Establishment and characterization of two novel patient-derived cell lines from myxofibrosarcoma: NCC-MFS7-C1 and NCC-MFS8-C1

Establishment and characterization of two novel patient-derived cell lines from myxofibrosarcoma: NCC-MFS7-C1 and NCC-MFS8-C1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Establishment and characterization of two novel patient-derived cell lines from myxofibrosarcoma: NCC-MFS7-C1 and NCC-MFS8-C1 Yuki Adachi, Rei Noguchi, Julia Osaki, Takuya Ono, Shuhei Iwata, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4251932/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Aug, 2024 Read the published version in Human Cell → Version 1 posted 5 You are reading this latest preprint version Abstract Myxofibrosarcoma (MFS), an aggressive soft tissue sarcoma, presents a significant challenge because of its high recurrence rate, distal metastasis, and complex genetic background. Although surgical resection is the standard treatment for MFS, the outcomes are unsatisfactory, and effective non-surgical treatment strategies, including drug therapy, are urgently warranted. MFS is a rare tumor that requires comprehensive preclinical research to develop promising drug therapies; however, only two MFS cell lines are publicly available worldwide. The present study reports two novel patient-derived MFS cell lines, NCC-MFS7-C1 and NCC-MFS8-C1. These cell lines have been extensively characterized for their genetic profile, proliferation, spheroid-forming capacity, and invasive behavior, confirming that they retain MFS hallmarks. Furthermore, we conducted comprehensive drug screening against these cell lines and six others previously established in our laboratory to identify potential therapeutic candidates for MFS. Among the screened agents, actinomycin D, bortezomib, and romidepsin demonstrated considerable antiproliferative effects that were superior to those of doxorubicin, a standard drug, highlighting their potential as novel drugs. In conclusion, NCC-MFS7-C1 and NCC-MFS8-C1 are valuable research resources that contribute to the understanding of the pathogenesis and development of novel therapies for MFS. Myxofibrosarcoma Soft tissue sarcoma Cell lines Patient-derived model Antitumor drug screening Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Myxofibrosarcoma (MFS), a notably aggressive soft tissue sarcoma, is a malignant fibroblastic neoplasm characterized by a variable myxoid stroma and pleomorphic cells [ 1 ]. Tumors are most commonly located in the limbs, with a particular propensity for the lower extremities [ 2 , 3 ]. MFS predominantly affects older adults, with a peak incidence in the sixth to eighth decades of life [ 4 , 5 ]. Notably, surgical resection is the cornerstone of curative treatment for MFS [ 2 , 6 ]. However, high rates of local recurrence (30–40%) and distant metastasis (15–35%) significantly affect the 5-year survival prospects, which hover around 60–70% [ 1 , 2 , 4 , 7 , 8 ]. These data highlight the need for comprehensive multidisciplinary treatment approaches that extend beyond surgery and include innovative drug therapies. Despite the clear necessity of such strategies, effective multidisciplinary methods, including established drug regimens, remain elusive. The intricate genetic landscape of MFS underpins its aggressive clinical behavior and contributes to significant challenges in the development of effective therapeutic strategies. Key genetic alterations observed in MFS include mutations and copy number alterations in pivotal tumor suppressor genes and cell cycle regulators, notably TP53, RB1, CDKN2A, and CDKN2B [ 9 , 10 ]. Moreover, frequent amplifications on chromosome 5p, which harbors oncogenes such as TRIO and RICTOR, have been implicated in driving the malignant potential of MFS [ 10 ]. These genetic aberrations not only confer a high degree of biological aggressiveness but also foster a heterogeneous tumor environment, complicating personalized treatment approaches. Cell lines are indispensable in cancer research as they provide a foundational platform for elucidating disease mechanisms and evaluating therapeutic interventions [ 11 , 12 ]. Their importance is amplified in the context of rare diseases such as MFS, where conducting extensive clinical trials is challenging owing to the limited patient population. This difficulty underscores the value of robust preclinical models, such as cell lines, for advancing our understanding and treatment of such diseases [ 13 ]. Despite the critical role of these models, the Cellosaurus database, a comprehensive resource for cell line information, lists only 16 MFS cell lines, with only two publicly available (Supplementary Table 1) [ 14 ]. This scarcity of MFS cell lines severely restricts research progress and the development of targeted therapies. Therefore, additional cell lines are strongly required to promote further research and improve the therapeutic outcomes of MFS. This study introduced two novel MFS cell lines, NCC-MFS7-C1 and NCC-MFS8-C1, established from tumor samples resected from two different patients. We meticulously characterized the NCC-MFS7-C1 and NCC-MFS8-C1 cell lines and examined their genetic profiles, proliferation rates, spheroid formation capabilities, and invasive behaviors. Additionally, we expanded our investigation to include drug screening across these novel cell lines and six others previously established in our laboratory with the aim of identifying potential therapeutic indications for MFS [ 15 – 20 ]. 2. Materials and methods 2.1 Patient data Case 1 The donor patient was a 74-year-old female with myxofibrosarcoma. She visited a previous hospital because of a soft mass on her left back. Magnetic resonance imaging (MRI) revealed a soft tissue mass 9 × 8 cm in size bordering the left thoracic back region (Fig. 1 A-C). The patient was referred to the National Cancer Center Hospital, Tokyo, Japan, for further treatment. The tumor was diagnosed as myxofibrosarcoma based on a needle biopsy. The patient underwent wide resection of the left thoracic back tumor, and tumor tissues obtained at the time of surgery were used to establish the cell line. Histologically, the tumor consisted of pleomorphic spindled cells with bizarre hyperchromatic nuclei in prominent myxoid stroma. High mitotic activity and abnormal mitosis were observed (Fig. 1 D). Case 2 The donor patient was a 79-year-old female with a recurrent myxofibrosarcoma. She had undergone a wide resection of a right knee tumor four years ago at the National Cancer Center Hospital in Tokyo, Japan. The tumor was diagnosed as a myxofibrosarcoma. Four years after surgery, she developed a soft mass in her right leg. MRI revealed an enhanced mass in the right leg (Fig. 2 A-C). The tumor was diagnosed as a recurrent myxofibrosarcoma based on a needle biopsy. The patient underwent wide resection of the right leg tumor, and tumor tissues obtained at the time of surgery was used to establish the cell line. Histologically, the tumor comprised of spindle-shaped cells with mild nuclear atypia in the myxoid stroma (Fig. 2 D). 2.2 Histological analysis Histology examination was performed of 4 µm-thick sections from a representative paraffinized tumor sample. The sections were deparaffinized and stained with hematoxylin and eosin (H&E). 2.3 Establishment of cell lines Surgically excised MFS tumor tissues were sectioned into smaller fragments. These samples were then processed for digestion and homogenization in the gentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany) utilizing the Tumor Cell Dissociation Kit human (Miltenyi Biotec) within the gentleMACS™ C Tube (Miltenyi Biotec) for 30 minutes at a temperature of 37°C. Subsequently, the cells were cultivated on 60 mm collagen-type I-coated dishes (Sumitomo Bakelite Co. Ltd., Tokyo, Japan) in a DMEM/F-12 mixture (Thermo Fisher Scientific Inc., MA, USA) supplemented with GlutaMAX (Thermo Fisher Scientific Inc.), 5% inactivated fetal bovine serum, and supplements including Y-27632 (ROCK inhibitor; Selleck Chemicals, Houston, TX, USA), basic fibroblast growth factor, epidermal growth factor, insulin, hydrocortisone, penicillin, and streptomycin. This culture environment was kept at 37°C with a 5% CO2 atmosphere, with periodic media changes and cell monitoring using microscopy. When the cells reached an appropriate density, they were washed, detached with Trypsin–EDTA (Nacalai Tesque Inc., Kyoto, Japan), and relocated to continue cultivation under the same optimal conditions. 2.4 Authentication of the cell lines DNA was isolated from the tumor tissues and corresponding cell cultures for authentication and quality assessment. The extraction was facilitated using the Wizard® Genomic DNA Purification Kit (Promega Co., Madison, WI, USA) and the Qiagen DNeasy Blood and Tissue Kit (QIAGEN N.V., Hilden, Germany), while the DNA quantity was evaluated with a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific Inc.). The authenticity of the cell lines was verified through an analysis of short tandem repeats (STR) across 10 specific loci, employing the GenePrint 10 system (Promega Co.) in conjunction with the 3500xL Genetic Analyzer (Thermo Fisher Scientific Inc.). 2.5 Quality control of the cell lines The presence of Mycoplasma was investigated by isolating and fragmenting DNA from cells, using an e-Myco Mycoplasma PCR Detection Kit (Intron Biotechnology, Gyeonggi-do, Korea). Following polymerase chain reaction amplification, the DNA fragments were separated via 1.5% agarose gel electrophoresis and visualized by staining with SYBR Safe (Invitrogen). Images were assessed using an Amersham Imager 600 (GE Healthcare Biosciences, Little Chalfont, UK). 2.6 Genetic analysis Single nucleotide polymorphism (SNP) array genotyping was performed using the Infinium OmniExpressExome-8 version 1.4 BeadChip (Illumina, San Diego, CA, USA), adhering to both the guidelines provided by the manufacturer and the methodologies outlined in previously published studies. Genomic DNA from both established cell lines and primary tumors was amplified before being subjected to hybridization on array slides using the iScan system (Illumina). The assessment of Log R ratios and B allele frequencies was carried out using Genome Studio 2011.1 and cnvPartition v3.2.0 software from Illumina, in conjunction the R software (version 4.0.3, provided by the R Foundation for Statistical Computing, http://www.R-project.org ) and the DNAcopy package (version 1.64.0, from Bioconductor, http://bioconductor.org ). Regions within chromosomes displaying copy numbers exceeding three or falling below one were identified as amplifications or deletions, respectively. Genes revealing copy number alterations (CNAs) were annotated using the biomaRt package (version 2.46.0, Bioconductor) and cross-referenced with data from the “Cancer Gene Census” in the Catalog of Somatic Mutations In Cancer database (GRCh 37 version 91). 2.7 Cell proliferation assay Cells were initially plated at a density of 2.5 × 10 4 cells/well in 24-well plates and monitored over 4 days. Cell proliferation was quantified at 24-hour intervals using a Cell Counting Kit-8 (DOJINDO LABORATORIES, Kumamoto, Japan). Cells' doubling time was calculated based on the growth curves. Each procedures was conducted in triplicate. 2.8 Spheroid formation assay The methodology for assessing spheroid formation aligned with that used inn previous studies. Initially, cells were plated at a density of 1 × 10 5 cells/well in 96-well clear round bottom ultra-low attachment microplates (Corning Inc., Corning, NY, USA), and their ability to form spheroids was verified through microscopy (KEYENCE Co., Osaka, Japan). Afrer a cultivation period of three days, the spheroids were extracted, encapsulated in iPGell (GenoStaff Co. Ltd., Tokyo, Japan), and fixed in a 10% neutral buffered formalin solution. For microscopic analysis, the encapsulated spheroids were embedded in paraffin, sectioned into slices 4 µm thick, stained with H&E, and examined under a microscope. 2.9 Invasion assay The invasive potential of the established MFS cell lines was investigated using the real-time cell analyzer xCELLigence (Agilent Technologies Inc., Sannta Clara, CA, USA). The upper chamber was prepared with a Matrigel coating (Corning Inc.) and seeded with 4.0 × 10 4 cells in a basal DMEM/F12 solution while the lower chamber was filled with a nutritive mix of DMEM/F12 with supplements. Electronic sensors, positioned underneath the membrane between the chambers, captured the impedance of migrating cells every 15 min and translated it into data over 72 h to quantify cell invasion. This method utilized the MG63 osteosarcoma cell line (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan) as a control. 2.10 Drug screening assay The antitumor potential of 214 agents, includinng drugs approved by the Food and Drug Administration, was evaluated (Supplementary Table 2). Cells were seeded in 384-well plates at a density of 5.0 × 10 3 cells per well and incubated for 1 day before treatment. Following this period, each drug was applied to duplicate wells and incubated for an additional 3 days. Cell growth and viability were determined using the Cell Counting Kit-8 (DOUJINDO LABORATORIES) by comparing the outcomes with those of a dimethyl sulfoxide control. These results integrated with the findings of previous studies on NCC-MFS1-C1, NCC-MFS2-C1, NCC-MFS3-C1, NCC-MFS4-C1, NCC-MFS5-C1, and NCC-MFS6-C1. The analysis involved quantile normalization and unsupervised hierarchical clustering using R (version 4.0.3, limma package version 3.46.0, Bioconductor) and the gplots package (version 3.1.0, CRAN, https://cran.r-project.org ). To ascertain the half-maximal inhibitory concentration (IC 50 ) values, 24 drugs showing promising results in preliminary screens were tested across a range of ten concentrations. IC 50 values were calculated by analyzing these concentration-survival relationships via logistic regression, utilizing GraphPad Prism 9.11 (GraphPad Software, San Diego, CA, USA). 3. Results 3.1 Authentication and quality control of the established MFS cell lines NCC-MFS7-C1 and NCC-MFS8-C1 cell lines originating from patients 1 and 2, respectively, were established and sustained for more than 20 passages over 3 months. Authentication of these cell lines was conducted by analyzing 10 microsatellite loci, which showed that the STR profiles were almost identical to those of primary tumor tissues (Table 1 and Supplementary Fig. 1). Additionally, no detection of Mycoplasma-specific DNA was detected, confirming the absence of mycoplasma contamination. Table 1 Results of short tandem repeat analysis of NCC-MFS7-C1, NCC-MFS8-C1, and each original tumor tissue Microsatellite NCC-MFS7-C1 NCC-MFS8-C1 (Chromosome) Cell line Tumor Tissue (Case 1 ) Cell line Tumor Tissue (Case 2 ) Amelogenin (X Y) X X X X TH01 (3) 6, 9.3 6, 9.3 8, 9 8, 9 D21S11 (21) 30 30 29, 30 29, 30 D5S818 (5) 9, 11 9, 11 10, 12 10, 12 D13S317 (13) 11, 12 11, 12 11 11, 12 D7S820 (7) 10, 12 10, 12 11 11 D16S539 (16) 12 9, 12 11 10, 11 CSF1PO (5) 10, 12 10, 12 11, 12 11, 12 vWA (12) 16 16 17, 18 17, 18 TPOX (2) 8 8 9, 11 9, 11 3.2 Characterization of the established MFS cell lines Analysis of SNP arrays for the established cell lines, NCC-MFS7-C1 and NCC-MFS8-C1, revealed specific chromosomal CNAs. In NCC-MFS7-C1, deletions were noted on chromosomes 5q, 6p, 16q, and 17p13.1, whereas amplifications were observed on chromosomes 7p, 7q, 17p11.2, 18q, and 19q. Conversely, NCC-MFS8-C1 exhibited deletions on chromosomes 1p, 3p, 6p, 9p, and 13q with no observed amplifications. Notably, deletions affected tumor suppressor genes such as TP53 on 17p13.1 in NCC-MFS7-C1 and both CDKN2A and CDKN2B on 9p21.3 in NCC-MFS8-C1 cells (Fig. 3 , Table 2, Supplementary Tables 3 and 4). The NCC-MFS7-C1 and NCC-MFS8-C1 cell lines showed an elongated spindle or polygonal appearance (Fig. 4 A, B) and formed spheroids when cultured on low-attachment plates (Fig. 4 C, D). The spheroids contained oval and pleomorphic cells with atypical nuclei. Growth curve analysis showed that the population doubling times for NCC-MFS7-C1 and NCC-MFS8-C1 cells were 29 h and 68 h, respectively (Fig. 4 E). Furthermore, both cell lines showed higher invasive capacity than did the MG63 osteosarcoma cell line (Fig. 4 F). 3.3 Sensitivity of the antitumor agents to the established MFS cell lines Extensive analysis was conducted to evaluate the effect of the 214 antitumor compounds on the proliferation of NCC-MFS7-C1 and NCC-MFS8-C1 cells (Supplementary Table 5). This included a comparison with data from six previously established MFS cell lines in our laboratory. Antitumor agents were categorized into effective (Cluster A) and ineffective (Cluster B) groups based on their efficacy (Fig. 5 A and Supplementary Table 6). Cluster A had a higher proportion of cytotoxic drugs than did cluster B, including among all topoisomerase inhibitors were included in cluster A (Fig. 5 B, C). The distribution of tyrosine kinase inhibitors across both clusters was similar; however, most anaplastic lymphoma kinase inhibitors were grouped into cluster A (Fig. 5 D). Molecular targeted drugs were predominantly distributed in cluster A, with all histone deacetylase (HDAC) inhibitors belonging to cluster A (Fig. 5 E). Further investigation into the screened antitumor agents involved calculating IC 50 values for each, along with values for six other MFS cell lines previously identified (Supplementary Table 7). Notably, actinomycin D (an RNA polymerase and topoisomerase inhibitor) and bortezomib (a proteasome inhibitor) showed lower IC 50 values than doxorubicin, the standard treatment agent for sarcomas, in the most cell lines (7/8). Moreover, romidepsin (an HDAC inhibitor) showed significantly lower IC 50 values than did doxorubicin in all cell lines (8/8), indicating its potent antitumor activity. Table 3 and Fig. 6 provide a comprehensive view of these IC 50 values and corresponding growth curves. 4. Discussion The establishment of the NCC-MFS7-C1 and NCC-MFS8-C1 cell lines represents a significant advancement in MFS research. Given the highly malignant nature of MFS and the challenges in conducting clinical trials owing to its rarity, the preclinical study is crucial. However, the scarcity of available cell lines poses major challenges. These cell lines offer a valuable resource for deepening our understanding of MFS and exploring novel therapeutic strategies. Genetic analysis of the newly established cell lines revealed significant alterations in key tumor suppressor genes, including TP53, CDKN2A, and CDKN2B. These findings align with those of previous studies highlighting the prevalence of such mutations in MFS, underscoring the relevance of these cell lines as models with representative genetic mutations in the disease [ 21 – 23 ]. However, the absence of CNA in genes such as RB1, ATRX, and HDLBP, which has been reported in other MFS studies, points to the genetic diversity inherent in MFS, further emphasizing the need for a broad range of patient-derived cancer models [ 9 , 24 ]. Functionally, NCC-MFS7-C1 and NCC-MFS8-C1 cell lines exhibited characteristics typical of MFS, including the ability to consistently proliferate and invade. NCC-MFS7-C1, in particular, demonstrated higher proliferative and invasive capacities than did NCC-MFS8-C1. With an average doubling time of 66.1 hours across previously established cell lines in our laboratory, NCC-MFS7-C1 emerged as a relatively fast-growing model, whereas NCC-MFS8-C1 presented a standard growth profile. Both cell lines showed spheroid formation capabilities, positioning them as promising tools for more advanced preclinical experiments. A comprehensive evaluation of 214 antitumor agents against these cell lines revealed promising results. In particular, actinomycin D, bortezomib, and romidepsin showed superior efficacy than did doxorubicin, the standard drug frequently used to treat MFS and other sarcomas. Although actinomycin D is sometimes utilized in VAC (vincristine, actinomycin D, cyclophosphamide) regimens for sarcoma treatment, bortezomib and romidepsin are rarely employed in managing sarcomas [ 25 , 26 ]. Initially, bortezomib was approved for multiple myeloma, and romidepsin for cutaneous T-cell lymphoma [ 27 – 30 ]. Despite a few reports suggesting the potential efficacy of bortezomib and HDAC inhibitors, OBP-801 (not romidepsin), in MFS models, these investigations are preclinical studies, confined to in vitro or in vivo data [ 31 , 32 ]. Therefore, these agents are promising candidates for MFS treatment that deserve further validation in large-scale drug screening of cell lines and clinical trials. Our study has three limitations. Although this study is the largest drug screening analysis conducted for MFS, the number of cell lines and drugs used in our study was insufficient, considering those for common cancers [ 11 , 33 ]. This limitation will be addressed by establishing a more diverse collection of MFS cell lines and conducting larger-scale drug sensitivity assays. Furthermore, although the treatment for metastatic tumors is challenging in MFS, our study did not include cell lines from metastatic lesions. We will require the cell lines from the metastatic sites to develop the novel therapy for the patients with metastasis. In addition, the metastatic potentials of our cell lines were not examined in this study. However, the conventional experiments using animal models are quite artificial as the cells or tumors are inoculated ectopically in the animal body, and subcutaneously transplanted tumors rarely metastasize in the mouse, even when derived from a highly metastatic patient tumor [ 34 ]. To address this issue, we require a novel cancer model or a protocol to recapitulate the metastasis in patients with MFS. In conclusion, this study introduced two novel fibrosarcoma (MFS) cell lines, NCC-MFS7-C1 and NCC-MFS8-C1. These cell lines are crucial for MFS research, as they will improve our understanding of the biological and genetic mechanisms of the disease and identify potential therapeutics. Sensitivity of various antitumor agents suggested that actinomycin D, bortezomib, and romidepsin are effective in treating MFS. These findings clearly indicates that the established cell lines will serve as valuable research models for future large-scale drug screening for MFS. Declarations Funding This study was supported by the Japan Agency for Medical Research and Development (grant number: 20ck0106537h). Conflicts of Interest The authors have no relevant financial or non-financial interests to disclose. Ethics approval The Ethics Committee of the National Cancer Center approved the use of clinical materials for this study (approval number: 2004-050). Informed consent Written informed consent was provided by the patients. Acknowledgments We thank Drs. S. Fukushima, S. Osaki, K. Ogura (Department of Musculoskeletal Oncology and Rehabilitation Medicine, National Cancer Center Hospital) ; Dr. T. Funada, and Dr. Y. Kobayashi (Department of Diagnostic Pathology, National Cancer Center Hospital); and the National Cancer Center Hospital for sampling the tumor tissue specimens from the surgically resected materials. We appreciate the technical assistance provided by Mr. Y. Ohno (Division of Rare Cancer Research, National Cancer Center Research Institute) and Mrs. Y. Shiotani (Central Animal Division, National Cancer Center Research Institute). We would like to thank Editage (www.editage.jp) for providing English language editing services and for their constructive comments on this manuscript. The Fundamental Innovative Oncology Core of the National Cancer Center provided technical assistance for this study. References board WHOcte. Soft tissue and bone tumours. 5th ed. World Health Organization classification of tumours. World Health Organization International Agency for Research on Cancer; 2020. Sanfilippo R, Miceli R, Grosso F, Fiore M, Puma E, Pennacchioli E et al. Myxofibrosarcoma: prognostic factors and survival in a series of patients treated at a single institution. Ann Surg Oncol. 2011;18(3):720-5. doi:10.1245/s10434-010-1341-4. Wakely PE, Jr. Cytopathology of myxofibrosarcoma: a study of 66 cases and literature review. J Am Soc Cytopathol. 2021;10(3):300-9. doi:10.1016/j.jasc.2020.09.004. Dewan V, Darbyshire A, Sumathi V, Jeys L, Grimer R. Prognostic and survival factors in myxofibrosarcomas. Sarcoma. 2012;2012:830879. doi:10.1155/2012/830879. Willems SM, Debiec-Rychter M, Szuhai K, Hogendoorn PC, Sciot R. Local recurrence of myxofibrosarcoma is associated with increase in tumour grade and cytogenetic aberrations, suggesting a multistep tumour progression model. Mod Pathol. 2006;19(3):407-16. doi:10.1038/modpathol.3800550. Ghazala CG, Agni NR, Ragbir M, Dildey P, Lee D, Rankin KS et al. Myxofibrosarcoma of the extremity and trunk: a multidisciplinary approach leads to good local rates of LOCAL control. Bone Joint J. 2016;98-b(12):1682-8. doi:10.1302/0301-620x.98b12.37568. van der Horst CAJ, Bongers SLM, Versleijen-Jonkers YMH, Ho VKY, Braam PM, Flucke UE et al. Overall Survival of Patients with Myxofibrosarcomas: An Epidemiological Study. Cancers. 2022;14(5):1102. Teurneau H, Engellau J, Ghanei I, Vult von Steyern F, Styring E. High Recurrence Rate of Myxofibrosarcoma: The Effect of Radiotherapy Is Not Clear. Sarcoma. 2019;2019:8517371. doi:10.1155/2019/8517371. Takeuchi Y, Yoshida K, Halik A, Kunitz A, Suzuki H, Kakiuchi N et al. The landscape of genetic aberrations in myxofibrosarcoma. Int J Cancer. 2022;151(4):565-77. doi:10.1002/ijc.34051. Heitzer E, Sunitsch S, Gilg MM, Lohberger B, Rinner B, Kashofer K et al. Expanded molecular profiling of myxofibrosarcoma reveals potentially actionable targets. Mod Pathol. 2017;30(12):1698-709. doi:10.1038/modpathol.2017.94. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012;483(7391):603-7. doi:10.1038/nature11003. Yang W, Soares J, Greninger P, Edelman EJ, Lightfoot H, Forbes S et al. Genomics of Drug Sensitivity in Cancer (GDSC): a resource for therapeutic biomarker discovery in cancer cells. Nucleic Acids Res. 2013;41(Database issue):D955-61. doi:10.1093/nar/gks1111. Kondo T. Current status and future outlook for patient-derived cancer models from a rare cancer research perspective. Cancer Sci. 2021;112(3):953-61. doi:10.1111/cas.14669. Bairoch A. The Cellosaurus, a Cell-Line Knowledge Resource. J Biomol Tech. 2018;29(2):25-38. doi:10.7171/jbt.18-2902-002. Kito F, Oyama R, Sakumoto M, Shiozawa K, Qiao Z, Toki S et al. Establishment and characterization of a novel cell line, NCC-MFS1-C1, derived from a patient with myxofibrosarcoma. Hum Cell. 2019;32(2):214-22. doi:10.1007/s13577-018-00233-1. Noguchi R, Yoshimatsu Y, Ono T, Sei A, Hirabayashi K, Ozawa I et al. Establishment and characterization of NCC-MFS2-C1: a novel patient-derived cancer cell line of myxofibrosarcoma. Hum Cell. 2021;34(1):246-53. doi:10.1007/s13577-020-00420-z. Tsuchiya R, Yoshimatsu Y, Noguchi R, Sin Y, Ono T, Sei A et al. Establishment and characterization of NCC-MFS3-C1: a novel patient-derived cell line of myxofibrosarcoma. Hum Cell. 2021;34(4):1266-73. doi:10.1007/s13577-021-00548-6. Yoshimatsu Y, Noguchi R, Tsuchiya R, Sin Y, Ono T, Sugaya J et al. Establishment and characterization of NCC-MFS4-C1: a novel patient-derived cell line of myxofibrosarcoma. Hum Cell. 2021;34(6):1911-8. doi:10.1007/s13577-021-00589-x. Tsuchiya R, Yoshimatsu Y, Noguchi R, Sin Y, Ono T, Akiyama T et al. Establishment and Characterization of NCC-MFS5-C1: A Novel Patient-Derived Cell Line of Myxofibrosarcoma. Cells. 2022;11(2). doi:10.3390/cells11020207. Yoshimatsu Y, Noguchi R, Sin Y, Tsuchiya R, Ono T, Akiyama T et al. Establishment and characterization of NCC-MFS6-C1: a novel patient-derived cell line of myxofibrosarcoma. Hum Cell. 2022;35(6):1993-2001. doi:10.1007/s13577-022-00749-7. Ogura K, Hosoda F, Arai Y, Nakamura H, Hama N, Totoki Y et al. Integrated genetic and epigenetic analysis of myxofibrosarcoma. Nat Commun. 2018;9(1):2765. doi:10.1038/s41467-018-03891-9. Yamashita A, Suehara Y, Hayashi T, Takagi T, Kubota D, Sasa K et al. Molecular and clinicopathological analysis revealed an immuno-checkpoint inhibitor as a potential therapeutic target in a subset of high-grade myxofibrosarcoma. Virchows Arch. 2022;481(4):1-17. doi:10.1007/s00428-022-03358-9. Sambri A, De Paolis M, Spinnato P, Donati DM, Bianchi G. The Biology of Myxofibrosarcoma: State of the Art and Future Perspectives. Oncol Res Treat. 2020;43(6):314-22. doi:10.1159/000507334. Li GZ, Okada T, Kim YM, Agaram NP, Sanchez-Vega F, Shen Y et al. Rb and p53-Deficient Myxofibrosarcoma and Undifferentiated Pleomorphic Sarcoma Require Skp2 for Survival. Cancer Res. 2020;80(12):2461-71. doi:10.1158/0008-5472.Can-19-1269. Nakano K, Ae K, Matsumoto S, Takahashi S. The VAC regimen for adult rhabdomyosarcoma: Differences between adolescent/young adult and older patients. Asia Pac J Clin Oncol. 2020;16(2):e47-e52. doi:10.1111/ajco.13279. Özkan A, Bayram İ, Sezgin G, Mirioğlu A, Küpeli S. Efficacy of replacing actinomycin-D with carboplatin in Ewing sarcoma consolidation treatment: Single-center experience. J Bone Oncol. 2022;35:100435. doi:10.1016/j.jbo.2022.100435. Sharma A, Preuss CV. Bortezomib. StatPearls. Treasure Island (FL): StatPearls Publishing Copyright © 2024, StatPearls Publishing LLC.; 2024. Cvek B. Proteasome inhibitors. Prog Mol Biol Transl Sci. 2012;109:161-226. doi:10.1016/b978-0-12-397863-9.00005-5. Grant C, Rahman F, Piekarz R, Peer C, Frye R, Robey RW et al. Romidepsin: a new therapy for cutaneous T-cell lymphoma and a potential therapy for solid tumors. Expert Rev Anticancer Ther. 2010;10(7):997-1008. doi:10.1586/era.10.88. Shimony S, Horowitz N, Ribakovsky E, Rozovski U, Avigdor A, Zloto K et al. Romidepsin treatment for relapsed or refractory peripheral and cutaneous T-cell lymphoma: Real-life data from a national multicenter observational study. Hematol Oncol. 2019;37(5):569-77. doi:10.1002/hon.2691. Li CF, Wang JM, Kang HY, Huang CK, Wang JW, Fang FM et al. Characterization of gene amplification-driven SKP2 overexpression in myxofibrosarcoma: potential implications in tumor progression and therapeutics. Clin Cancer Res. 2012;18(6):1598-610. doi:10.1158/1078-0432.Ccr-11-3077. Kawarazaki A, Horinaka M, Yasuda S, Kawashima H, Numajiri T, Sakai T. The HDAC inhibitor OBP-801 suppresses the growth of myxofibrosarcoma cells. J buon. 2020;25(1):464-71. Bashi AC, Coker EA, Bulusu KC, Jaaks P, Crafter C, Lightfoot H et al. Large-scale Pan-cancer Cell Line Screening Identifies Actionable and Effective Drug Combinations. Cancer Discov. 2024:Of1-of20. doi:10.1158/2159-8290.Cd-23-0388. Fidler IJ. Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award lecture. Cancer Res. 1990;50(19):6130-8. Table 2 and 3 Table 2 and 3 are available in the Supplementary Files section. Supplementary Files SupplementaryFig.1STRofcelllinesandoriginaltumors.tif SupplementaryTable1ListofMFScelllines.xlsx SupplementaryTable2Listofthe214anticancerdrugs.xlsx SupplementaryTable3ListofCNAsofNCCMFS7C1.xlsx SupplementaryTable4ListofCNAsofNCCMFS8C1.xlsx SupplementaryTable5Cellviabilitywith214drugs.xlsx SupplementaryTable6Drugtypeandclassification.xlsx SupplementaryTable7IC50valuesinMFScelllines.xlsx Table2RepresentativeCNAsofMFScelllines.xlsx Table3SummaryofIC50valuesinMFScelllines.xlsx Cite Share Download PDF Status: Published Journal Publication published 31 Aug, 2024 Read the published version in Human Cell → Version 1 posted Editorial decision: Major Revisions Needed 03 May, 2024 Reviewers agreed at journal 15 Apr, 2024 Reviewers invited by journal 14 Apr, 2024 Editor assigned by journal 14 Apr, 2024 First submitted to journal 11 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4251932","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":291129052,"identity":"e6caf065-dce6-4c7b-be19-1b9440da802f","order_by":0,"name":"Yuki Adachi","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Adachi","suffix":""},{"id":291129053,"identity":"d0c1d943-a3dd-4a03-9f93-ff7001ab42ff","order_by":1,"name":"Rei Noguchi","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Rei","middleName":"","lastName":"Noguchi","suffix":""},{"id":291129054,"identity":"ea685ed3-5938-4bc0-bc25-ebd50f0cd853","order_by":2,"name":"Julia Osaki","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Osaki","suffix":""},{"id":291129055,"identity":"c19fa4c0-6686-44ea-a406-00898541a7ec","order_by":3,"name":"Takuya Ono","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Takuya","middleName":"","lastName":"Ono","suffix":""},{"id":291129056,"identity":"6891eca2-f06f-4409-a507-e74fb46444e2","order_by":4,"name":"Shuhei Iwata","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Shuhei","middleName":"","lastName":"Iwata","suffix":""},{"id":291129057,"identity":"cda7d71c-fb5d-4a3b-9ae9-4ae5d4e4b584","order_by":5,"name":"Taro Akiyama","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Taro","middleName":"","lastName":"Akiyama","suffix":""},{"id":291129058,"identity":"f9e1ebc5-2f7b-44f3-9b31-96110d762d75","order_by":6,"name":"Ryuto Tsuchiya","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Ryuto","middleName":"","lastName":"Tsuchiya","suffix":""},{"id":291129059,"identity":"986f11c5-a375-458a-92bb-4ff56d9e7a0c","order_by":7,"name":"Yu Toda","email":"","orcid":"","institution":"National Cancer Center Hospital: Kokuritsu Gan Kenkyu Center Chuo Byoin","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Toda","suffix":""},{"id":291129060,"identity":"f1af6c81-f6ba-45fe-962f-15a46f4b8d63","order_by":8,"name":"Tetsuya Sekita","email":"","orcid":"","institution":"National Cancer Center Hospital: Kokuritsu Gan Kenkyu Center Chuo Byoin","correspondingAuthor":false,"prefix":"","firstName":"Tetsuya","middleName":"","lastName":"Sekita","suffix":""},{"id":291129061,"identity":"671a38b6-7445-4f25-b946-90573d381aed","order_by":9,"name":"Shintaro Iwata","email":"","orcid":"","institution":"National Cancer Center Hospital: Kokuritsu Gan Kenkyu Center Chuo Byoin","correspondingAuthor":false,"prefix":"","firstName":"Shintaro","middleName":"","lastName":"Iwata","suffix":""},{"id":291129062,"identity":"d05a4dbe-b91d-4ea1-a6d2-1b5333939f93","order_by":10,"name":"Eisuke Kobayashi","email":"","orcid":"","institution":"National Cancer Center Hospital: Kokuritsu Gan Kenkyu Center Chuo Byoin","correspondingAuthor":false,"prefix":"","firstName":"Eisuke","middleName":"","lastName":"Kobayashi","suffix":""},{"id":291129063,"identity":"f4439804-b606-4561-9231-6a8df8d3e8a9","order_by":11,"name":"Naoki Kojima","email":"","orcid":"","institution":"National Cancer Center Hospital: Kokuritsu Gan Kenkyu Center Chuo Byoin","correspondingAuthor":false,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Kojima","suffix":""},{"id":291129064,"identity":"4c5fc73c-c978-4d7a-ac35-3417ee617a74","order_by":12,"name":"Akihiko Yoshida","email":"","orcid":"","institution":"National Cancer Center Hospital: Kokuritsu Gan Kenkyu Center Chuo Byoin","correspondingAuthor":false,"prefix":"","firstName":"Akihiko","middleName":"","lastName":"Yoshida","suffix":""},{"id":291129065,"identity":"f30bd783-ede5-486d-bff2-4b682981da8e","order_by":13,"name":"Hideki Yokoo","email":"","orcid":"","institution":"Asahikawa Medical University: Asahikawa Ika Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Hideki","middleName":"","lastName":"Yokoo","suffix":""},{"id":291129066,"identity":"0294a07a-e358-4ad7-a4d4-4ef45a3074f2","order_by":14,"name":"Akira Kawai","email":"","orcid":"","institution":"National Cancer Center Hospital: Kokuritsu Gan Kenkyu Center Chuo Byoin","correspondingAuthor":false,"prefix":"","firstName":"Akira","middleName":"","lastName":"Kawai","suffix":""},{"id":291129067,"identity":"4957c776-8b29-437c-aa95-e7e018affdbb","order_by":15,"name":"Tadashi Kondo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYFACNjCWg3GZCWrggWoxJl1LYgPRzrJnP5b4uKDMJn07++EHjF8qGNjNCdrCk3bYeMa5tNydPWkGzDJnGJgtCdnHw5DeJs3bdjh3ww0eBmbJNgZmgwOEtPA/B2tJNyBei0TaMZCWBJAWxo9EabnxLNmY51ya4YYzaQaHGc5IEPYLe3+a4WOeMht5g+OHHz78UWGTTDDEUMBhHgaJZAOStDD+YGCwI03LKBgFo2AUjAQAAC9ZNwgyuRd+AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-6405-7792","institution":"National Cancer Center Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Tadashi","middleName":"","lastName":"Kondo","suffix":""}],"badges":[],"createdAt":"2024-04-11 10:51:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4251932/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4251932/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13577-024-01124-4","type":"published","date":"2024-08-31T15:58:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55010087,"identity":"72d8af5a-f5e3-4fa9-87f8-2082a81dfd1b","added_by":"auto","created_at":"2024-04-19 19:17:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":816136,"visible":true,"origin":"","legend":"\u003cp\u003eClinical and pathological data of NCC-MFS7-C1\u003c/p\u003e\n\u003cp\u003eMagnetic resonance imaging demonstrated an enhanced mass of the left thoracic back (A: T2-weighted axial view, B: gadolinium-enhanced T1-weighted axial view, C: gadolinium-enhanced T1-weighted sagittal view). The histological image of the tumor showed morphology of myxofibrosarcoma (D: H\u0026amp;E).\u003c/p\u003e","description":"","filename":"Fig.1ClinicalandpathologicaldataofNCCMFS7C1.png","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/9054d7863e0d8670de9a6eb6.png"},{"id":55010086,"identity":"d340355d-6f6e-43c4-b69b-ce3d692a24fd","added_by":"auto","created_at":"2024-04-19 19:17:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":707932,"visible":true,"origin":"","legend":"\u003cp\u003eClinical and pathological data of NCC-MFS8-C1\u003c/p\u003e\n\u003cp\u003eMagnetic resonance imaging demonstrated an enhanced mass of the right leg (A: T2-weighted axial view, B: gadolinium-enhanced T1-weighted axial view, C: gadolinium-enhanced T1-weighted sagittal view).\u003c/p\u003e\n\u003cp\u003eThe histological image of the tumor showed morphology of myxofibrosarcoma (D: H\u0026amp;E).\u003c/p\u003e","description":"","filename":"Fig.2ClinicalandpathologicaldataofNCCMFS8C1.png","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/b14247ca8571c78150055d49.png"},{"id":55010495,"identity":"d35e3e97-8fa0-4d5d-8d12-8fc48844b94a","added_by":"auto","created_at":"2024-04-19 19:25:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":210969,"visible":true,"origin":"","legend":"\u003cp\u003eSingle-nucleotide polymorphism array analysis.\u003c/p\u003e\n\u003cp\u003eFocal copy number alterations were identified in the NCC-MFS7-C1 and NCC-MFS8-C1 cells. X- and Y-axes indicate the chromosomal location and log ratio of copy number alterations, respectively.\u003c/p\u003e","description":"","filename":"Fig.3Singlenucleotidepolymorphismarrayanalysis.png","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/1bfefe3c70742ee68cbbd011.png"},{"id":55010092,"identity":"c1db6c66-87c8-4629-ad1c-161a483683c6","added_by":"auto","created_at":"2024-04-19 19:17:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1347350,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of NCC-MFS7-C1 and NCC-MFS8-C1\u003c/p\u003e\n\u003cp\u003eMicroscopic appearance of (A) NCC-MFS7-C1 and (B) NCC-MFS8-C1 cells in 2-dimensional culture conditions. Hematoxylin and eosin–stained spheroid sections of (C) NCC-MFS7-C1 and (D) NCC-MFS8-C1 cells were fabricated in 96-well low–attachment round– bottom plates. Growth curves of NCC-MFS7-C1 and NCC-MFS8-C1 cells (E). Each point represents the mean ± standard deviation (n = 3). The invasion capabilitiees of NCC-MFS7-C1 and NCC-MFS8-C1 cells were observed using a Real-Time Cell Analyzer (F). The MG63 osteosarcoma cell line was used as the control.\u003c/p\u003e","description":"","filename":"Fig.4CharacterizationofNCCMFS7C1andNCCMFS8C1.png","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/d5ab701d53418bb12929ee51.png"},{"id":55010497,"identity":"a646c75a-f167-4fcc-bc9e-7adc4e2548b2","added_by":"auto","created_at":"2024-04-19 19:25:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":177606,"visible":true,"origin":"","legend":"\u003cp\u003eDrug screening assay of myxofibrosarcoma cell lines\u003c/p\u003e\n\u003cp\u003e(A) The antitumor agents were categorized into two groups according to their antitumor effect: cluster A as the effective group and cluster B as the ineffective group. (B–E) The proportion of each agent type belonging to each cluster. The graphs were obtained after the normalization of the number of agents. Data regarding NCC-MFS1-C1, NCC-MFS2-C1, NCC-MFS3-C1, NCC-MFS4-C1, NCC-MFS5-C1, and NCC-MFS6-C1 were previously reported. MFS, myxofibrosarcoma.\u003c/p\u003e","description":"","filename":"Fig.5Drugscreeningassayofmyxofibrosarcomacelllines.png","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/214ecbb067f8a9fa74407ff2.png"},{"id":55010093,"identity":"6f3989e5-3d87-462d-8ad0-5ddc17e39827","added_by":"auto","created_at":"2024-04-19 19:17:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":585226,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of myxofibrosarcoma cell lines at different concentrations of antitumor agents\u003c/p\u003e\n\u003cp\u003eAntiproliferative effects of romidepsin (A), actinomycin D (B), bortezomib (C), and doxorubicin (D) on NCC-MFS1-C1, NCC-MFS2-C1, NCC-MFS3-C1, NCC-MFS4-C1, NCC-MFS5-C1, NCC-MFS6-C1, NCC-MFS7-C1, and NCC-MFS8-C1 cells are shown. Half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values are summarized in Table 3. MFS, myxofibrosarcoma.\u003c/p\u003e","description":"","filename":"Fig.6IC50valuesof8MFScelllines.png","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/a5107543bf736dc0059a2b8f.png"},{"id":63821139,"identity":"c9356bf7-82d9-4b95-807f-9bea45091fd8","added_by":"auto","created_at":"2024-09-02 16:12:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5213243,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/a0521dc5-4265-4e76-98ee-352225cd5322.pdf"},{"id":55010091,"identity":"ac8db441-5cb3-4c96-847e-9acb676e5033","added_by":"auto","created_at":"2024-04-19 19:17:03","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":252923,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.1STRofcelllinesandoriginaltumors.tif","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/321ffba4a7d5828712dfc244.tif"},{"id":55010496,"identity":"2420d234-3e18-4f66-91b4-7322b5b11f8a","added_by":"auto","created_at":"2024-04-19 19:25:03","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10756,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1ListofMFScelllines.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/87a404b69b5fbaa99f703c93.xlsx"},{"id":55010090,"identity":"3124aa01-fd42-4b8c-9499-17c86bc65f49","added_by":"auto","created_at":"2024-04-19 19:17:03","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":22937,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2Listofthe214anticancerdrugs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/b7c67e46dfd242d73d94e3a4.xlsx"},{"id":55010097,"identity":"934b4500-375b-4430-9db6-ad93416dbfec","added_by":"auto","created_at":"2024-04-19 19:17:04","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13511,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3ListofCNAsofNCCMFS7C1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/655dfaa69cfae8b97c4e0837.xlsx"},{"id":55010128,"identity":"56fd543f-e3dc-459f-9d13-38e1022d28f1","added_by":"auto","created_at":"2024-04-19 19:17:04","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":13647,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable4ListofCNAsofNCCMFS8C1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/047e4fb50c542652c295cc73.xlsx"},{"id":55010098,"identity":"e03fef77-d6b9-41f1-8c97-7a5ab999b37a","added_by":"auto","created_at":"2024-04-19 19:17:04","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":70938,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable5Cellviabilitywith214drugs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/2de158256f26130ee2851709.xlsx"},{"id":55010101,"identity":"da4fcb45-1f1d-47e1-bb6b-bb346ee81aa7","added_by":"auto","created_at":"2024-04-19 19:17:04","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":48158,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable6Drugtypeandclassification.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/e6bfb9d286e3455249dc9a77.xlsx"},{"id":55010115,"identity":"c908b3aa-66d2-4e7a-93ca-ae6b8f1922b3","added_by":"auto","created_at":"2024-04-19 19:17:04","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":13242,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable7IC50valuesinMFScelllines.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/3c8b515de02a35a9f9022e38.xlsx"},{"id":55010498,"identity":"40a04683-e316-4f11-97af-318afc46333a","added_by":"auto","created_at":"2024-04-19 19:25:04","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":11534,"visible":true,"origin":"","legend":"","description":"","filename":"Table2RepresentativeCNAsofMFScelllines.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/b54ba77bd79239a66db6eebf.xlsx"},{"id":55010099,"identity":"4f6d790d-6a11-4d9d-b749-c27b05d5c3ad","added_by":"auto","created_at":"2024-04-19 19:17:04","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":11031,"visible":true,"origin":"","legend":"","description":"","filename":"Table3SummaryofIC50valuesinMFScelllines.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4251932/v1/f9f62a6d6db68ecfdb09478a.xlsx"}],"financialInterests":"","formattedTitle":"Establishment and characterization of two novel patient-derived cell lines from myxofibrosarcoma: NCC-MFS7-C1 and NCC-MFS8-C1","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMyxofibrosarcoma (MFS), a notably aggressive soft tissue sarcoma, is a malignant fibroblastic neoplasm characterized by a variable myxoid stroma and pleomorphic cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Tumors are most commonly located in the limbs, with a particular propensity for the lower extremities [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. MFS predominantly affects older adults, with a peak incidence in the sixth to eighth decades of life [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Notably, surgical resection is the cornerstone of curative treatment for MFS [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, high rates of local recurrence (30\u0026ndash;40%) and distant metastasis (15\u0026ndash;35%) significantly affect the 5-year survival prospects, which hover around 60\u0026ndash;70% [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These data highlight the need for comprehensive multidisciplinary treatment approaches that extend beyond surgery and include innovative drug therapies. Despite the clear necessity of such strategies, effective multidisciplinary methods, including established drug regimens, remain elusive.\u003c/p\u003e \u003cp\u003eThe intricate genetic landscape of MFS underpins its aggressive clinical behavior and contributes to significant challenges in the development of effective therapeutic strategies. Key genetic alterations observed in MFS include mutations and copy number alterations in pivotal tumor suppressor genes and cell cycle regulators, notably TP53, RB1, CDKN2A, and CDKN2B [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Moreover, frequent amplifications on chromosome 5p, which harbors oncogenes such as TRIO and RICTOR, have been implicated in driving the malignant potential of MFS [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These genetic aberrations not only confer a high degree of biological aggressiveness but also foster a heterogeneous tumor environment, complicating personalized treatment approaches.\u003c/p\u003e \u003cp\u003eCell lines are indispensable in cancer research as they provide a foundational platform for elucidating disease mechanisms and evaluating therapeutic interventions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Their importance is amplified in the context of rare diseases such as MFS, where conducting extensive clinical trials is challenging owing to the limited patient population. This difficulty underscores the value of robust preclinical models, such as cell lines, for advancing our understanding and treatment of such diseases [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Despite the critical role of these models, the Cellosaurus database, a comprehensive resource for cell line information, lists only 16 MFS cell lines, with only two publicly available (Supplementary Table\u0026nbsp;1) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This scarcity of MFS cell lines severely restricts research progress and the development of targeted therapies. Therefore, additional cell lines are strongly required to promote further research and improve the therapeutic outcomes of MFS.\u003c/p\u003e \u003cp\u003eThis study introduced two novel MFS cell lines, NCC-MFS7-C1 and NCC-MFS8-C1, established from tumor samples resected from two different patients. We meticulously characterized the NCC-MFS7-C1 and NCC-MFS8-C1 cell lines and examined their genetic profiles, proliferation rates, spheroid formation capabilities, and invasive behaviors. Additionally, we expanded our investigation to include drug screening across these novel cell lines and six others previously established in our laboratory with the aim of identifying potential therapeutic indications for MFS [\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Patient data\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eCase 1\u003c/strong\u003e \u003cp\u003eThe donor patient was a 74-year-old female with myxofibrosarcoma. She visited a previous hospital because of a soft mass on her left back. Magnetic resonance imaging (MRI) revealed a soft tissue mass 9 \u0026times; 8 cm in size bordering the left thoracic back region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). The patient was referred to the National Cancer Center Hospital, Tokyo, Japan, for further treatment. The tumor was diagnosed as myxofibrosarcoma based on a needle biopsy. The patient underwent wide resection of the left thoracic back tumor, and tumor tissues obtained at the time of surgery were used to establish the cell line. Histologically, the tumor consisted of pleomorphic spindled cells with bizarre hyperchromatic nuclei in prominent myxoid stroma. High mitotic activity and abnormal mitosis were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCase 2\u003c/strong\u003e \u003cp\u003eThe donor patient was a 79-year-old female with a recurrent myxofibrosarcoma. She had undergone a wide resection of a right knee tumor four years ago at the National Cancer Center Hospital in Tokyo, Japan. The tumor was diagnosed as a myxofibrosarcoma. Four years after surgery, she developed a soft mass in her right leg. MRI revealed an enhanced mass in the right leg (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). The tumor was diagnosed as a recurrent myxofibrosarcoma based on a needle biopsy. The patient underwent wide resection of the right leg tumor, and tumor tissues obtained at the time of surgery was used to establish the cell line. Histologically, the tumor comprised of spindle-shaped cells with mild nuclear atypia in the myxoid stroma (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Histological analysis\u003c/h2\u003e \u003cp\u003eHistology examination was performed of 4 \u0026micro;m-thick sections from a representative paraffinized tumor sample. The sections were deparaffinized and stained with hematoxylin and eosin (H\u0026amp;E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Establishment of cell lines\u003c/h2\u003e \u003cp\u003eSurgically excised MFS tumor tissues were sectioned into smaller fragments. These samples were then processed for digestion and homogenization in the gentleMACS\u0026trade; Octo Dissociator with Heaters (Miltenyi Biotec B.V. \u0026amp; Co. KG, Bergisch Gladbach, Germany) utilizing the Tumor Cell Dissociation Kit human (Miltenyi Biotec) within the gentleMACS\u0026trade; C Tube (Miltenyi Biotec) for 30 minutes at a temperature of 37\u0026deg;C. Subsequently, the cells were cultivated on 60 mm collagen-type I-coated dishes (Sumitomo Bakelite Co. Ltd., Tokyo, Japan) in a DMEM/F-12 mixture (Thermo Fisher Scientific Inc., MA, USA) supplemented with GlutaMAX (Thermo Fisher Scientific Inc.), 5% inactivated fetal bovine serum, and supplements including Y-27632 (ROCK inhibitor; Selleck Chemicals, Houston, TX, USA), basic fibroblast growth factor, epidermal growth factor, insulin, hydrocortisone, penicillin, and streptomycin. This culture environment was kept at 37\u0026deg;C with a 5% CO2 atmosphere, with periodic media changes and cell monitoring using microscopy. When the cells reached an appropriate density, they were washed, detached with Trypsin\u0026ndash;EDTA (Nacalai Tesque Inc., Kyoto, Japan), and relocated to continue cultivation under the same optimal conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Authentication of the cell lines\u003c/h2\u003e \u003cp\u003eDNA was isolated from the tumor tissues and corresponding cell cultures for authentication and quality assessment. The extraction was facilitated using the Wizard\u0026reg; Genomic DNA Purification Kit (Promega Co., Madison, WI, USA) and the Qiagen DNeasy Blood and Tissue Kit (QIAGEN N.V., Hilden, Germany), while the DNA quantity was evaluated with a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific Inc.). The authenticity of the cell lines was verified through an analysis of short tandem repeats (STR) across 10 specific loci, employing the GenePrint 10 system (Promega Co.) in conjunction with the 3500xL Genetic Analyzer (Thermo Fisher Scientific Inc.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Quality control of the cell lines\u003c/h2\u003e \u003cp\u003eThe presence of Mycoplasma was investigated by isolating and fragmenting DNA from cells, using an e-Myco Mycoplasma PCR Detection Kit (Intron Biotechnology, Gyeonggi-do, Korea). Following polymerase chain reaction amplification, the DNA fragments were separated via 1.5% agarose gel electrophoresis and visualized by staining with SYBR Safe (Invitrogen). Images were assessed using an Amersham Imager 600 (GE Healthcare Biosciences, Little Chalfont, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Genetic analysis\u003c/h2\u003e \u003cp\u003eSingle nucleotide polymorphism (SNP) array genotyping was performed using the Infinium OmniExpressExome-8 version 1.4 BeadChip (Illumina, San Diego, CA, USA), adhering to both the guidelines provided by the manufacturer and the methodologies outlined in previously published studies. Genomic DNA from both established cell lines and primary tumors was amplified before being subjected to hybridization on array slides using the iScan system (Illumina). The assessment of Log R ratios and B allele frequencies was carried out using Genome Studio 2011.1 and cnvPartition v3.2.0 software from Illumina, in conjunction the R software (version 4.0.3, provided by the R Foundation for Statistical Computing, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.R-project.org\u003c/span\u003e\u003cspan address=\"http://www.R-project.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the DNAcopy package (version 1.64.0, from Bioconductor, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioconductor.org\u003c/span\u003e\u003cspan address=\"http://bioconductor.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Regions within chromosomes displaying copy numbers exceeding three or falling below one were identified as amplifications or deletions, respectively. Genes revealing copy number alterations (CNAs) were annotated using the biomaRt package (version 2.46.0, Bioconductor) and cross-referenced with data from the \u0026ldquo;Cancer Gene Census\u0026rdquo; in the Catalog of Somatic Mutations In Cancer database (GRCh 37 version 91).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell proliferation assay\u003c/h2\u003e \u003cp\u003eCells were initially plated at a density of 2.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well in 24-well plates and monitored over 4 days. Cell proliferation was quantified at 24-hour intervals using a Cell Counting Kit-8 (DOJINDO LABORATORIES, Kumamoto, Japan). Cells' doubling time was calculated based on the growth curves. Each procedures was conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Spheroid formation assay\u003c/h2\u003e \u003cp\u003eThe methodology for assessing spheroid formation aligned with that used inn previous studies. Initially, cells were plated at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in 96-well clear round bottom ultra-low attachment microplates (Corning Inc., Corning, NY, USA), and their ability to form spheroids was verified through microscopy (KEYENCE Co., Osaka, Japan). Afrer a cultivation period of three days, the spheroids were extracted, encapsulated in iPGell (GenoStaff Co. Ltd., Tokyo, Japan), and fixed in a 10% neutral buffered formalin solution. For microscopic analysis, the encapsulated spheroids were embedded in paraffin, sectioned into slices 4 \u0026micro;m thick, stained with H\u0026amp;E, and examined under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Invasion assay\u003c/h2\u003e \u003cp\u003eThe invasive potential of the established MFS cell lines was investigated using the real-time cell analyzer xCELLigence (Agilent Technologies Inc., Sannta Clara, CA, USA). The upper chamber was prepared with a Matrigel coating (Corning Inc.) and seeded with 4.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells in a basal DMEM/F12 solution while the lower chamber was filled with a nutritive mix of DMEM/F12 with supplements. Electronic sensors, positioned underneath the membrane between the chambers, captured the impedance of migrating cells every 15 min and translated it into data over 72 h to quantify cell invasion. This method utilized the MG63 osteosarcoma cell line (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan) as a control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Drug screening assay\u003c/h2\u003e \u003cp\u003eThe antitumor potential of 214 agents, includinng drugs approved by the Food and Drug Administration, was evaluated (Supplementary Table\u0026nbsp;2). Cells were seeded in 384-well plates at a density of 5.0 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well and incubated for 1 day before treatment. Following this period, each drug was applied to duplicate wells and incubated for an additional 3 days. Cell growth and viability were determined using the Cell Counting Kit-8 (DOUJINDO LABORATORIES) by comparing the outcomes with those of a dimethyl sulfoxide control. These results integrated with the findings of previous studies on NCC-MFS1-C1, NCC-MFS2-C1, NCC-MFS3-C1, NCC-MFS4-C1, NCC-MFS5-C1, and NCC-MFS6-C1. The analysis involved quantile normalization and unsupervised hierarchical clustering using R (version 4.0.3, limma package version 3.46.0, Bioconductor) and the gplots package (version 3.1.0, CRAN, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cran.r-project.org\u003c/span\u003e\u003cspan address=\"https://cran.r-project.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo ascertain the half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values, 24 drugs showing promising results in preliminary screens were tested across a range of ten concentrations. IC\u003csub\u003e50\u003c/sub\u003e values were calculated by analyzing these concentration-survival relationships via logistic regression, utilizing GraphPad Prism 9.11 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Authentication and quality control of the established MFS cell lines\u003c/h2\u003e \u003cp\u003eNCC-MFS7-C1 and NCC-MFS8-C1 cell lines originating from patients 1 and 2, respectively, were established and sustained for more than 20 passages over 3 months. Authentication of these cell lines was conducted by analyzing 10 microsatellite loci, which showed that the STR profiles were almost identical to those of primary tumor tissues (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Fig.\u0026nbsp;1). Additionally, no detection of Mycoplasma-specific DNA was detected, confirming the absence of mycoplasma contamination.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of short tandem repeat analysis of NCC-MFS7-C1, NCC-MFS8-C1, and each original tumor tissue\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMicrosatellite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eNCC-MFS7-C1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eNCC-MFS8-C1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(Chromosome)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell line\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor Tissue (Case \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell line\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTumor Tissue\u003c/p\u003e \u003cp\u003e(Case \u003cspan refid=\"FPar2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmelogenin (X Y)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTH01 (3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6, 9.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6, 9.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8, 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8, 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD21S11 (21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29, 30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29, 30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD5S818 (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9, 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9, 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10, 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10, 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD13S317 (13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11, 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11, 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11, 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD7S820 (7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10, 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10, 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD16S539 (16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9, 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10, 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCSF1PO (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10, 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10, 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11, 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11, 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003evWA (12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17, 18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17, 18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPOX (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9, 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9, 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Characterization of the established MFS cell lines\u003c/h2\u003e \u003cp\u003eAnalysis of SNP arrays for the established cell lines, NCC-MFS7-C1 and NCC-MFS8-C1, revealed specific chromosomal CNAs. In NCC-MFS7-C1, deletions were noted on chromosomes 5q, 6p, 16q, and 17p13.1, whereas amplifications were observed on chromosomes 7p, 7q, 17p11.2, 18q, and 19q. Conversely, NCC-MFS8-C1 exhibited deletions on chromosomes 1p, 3p, 6p, 9p, and 13q with no observed amplifications. Notably, deletions affected tumor suppressor genes such as \u003cem\u003eTP53\u003c/em\u003e on 17p13.1 in NCC-MFS7-C1 and both \u003cem\u003eCDKN2A\u003c/em\u003e and \u003cem\u003eCDKN2B\u003c/em\u003e on 9p21.3 in NCC-MFS8-C1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;2, Supplementary Tables\u0026nbsp;3 and 4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe NCC-MFS7-C1 and NCC-MFS8-C1 cell lines showed an elongated spindle or polygonal appearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B) and formed spheroids when cultured on low-attachment plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). The spheroids contained oval and pleomorphic cells with atypical nuclei. Growth curve analysis showed that the population doubling times for NCC-MFS7-C1 and NCC-MFS8-C1 cells were 29 h and 68 h, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Furthermore, both cell lines showed higher invasive capacity than did the MG63 osteosarcoma cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Sensitivity of the antitumor agents to the established MFS cell lines\u003c/h2\u003e \u003cp\u003eExtensive analysis was conducted to evaluate the effect of the 214 antitumor compounds on the proliferation of NCC-MFS7-C1 and NCC-MFS8-C1 cells (Supplementary Table\u0026nbsp;5). This included a comparison with data from six previously established MFS cell lines in our laboratory. Antitumor agents were categorized into effective (Cluster A) and ineffective (Cluster B) groups based on their efficacy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Supplementary Table\u0026nbsp;6). Cluster A had a higher proportion of cytotoxic drugs than did cluster B, including among all topoisomerase inhibitors were included in cluster A (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). The distribution of tyrosine kinase inhibitors across both clusters was similar; however, most anaplastic lymphoma kinase inhibitors were grouped into cluster A (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Molecular targeted drugs were predominantly distributed in cluster A, with all histone deacetylase (HDAC) inhibitors belonging to cluster A (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther investigation into the screened antitumor agents involved calculating IC\u003csub\u003e50\u003c/sub\u003e values for each, along with values for six other MFS cell lines previously identified (Supplementary Table\u0026nbsp;7). Notably, actinomycin D (an RNA polymerase and topoisomerase inhibitor) and bortezomib (a proteasome inhibitor) showed lower IC\u003csub\u003e50\u003c/sub\u003e values than doxorubicin, the standard treatment agent for sarcomas, in the most cell lines (7/8). Moreover, romidepsin (an HDAC inhibitor) showed significantly lower IC\u003csub\u003e50\u003c/sub\u003e values than did doxorubicin in all cell lines (8/8), indicating its potent antitumor activity. Table\u0026nbsp;3 and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e provide a comprehensive view of these IC\u003csub\u003e50\u003c/sub\u003e values and corresponding growth curves.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe establishment of the NCC-MFS7-C1 and NCC-MFS8-C1 cell lines represents a significant advancement in MFS research. Given the highly malignant nature of MFS and the challenges in conducting clinical trials owing to its rarity, the preclinical study is crucial. However, the scarcity of available cell lines poses major challenges. These cell lines offer a valuable resource for deepening our understanding of MFS and exploring novel therapeutic strategies.\u003c/p\u003e \u003cp\u003eGenetic analysis of the newly established cell lines revealed significant alterations in key tumor suppressor genes, including TP53, CDKN2A, and CDKN2B. These findings align with those of previous studies highlighting the prevalence of such mutations in MFS, underscoring the relevance of these cell lines as models with representative genetic mutations in the disease [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the absence of CNA in genes such as RB1, ATRX, and HDLBP, which has been reported in other MFS studies, points to the genetic diversity inherent in MFS, further emphasizing the need for a broad range of patient-derived cancer models [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFunctionally, NCC-MFS7-C1 and NCC-MFS8-C1 cell lines exhibited characteristics typical of MFS, including the ability to consistently proliferate and invade. NCC-MFS7-C1, in particular, demonstrated higher proliferative and invasive capacities than did NCC-MFS8-C1. With an average doubling time of 66.1 hours across previously established cell lines in our laboratory, NCC-MFS7-C1 emerged as a relatively fast-growing model, whereas NCC-MFS8-C1 presented a standard growth profile. Both cell lines showed spheroid formation capabilities, positioning them as promising tools for more advanced preclinical experiments.\u003c/p\u003e \u003cp\u003eA comprehensive evaluation of 214 antitumor agents against these cell lines revealed promising results. In particular, actinomycin D, bortezomib, and romidepsin showed superior efficacy than did doxorubicin, the standard drug frequently used to treat MFS and other sarcomas. Although actinomycin D is sometimes utilized in VAC (vincristine, actinomycin D, cyclophosphamide) regimens for sarcoma treatment, bortezomib and romidepsin are rarely employed in managing sarcomas [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Initially, bortezomib was approved for multiple myeloma, and romidepsin for cutaneous T-cell lymphoma [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Despite a few reports suggesting the potential efficacy of bortezomib and HDAC inhibitors, OBP-801 (not romidepsin), in MFS models, these investigations are preclinical studies, confined to in vitro or in vivo data [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, these agents are promising candidates for MFS treatment that deserve further validation in large-scale drug screening of cell lines and clinical trials.\u003c/p\u003e \u003cp\u003eOur study has three limitations. Although this study is the largest drug screening analysis conducted for MFS, the number of cell lines and drugs used in our study was insufficient, considering those for common cancers [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This limitation will be addressed by establishing a more diverse collection of MFS cell lines and conducting larger-scale drug sensitivity assays. Furthermore, although the treatment for metastatic tumors is challenging in MFS, our study did not include cell lines from metastatic lesions. We will require the cell lines from the metastatic sites to develop the novel therapy for the patients with metastasis. In addition, the metastatic potentials of our cell lines were not examined in this study. However, the conventional experiments using animal models are quite artificial as the cells or tumors are inoculated ectopically in the animal body, and subcutaneously transplanted tumors rarely metastasize in the mouse, even when derived from a highly metastatic patient tumor [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To address this issue, we require a novel cancer model or a protocol to recapitulate the metastasis in patients with MFS.\u003c/p\u003e \u003cp\u003eIn conclusion, this study introduced two novel fibrosarcoma (MFS) cell lines, NCC-MFS7-C1 and NCC-MFS8-C1. These cell lines are crucial for MFS research, as they will improve our understanding of the biological and genetic mechanisms of the disease and identify potential therapeutics. Sensitivity of various antitumor agents suggested that actinomycin D, bortezomib, and romidepsin are effective in treating MFS. These findings clearly indicates that the established cell lines will serve as valuable research models for future large-scale drug screening for MFS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Japan Agency for Medical Research and Development (grant number: 20ck0106537h).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Ethics Committee of the National Cancer Center approved the use of clinical materials for this study (approval number: 2004-050).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was provided by the patients.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Drs. S. Fukushima, S. Osaki, K. Ogura (Department of Musculoskeletal Oncology and Rehabilitation Medicine, National Cancer Center Hospital) ; Dr. T. Funada, and Dr. Y. Kobayashi (Department of Diagnostic Pathology, National Cancer Center Hospital); and the National Cancer Center Hospital for sampling the tumor tissue specimens from the surgically resected materials. We appreciate the technical assistance provided by Mr. Y. Ohno (Division of Rare Cancer Research, National Cancer Center Research Institute) and Mrs. Y. Shiotani (Central Animal Division, National Cancer Center Research Institute). We would like to thank Editage (www.editage.jp) for providing English language editing services and for their constructive comments on this manuscript. The Fundamental Innovative Oncology Core of the National Cancer Center provided technical assistance for this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eboard WHOcte. Soft tissue and bone tumours. 5th ed. World Health Organization classification of tumours. World Health Organization International Agency for Research on Cancer; 2020.\u003c/li\u003e\n\u003cli\u003eSanfilippo R, Miceli R, Grosso F, Fiore M, Puma E, Pennacchioli E et al. Myxofibrosarcoma: prognostic factors and survival in a series of patients treated at a single institution. Ann Surg Oncol. 2011;18(3):720-5. doi:10.1245/s10434-010-1341-4.\u003c/li\u003e\n\u003cli\u003eWakely PE, Jr. Cytopathology of myxofibrosarcoma: a study of 66 cases and literature review. J Am Soc Cytopathol. 2021;10(3):300-9. doi:10.1016/j.jasc.2020.09.004.\u003c/li\u003e\n\u003cli\u003eDewan V, Darbyshire A, Sumathi V, Jeys L, Grimer R. Prognostic and survival factors in myxofibrosarcomas. Sarcoma. 2012;2012:830879. doi:10.1155/2012/830879.\u003c/li\u003e\n\u003cli\u003eWillems SM, Debiec-Rychter M, Szuhai K, Hogendoorn PC, Sciot R. Local recurrence of myxofibrosarcoma is associated with increase in tumour grade and cytogenetic aberrations, suggesting a multistep tumour progression model. Mod Pathol. 2006;19(3):407-16. doi:10.1038/modpathol.3800550.\u003c/li\u003e\n\u003cli\u003eGhazala CG, Agni NR, Ragbir M, Dildey P, Lee D, Rankin KS et al. Myxofibrosarcoma of the extremity and trunk: a multidisciplinary approach leads to good local rates of LOCAL control. Bone Joint J. 2016;98-b(12):1682-8. doi:10.1302/0301-620x.98b12.37568.\u003c/li\u003e\n\u003cli\u003evan der Horst CAJ, Bongers SLM, Versleijen-Jonkers YMH, Ho VKY, Braam PM, Flucke UE et al. Overall Survival of Patients with Myxofibrosarcomas: An Epidemiological Study. Cancers. 2022;14(5):1102.\u003c/li\u003e\n\u003cli\u003eTeurneau H, Engellau J, Ghanei I, Vult von Steyern F, Styring E. High Recurrence Rate of Myxofibrosarcoma: The Effect of Radiotherapy Is Not Clear. Sarcoma. 2019;2019:8517371. doi:10.1155/2019/8517371.\u003c/li\u003e\n\u003cli\u003eTakeuchi Y, Yoshida K, Halik A, Kunitz A, Suzuki H, Kakiuchi N et al. The landscape of genetic aberrations in myxofibrosarcoma. Int J Cancer. 2022;151(4):565-77. doi:10.1002/ijc.34051.\u003c/li\u003e\n\u003cli\u003eHeitzer E, Sunitsch S, Gilg MM, Lohberger B, Rinner B, Kashofer K et al. Expanded molecular profiling of myxofibrosarcoma reveals potentially actionable targets. Mod Pathol. 2017;30(12):1698-709. doi:10.1038/modpathol.2017.94.\u003c/li\u003e\n\u003cli\u003eBarretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012;483(7391):603-7. doi:10.1038/nature11003.\u003c/li\u003e\n\u003cli\u003eYang W, Soares J, Greninger P, Edelman EJ, Lightfoot H, Forbes S et al. Genomics of Drug Sensitivity in Cancer (GDSC): a resource for therapeutic biomarker discovery in cancer cells. Nucleic Acids Res. 2013;41(Database issue):D955-61. doi:10.1093/nar/gks1111.\u003c/li\u003e\n\u003cli\u003eKondo T. Current status and future outlook for patient-derived cancer models from a rare cancer research perspective. Cancer Sci. 2021;112(3):953-61. doi:10.1111/cas.14669.\u003c/li\u003e\n\u003cli\u003eBairoch A. The Cellosaurus, a Cell-Line Knowledge Resource. J Biomol Tech. 2018;29(2):25-38. doi:10.7171/jbt.18-2902-002.\u003c/li\u003e\n\u003cli\u003eKito F, Oyama R, Sakumoto M, Shiozawa K, Qiao Z, Toki S et al. Establishment and characterization of a novel cell line, NCC-MFS1-C1, derived from a patient with myxofibrosarcoma. Hum Cell. 2019;32(2):214-22. doi:10.1007/s13577-018-00233-1.\u003c/li\u003e\n\u003cli\u003eNoguchi R, Yoshimatsu Y, Ono T, Sei A, Hirabayashi K, Ozawa I et al. Establishment and characterization of NCC-MFS2-C1: a novel patient-derived cancer cell line of myxofibrosarcoma. Hum Cell. 2021;34(1):246-53. doi:10.1007/s13577-020-00420-z.\u003c/li\u003e\n\u003cli\u003eTsuchiya R, Yoshimatsu Y, Noguchi R, Sin Y, Ono T, Sei A et al. Establishment and characterization of NCC-MFS3-C1: a novel patient-derived cell line of myxofibrosarcoma. Hum Cell. 2021;34(4):1266-73. doi:10.1007/s13577-021-00548-6.\u003c/li\u003e\n\u003cli\u003eYoshimatsu Y, Noguchi R, Tsuchiya R, Sin Y, Ono T, Sugaya J et al. Establishment and characterization of NCC-MFS4-C1: a novel patient-derived cell line of myxofibrosarcoma. Hum Cell. 2021;34(6):1911-8. doi:10.1007/s13577-021-00589-x.\u003c/li\u003e\n\u003cli\u003eTsuchiya R, Yoshimatsu Y, Noguchi R, Sin Y, Ono T, Akiyama T et al. Establishment and Characterization of NCC-MFS5-C1: A Novel Patient-Derived Cell Line of Myxofibrosarcoma. Cells. 2022;11(2). doi:10.3390/cells11020207.\u003c/li\u003e\n\u003cli\u003eYoshimatsu Y, Noguchi R, Sin Y, Tsuchiya R, Ono T, Akiyama T et al. Establishment and characterization of NCC-MFS6-C1: a novel patient-derived cell line of myxofibrosarcoma. Hum Cell. 2022;35(6):1993-2001. doi:10.1007/s13577-022-00749-7.\u003c/li\u003e\n\u003cli\u003eOgura K, Hosoda F, Arai Y, Nakamura H, Hama N, Totoki Y et al. Integrated genetic and epigenetic analysis of myxofibrosarcoma. Nat Commun. 2018;9(1):2765. doi:10.1038/s41467-018-03891-9.\u003c/li\u003e\n\u003cli\u003eYamashita A, Suehara Y, Hayashi T, Takagi T, Kubota D, Sasa K et al. Molecular and clinicopathological analysis revealed an immuno-checkpoint inhibitor as a potential therapeutic target in a subset of high-grade myxofibrosarcoma. Virchows Arch. 2022;481(4):1-17. doi:10.1007/s00428-022-03358-9.\u003c/li\u003e\n\u003cli\u003eSambri A, De Paolis M, Spinnato P, Donati DM, Bianchi G. The Biology of Myxofibrosarcoma: State of the Art and Future Perspectives. Oncol Res Treat. 2020;43(6):314-22. doi:10.1159/000507334.\u003c/li\u003e\n\u003cli\u003eLi GZ, Okada T, Kim YM, Agaram NP, Sanchez-Vega F, Shen Y et al. Rb and p53-Deficient Myxofibrosarcoma and Undifferentiated Pleomorphic Sarcoma Require Skp2 for Survival. Cancer Res. 2020;80(12):2461-71. doi:10.1158/0008-5472.Can-19-1269.\u003c/li\u003e\n\u003cli\u003eNakano K, Ae K, Matsumoto S, Takahashi S. The VAC regimen for adult rhabdomyosarcoma: Differences between adolescent/young adult and older patients. Asia Pac J Clin Oncol. 2020;16(2):e47-e52. doi:10.1111/ajco.13279.\u003c/li\u003e\n\u003cli\u003e\u0026Ouml;zkan A, Bayram İ, Sezgin G, Mirioğlu A, K\u0026uuml;peli S. Efficacy of replacing actinomycin-D with carboplatin in Ewing sarcoma consolidation treatment: Single-center experience. J Bone Oncol. 2022;35:100435. doi:10.1016/j.jbo.2022.100435.\u003c/li\u003e\n\u003cli\u003eSharma A, Preuss CV. Bortezomib. StatPearls. Treasure Island (FL): StatPearls Publishing Copyright \u0026copy; 2024, StatPearls Publishing LLC.; 2024.\u003c/li\u003e\n\u003cli\u003eCvek B. Proteasome inhibitors. Prog Mol Biol Transl Sci. 2012;109:161-226. doi:10.1016/b978-0-12-397863-9.00005-5.\u003c/li\u003e\n\u003cli\u003eGrant C, Rahman F, Piekarz R, Peer C, Frye R, Robey RW et al. Romidepsin: a new therapy for cutaneous T-cell lymphoma and a potential therapy for solid tumors. Expert Rev Anticancer Ther. 2010;10(7):997-1008. doi:10.1586/era.10.88.\u003c/li\u003e\n\u003cli\u003eShimony S, Horowitz N, Ribakovsky E, Rozovski U, Avigdor A, Zloto K et al. Romidepsin treatment for relapsed or refractory peripheral and cutaneous T-cell lymphoma: Real-life data from a national multicenter observational study. Hematol Oncol. 2019;37(5):569-77. doi:10.1002/hon.2691.\u003c/li\u003e\n\u003cli\u003eLi CF, Wang JM, Kang HY, Huang CK, Wang JW, Fang FM et al. Characterization of gene amplification-driven SKP2 overexpression in myxofibrosarcoma: potential implications in tumor progression and therapeutics. Clin Cancer Res. 2012;18(6):1598-610. doi:10.1158/1078-0432.Ccr-11-3077.\u003c/li\u003e\n\u003cli\u003eKawarazaki A, Horinaka M, Yasuda S, Kawashima H, Numajiri T, Sakai T. The HDAC inhibitor OBP-801 suppresses the growth of myxofibrosarcoma cells. J buon. 2020;25(1):464-71.\u003c/li\u003e\n\u003cli\u003eBashi AC, Coker EA, Bulusu KC, Jaaks P, Crafter C, Lightfoot H et al. Large-scale Pan-cancer Cell Line Screening Identifies Actionable and Effective Drug Combinations. Cancer Discov. 2024:Of1-of20. doi:10.1158/2159-8290.Cd-23-0388.\u003c/li\u003e\n\u003cli\u003eFidler IJ. Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award lecture. Cancer Res. 1990;50(19):6130-8.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 2 and 3","content":"\u003cp\u003eTable 2 and 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Myxofibrosarcoma, Soft tissue sarcoma, Cell lines, Patient-derived model, Antitumor drug screening","lastPublishedDoi":"10.21203/rs.3.rs-4251932/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4251932/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMyxofibrosarcoma (MFS), an aggressive soft tissue sarcoma, presents a significant challenge because of its high recurrence rate, distal metastasis, and complex genetic background. Although surgical resection is the standard treatment for MFS, the outcomes are unsatisfactory, and effective non-surgical treatment strategies, including drug therapy, are urgently warranted. MFS is a rare tumor that requires comprehensive preclinical research to develop promising drug therapies; however, only two MFS cell lines are publicly available worldwide. The present study reports two novel patient-derived MFS cell lines, NCC-MFS7-C1 and NCC-MFS8-C1. These cell lines have been extensively characterized for their genetic profile, proliferation, spheroid-forming capacity, and invasive behavior, confirming that they retain MFS hallmarks. Furthermore, we conducted comprehensive drug screening against these cell lines and six others previously established in our laboratory to identify potential therapeutic candidates for MFS. Among the screened agents, actinomycin D, bortezomib, and romidepsin demonstrated considerable antiproliferative effects that were superior to those of doxorubicin, a standard drug, highlighting their potential as novel drugs. In conclusion, NCC-MFS7-C1 and NCC-MFS8-C1 are valuable research resources that contribute to the understanding of the pathogenesis and development of novel therapies for MFS.\u003c/p\u003e","manuscriptTitle":"Establishment and characterization of two novel patient-derived cell lines from myxofibrosarcoma: NCC-MFS7-C1 and NCC-MFS8-C1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-19 19:16:58","doi":"10.21203/rs.3.rs-4251932/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2024-05-03T05:17:38+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-04-15T22:03:20+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-15T02:27:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-14T14:04:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Cell","date":"2024-04-11T06:50:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"36b1c420-fd1c-40e4-9697-dcbf9e131bc2","owner":[],"postedDate":"April 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-02T16:05:02+00:00","versionOfRecord":{"articleIdentity":"rs-4251932","link":"https://doi.org/10.1007/s13577-024-01124-4","journal":{"identity":"human-cell","isVorOnly":false,"title":"Human Cell"},"publishedOn":"2024-08-31 15:58:12","publishedOnDateReadable":"August 31st, 2024"},"versionCreatedAt":"2024-04-19 19:16:58","video":"","vorDoi":"10.1007/s13577-024-01124-4","vorDoiUrl":"https://doi.org/10.1007/s13577-024-01124-4","workflowStages":[]},"version":"v1","identity":"rs-4251932","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4251932","identity":"rs-4251932","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}