock inhibitor) at 37 °C in a humidi- fied atmosphere with 5% CO2. The cells were maintained for more than 3 months under tissue culture conditions and were passaged more than 20 times.

Authentication and quality control of the established cell line
The established cell line was authenticated by examining short tandem repeats (STRs) at 10 loci using the GenePrint 10 system (Promega, Madison, WI, USA) according to themanufacturer’s instructions and the procedure described in a previous study [23]. The STR pattern was analyzed using the GeneMapper software (Thermo Fisher Scientific, Waltham, MA, USA) and matched to the data in the public cell banks using a function of Cellosaurus with a standard match threshold of 80% [24].
Mycoplasma contamination was examined using the DNA in the tissue culture medium of the cell line as previ- ously reported [23].

Genetic analysis
The mutation of MYOD1 in the established cell line was examined as previously reported [20]. Briefly, total RNA was used for reverse transcription with the Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The MYOD1 was amplified with the forward primer MYOD1 (L122) F (5′- CAAGCGCAAGACCACCAAC-3′), and the reverse primer MYOD1 (L122) R (5′- GGTTTGGATTGCTCGACG
TG-3′), using Platinum Taq DNA Polymerase High Fidelity (Thermo Fisher Scientific). Sanger sequence analysis was performed with the identical primer set for junction and BigDye v3.1 Cycle Sequencing Kit. The sequence analysis was conducted using the Applied Biosystems 3130xL by GENEWIZ (GENEWIZ, South Plainfield, NJ, USA). The sequence data were matched against the MYOD1 sequence (NCBI Reference Sequence: NM_002478.5).

Single‑nucleotide polymorphism (SNP) array
Single-nucleotide polymorphism (SNP) array genotyping was conducted with the Infinium OmniExpressExome-8 version 1.4 BeadChip (Illumina, San Diego, CA, USA) fol- lowing the manufacturer’s instructions and the procedure described in a previous study [25]. The SNP array data were analyzed using the R version 4.0.3 (R Foundation for Statis- tical Computing, and DNAcopy package version 1.64.0 (Bioconductor, https://bioconductor. org/). Chromosome regions with copy numbers > 3 and < 1 were defined as amplifications and deletions, respectively. Genes that showed copy number alterations (CNAs) were annotated using the biomaRt package version 2.46.0 (Bio- conductor) and “Cancer Gene Census” in the Catalogue of Somatic Mutations in Cancer database (GRCh 37 version 91).

Cell proliferation assay
Cell proliferation assays were performed as described pre- viously [25]. Briefly, the cells were seeded at a density of
2.5 × 104 cells/well in 24-well culture plates at day 0. Thenumber of cells was counted at multiple time points. Thedoubling time was calculated based on the growth curve. All the experiments were performed in triplicates.

Spheroid formation assay
Spheroid formation was examined as described previously [23]. The obtained spherical colonies were prepared for par- affin sections using iPGell (Genostaff, Tokyo, Japan) accord- ing to the manufacturer’s instructions. Cell blocks were fixed with 10% formalin neutral buffer solution and embedded in paraffin. Four-micrometer-thick paraffin sections were pre- pared and stained with H&E. Immunohistochemistry using MyoD1 antibody was also performed on the specimens.

Invasion assay using real‑time cell analyzer
Invasion potential was examined using a real-time cell analyzer (xCELLigence, Agilent, Santa Clara, CA, USA) according to the manufacturer’s instructions and the proce- dure described in a previous study [23]. MG63 osteosarcoma cells (JCRB; Ibaraki Osaka, Japan) were used as controls [26]. Monitoring was done every 15 min for 72 h and rela- tive cell proliferation was plotted as a function of time after seeding.

Assessment of tumorigenesis in nude mice
Mice tumorigenesis in this study was assessed in compli- ance with the guidelines of the Institute for Laboratory Ani- mal Research, National Cancer Center Research Institute as previously described [25]. Briefly, we used female BALB/c nude mice purchased from CLEA Japan, Inc. (Tokyo, Japan). A 100-μL volume of cells in a 1:1 mixture of Matrigel (BD Biosciences) was injected subcutaneously into the mice (1 × 106 cells). Subsequently, the tumor size was measured weekly. The tumor volumes were calculated according to the following formula: volume = (length × width2)/2. After 2 months, the tumors were surgically resected, and the specimens were stained with H&E. Additionally, immuno- histochemistry using MyoD1 antibody was performed on the specimens.

Screening for the anti‑proliferative effects of anti‑cancer agents
The anti-proliferative effects of 214 anti-cancer agents, including FDA-approved drugs (Selleck Chemicals, Hou- ston, TX, USA), were assessed according to the manufac- turer’s instructions and the procedure described in a previ- ous study [23]. A list of the anti-cancer agents utilized is provided in Supplementary Table 1.Dose–response experiments were also performed to vali- date the available hits in the pilot screening according to
the methods as we previously reported. The IC50, defined as the sample concentration required to inhibit cell growth by 50% in comparison with the growth of the control cells, was determined from the dose–response curves.

Authentication of the established cell line

The NCC-ssRMS2-C1 cell line was authenticated by analyz- ing the STR status of 10 microsatellites. STR allele patterns of NCC-ssRMS2-C1 cells and the corresponding original tumor tissue (Table 1, Supplementary Fig. 1) were con- firmed to be identical. The STR patterns of NCC-ssRMS2- C1 cells were found to be unique and did not match those of cell lines in the public cell banks examined using Cel- losaurus. Thus, we concluded that NCC-ssRMS2-C1 was a novel cell line. We verified that the NCC-ssRMS2-C1 cells were not contaminated with Mycoplasma (data not shown).
Characterization of the cell line

MYOD1 mutation was not detected in both the original tumor tissue and NCC-ssRMS2-C1 cells (Supplementary Fig. 2). SNP array analysis revealed CNAs in NCC-ssRMS2- C1 cells, corresponding to the original tumor. Partial allelic amplification was identified in chromosomes 2q and 3q, while deletions were found in 1p, 7q, 8p, 12q, 13q, and 15q. The deletion in chromosome 13q included the tumor sup- pressor gene RB1 (Fig. 2, Supplementary Table 2).
NCC-ssRMS2-C1 cells were composed of atypical spindle cells in monolayer culture conditions (Fig. 3A, B). The population doubling time of NCC-ssRMS2- C1 cells was approximately 41 h based on the growth curve (Fig. 3C). NCC-ssRMS2-C1 cells also had the potential to form spheroids when they were seeded on a low-attachment microplate. The H&E-stained spheroid

Table 1 Short tandem repeat analysis

Microsatellite (Chromosome) NCC-ssRMS2-C1 Tumor tissue
Amelogenin (X Y) X,Y X,Y
TH01 (3) 9 9
D21S11 (21) 30, 32.2 30, 32.2
D5S818 (5) 10, 11 10, 11
D13S317 (13) 11 11
D7S820 (7) 11 11
D16S539 (16) 9, 11 9, 11
CSF1PO (5) 11, 13 11, 13
vWA (12) 14, 17 14, 17
TPOX (2) 8, 11 8, 11

ImageFig. 2 Analysis of the single- nucleotide polymorphism array. Allele-specific copy number analysis revealed DNA copy number alterations in the A normal tissue, B original tumor, and C NCC-ssRMS2-C1 cells (passage 16). The X-axis and
Y-axis indicate the log ratio of copy number and chromosome number, respectivelyction showed pleomorphic atypical cells (Fig. 3D). Immunohistochemically, these cells were negative for MyoD1 (Fig. 3E). The invasion assay revealed that NCC- ssRMS2-C1 cells had an increased invasiveness compared to MG63 cells. The invasion ability of NCC-ssRMS2-C1 cells depended on the time after seeding (Fig. 3F).

Tumorigenesis in nude mice

NCC-ssRMS2-C1 cells transplanted into BALB/c nude mice formed small tumor masses under the described con- ditions (Fig. 4A). The tumor showed intersecting fascicles of atypical spindle cell proliferation against a collagenous background in H&E sections (Fig. 4B) and was diffusely positive for MyoD1 on immunohistochemistry (Fig. 4C).
However, the tumor growth was not sufficient (Fig. 4D).
Sensitivity to anti‑cancer agents

The cell viability of NCC-ssRMS2-C1 after treatment with 214 anti-cancer agents at a fixed concentration of 10 µM is shown in Supplementary Table 3. Among the 214 anti-cancer agents examined, 24 agents that either showed significant anti-proliferative effects on NCC-ssRMS2-C1 cells or are frequently used as standard chemotherapy for sarcomas were further examined to calculate their IC50 values. The four agents with the lowest IC50 values are shown in Table 2, and the IC50 values of 24 agents are in Supplementary Table 4. Growth curves that served as the basis for the calculation of the IC50 values are shown in Fig. 5 and Supplementary Fig. 3.

Fig. 3 Characterization of NCC-ssRMS2-C1 cells. A and B NCC-ssRMS2-C1 cells (passage 24) showing atypical spindle form under 2D culture conditions. C Growth curve of NCC-ssRMS2-C1 cells (pas- sage 20). The Y-axis indicates
the relative cell proliferation of NCC-ssRMS2-C1 cells, and the X-axis represents the day after seeding. D The H&E-stained spheroid section of NCC- ssRMS2-C1 cells (passage 26) showing dense proliferation of pleomorphic atypical cells. E Immunohistochemically, the spheroid section of NCC- ssRMS2-C1 cells were negative for MyoD1. F Real-time cell analyzer invasion assay illustrat- ing the invasion ability of NCC- ssRMS2-C1 cells (passage 28) compared to that of MG63 osteosarcoma cells

ImageFig. 4 Tumorigenesis in nude mice. A NCC-ssRMS2-C1 cells transplanted into BALB/c nude mice formed a small tumor mass under the described condition. B The tumor showed intersecting fascicles of atypi- cal spindle cell proliferation
in a collagenous background in H&E staining sections. C Immunohistochemically, the
tumor was diffusely positive for MyoD1. D The graph illustrat- ing estimated tumor volume.
Bars represent the mean stand- ard error

Table 2 Summary of half-maximal inhibitory concentration (IC50) values in the cells

CAS# Name of drugs NCC-ssRMS2-C1 IC50 (μM)
128517-07-7 Romidepsin (FK228, Dep- sipeptide) 0.01479
114899-77-3 Trabectedin 0.01553
50-76-0 ActinomycinD 0.06973
179324-69-7 Bortezomib (PS-341) 0.08594

SsRMS is a rare subtype of RMS. Although conventional chemotherapy for other subtypes of RMS is utilized, opti- mal treatment methods for ssRMS have not been deter- mined to date [9, 10]. In recent years, genomic profiling and drug screening tests using cell lines have enabled identification of novel candidate drugs for cancer [15–19]. However, only two ssRMS cell lines have been reported [20, 21], precluding ssRMS from benefiting from these

Growth curves for the IC50 value calculation of the investigated anti-cancer agents. A–D Viability of NCC-ssRMS2-C1 cells (passage 30) treated with anti-cancer agents at different concentrations. The name of each anti-cancer agent is labeled under the graphscreening analyses. Hence, we established a novel ssRMS cell line, NCC-ssRMS2-C1.The NCC-ssRMS2-C1 cells did not harbor the MYOD1 mutation, which is observed in 40–60% of ssRMS [5, 7, 8]. In contrast, SNP array analysis revealed multiple CNAs including deletion of the tumor suppressor gene RB1. The studies on CNAs of ssRMS are quite limited, and their bio- logical significance remains unclear [27]. Since the NCC- ssRMS2-C1 cell line was established from a tumor derived from a 40-year-old ssRMS patient, this does not belong to the congenital/infant group which has a fusion gene and is instead classified under the group with no recurrent identifi- able genetic alterations. The two previously reported ssRMS cell lines are classified under the group with MYOD1 muta- tion [20, 21]. Therefore, the NCC-ssRMS2-C1 cell line is the first ssRMS cell line which belongs to the group with no recurrent identifiable genetic alterations and may serve as a useful tool to unravel the diverse molecular background of ssRMS.
NCC-ssRMS2-C1 cells exhibited a spindle cell mor- phology and demonstrated constant growth and aggres- sive invasion, which may reflect the characteristics of the original tumor. Its spheroid formation ability contributes to the understanding of the behavior of ssRMS in a 3D envi- ronment. While NCC-ssRMS2-C1 cells had these features reflecting the original tumor, the immunohistochemical staining for MyoD1 was lost in vitro. The cultured cells were exposed to different oxygen concentrations in the arti- ficial culture environment compared to those in the human body. This may have led to fluctuations in the expression of HIF1 and other genes, resulting in the loss of MyoD1
[28]. Meanwhile, the cells transplanted into the animals were immunohistochemically positive for MyoD1. This result may be due to the fact that the cells were placed in an envi- ronment similar to that of the original tumor. Although the in vivo findings reflected the original tumor, NCC-ssRMS2- C1 cells did not sufficiently grow in nude mice under the described conditions, indicating that NCC-ssRMS2-C1 cells may not be suitable for xenograft experiments.

In the drug screening using 214 anti-cancer agents, the four agents with the lowest IC50 values were romidepsin, trabectedin, actinomycin D, and bortezomib. Actinomycin D is used as a standard drug in other subtypes of RMS [3]. The ssRMS cell line NCC-ssRMS1-C1 [20] also demon- strated a significant response to actinomycin D, suggesting the sensitivity of ssRMS to actinomycin D. Trabectedin has shown good efficacy on soft tissue sarcomas [29], and is expected to be similarly effective on ssRMS. In addition to these two drugs, romidepsin and bortezomib exhibited remarkable anti-proliferative effects. Romidepsin is a histone deacetylase (HDAC) inhibitor usually used for the treatment of peripheral T-cell lymphoma [30]. The efficacy of HDAC inhibitors in other subtypes of RMS cell lines has been reported [31–33] supporting the possible utility of romidep- sin as a novel candidate drug for the treatment of ssRMS. Bortezomib is a proteasome inhibitor used for blood cancers such as multiple myeloma [34]. The efficacy of bortezomib on other subtypes of RMS has also been reported in several studies [35, 36] making it a potential candidate drug.

In this study, we successfully established the first ssRMScell line which belongs to the group with no recurrent iden- tifiable genetic alterations to be reported. However, thenumber of ssRMS cell lines remains insufficient. Consid- ering the diversity of the disease, there is still a need to further establish ssRMS cell lines to be shared widely in the research community. We conclude that NCC-ssRMS2-C1 cell line is useful for basic and translational research and for elucidating the molecular background of ssRMS.
Supplementary Information The online version contains supplemen- tary material available at

Acknowledgements We thank Drs. E. Kobayashi, S. Iwata, S. Fuku- shima, M. Nakagawa, T. Komatsubara, C. Sato (Department of Mus- culoskeletal Oncology), and Drs. N. Kojima, J. Kashima, M. Arakaki (Department of Diagnostic Pathology), National Cancer Center Hos- pital, for sampling tumor tissue specimens from surgically resected materials. We also appreciate the technical assistance provided by Ms.
Y. Kuwata (Division of Rare Cancer Research). We appreciate the technical support provided by Ms. Y. Shiotani, Mr. N. Uchiya, and Dr.
T. Imai (Central Animal Division, National Cancer Center Research Institute). We would also like to thank Editage ( for their help with English language editing and their constructive com- ments on the manuscript. This research was technically assisted by the Fundamental Innovative Oncology Core in the National Cancer Center.

Funding This research was supported by the Japan Agency for Medi- cal Research and Development (Grant number 20ck0106537h0001).


Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval The ethical committee of the National Cancer Center approved the use of clinical materials for this study (Approval number 2004–050).
Informed consent Written informed consent for publication was pro- vided by the patient.

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