ESTABLISHMENT AND CHARACTERIZATION OF PATIENT-DERIVED PLEOMORPHIC RHABDOMYOSARCOMA MODELS

Abstract

Background: Pleomorphic rhabdomyosarcoma (pRMS) is an aggressive mesenchymal malignancy affecting adults, and its characteristics and clinical behaviors differ considerably from those of embryonal and alveolar RMS subtypes. A therapeutic strategy for pRMS has not been established, and its prognosis remains poor. Further investigations are therefore required to improve the clinical outcomes associated with this disease. Patient-derived cancer models are essential tools for basic and translational research, and numerous models of different RMS subtypes have been established. However, only two pRMS cell lines are available, and no xenograft model of this disease has been developed. Hence, the objective of this study was to establish patient-derived pRMS models.

Methods: We obtained tumor tissues from a 73-year-old pRMS patient who had not received chemotherapy or radiotherapy. We prepared patient-derived xenografts (PDXs) from these tumor tissues and stable patient-derived cell lines from both the original tumor and a PDX. The established models were then characterized, and their novelty was confirmed by short tandem repeat analysis.

Results: The PDX tumors were histologically similar to the original source tumor. Moreover, the established cell lines exhibited morphological features resembling those of RMS, the ability to form spheroids, constant growth, and invasive behavior. By screening an anti-cancer drug library, we identified mitoxantrone, ponatinib, romidepsin, vandetanib, belinostat, bortezomib, and vorinostat as potential drugs for pRMS treatment.

Conclusions: Our novel pRMS models will be useful research resources, providing an opportunity for in-depth investigations of the molecular basis and treatment of this disease. Clinical trials for the drugs showing anti-proliferative effects on pRMS cells may be worth considering in further studies.

Introduction

Rhabdomyosarcomas (RMSs) are diverse mesenchymal malignancies that arise from skeletal muscle precursor cells¹⁾. RMSs are histologically classified into four types with remarkably different clinical outcomes: embryonal RMS (eRMS), alveolar RMS (aRMS), spindle/sclerosing RMS (sRMS), and pleomorphic RMS (pRMS)¹⁾. eRMS and aRMS are the most common types of soft-tissue sarcoma in children and adolescents. Although RMSs are characterized by high grades of malignancy and a propensity for metastasis, eRMS and aRMS respond well to chemotherapy^2,3,4,5,6), and approximately 70% of patients with localized disease can be cured with multidisciplinary treatment protocols^4,5). In contrast, pRMS, which occurs among the adult population and is the most common RMS subtype in this group, has an aggressive phenotype and markedly poor prognosis, with 1-year to 20-month disease-free survival rates ranging from 12.5 to 50%^{7,8,9,10,11,12,13,14,15,16,17,18)}. Moreover, in a study of 38 pRMS patients, Furlong et al. recorded a 5-year survival rate of only 27%¹⁹⁾. Thus, further investigation of this disease and novel therapeutic strategies are required.

Patient-derived cancer models are indispensable tools for investigating the molecular backgrounds of malignant phenotypes and ascertaining the effects and modes of action of anti-cancer drugs²⁰⁾. The utility of patient-derived models has been demonstrated for several types of malignancies²¹⁾, including eRMS²²⁾. Numerous cell lines have been successfully established from RMS tissues and utilized in cancer research²³⁾. However, only two pRMS cell lines, HS-RMS-1²⁴⁾ and HS-RMS-2²⁵⁾, have been generated, and the development of a patient-derived xenograft (PDX) model of pRMS has not been reported to date. Considering the urgent requirement for novel treatments for this disease, more patient-derived pRMS models need to be established.

In this study, we established PDXs and two patient-derived pRMS cell lines and examined their morphological features and biological characteristics. Our data suggest that these preclinical models will be useful in elucidating the molecular mechanisms underlying the aggressive clinical features of pRMS.

Materials and Methods

Patient

The donor was a 73-year-old man who had presented himself at the National Cancer Center Hospital, Tokyo, Japan, for a soft-tissue tumor of the right thigh, which had been increasing in size for a number of months (Figure 1A). Based on a biopsy diagnosis of spindle cell sarcoma, a wide excision of the tumor was performed, from which the samples used to establish the models in this study were taken. Histological evaluation of the resected specimen demonstrated a fascicular to storiform proliferation of pleomorphic spindle cells within a fibrous to focally myxoid background, associated with necrosis and brisk mitotic activity (Figure 1B and C). The tumor cells were immunohistochemically positive for myogenin (Figure 1D), cytokeratin (rare), smooth muscle actin, and desmin, whereas they were negative for S100 protein, MDM2, and CDK4 (data not shown). The final diagnosis of pRMS was rendered. The resection margin was negative, and the patient did not receive adjuvant chemotherapy and has been disease-free for 1 year and 10 months as of publication of this study. This study was approved by the ethics committee of the National Cancer Center, and written informed consent was obtained from the patient.

Figure 1.

Characteristics of the original tumor in this study.

An axial view obtained by gadolinium-enhanced fat-suppressed magnetic resonance imaging (A). The tumor was located in the subcutaneous tissue of the medial side of the right thigh and was 74 mm in size. It was multinodular and hypervascular and exhibited some myxoid features. Images of hematoxylin and eosin (H&E) staining are shown (B and C). Expression of myogenin was confirmed by immunohistochemistry (D). Original magnification: 200× (B) and 400× (C, D). Scale bars indicate 100 μm (B) or 50 μm (C and D).

Histology

A representative paraffinized block of the tumor was cut into 4-μm-thick sections, which were then deparaffinized and stained with hematoxylin and eosin (HE) (Figure 1B and C).

PDXs

Several pieces of pRMS tissue obtained by surgical resection were subcutaneously engrafted using a 13-gauge transplant needle into the bilateral hind flanks of 6–12-week-old severely immunodeficient female NOD.Cg-Prkdc^scid Il2rg^tm1Sug/Jic (also known as NOD/Shi-scid IL-2Rγ^null or NOG) mice (Central Institute for Experimental Animals, Tokyo, Japan). When the tumors reached 500–1,000 mm³, they were transplanted into another recipient mouse. Tumor size was measured periodically using digital calipers (SuperCaliper, Mitutoyo, Kanagawa, Japan), with tumor volume being calculated as π/6 × length × width × thickness²⁶⁾. After two passages, the tumors were treated with CELLBANKER 1 plus (Takara Bio, Shiga, Japan) at –80ºC overnight and cryopreserved in liquid nitrogen. All animal experiments were performed in accordance with the Guidelines for Animal Experiments of the National Cancer Center and approved by the Institutional Committee for Ethics of Animal Experimentation (T13–016).

Primary tissue culture

Tumor tissue was cut into small pieces and treated with collagenase 2 (Worthington, Lakewood, NJ, USA) and hyaluronidase from bovine testes (H3506, Sigma-Aldrich, St. Louis, MO, USA) at 37ºC for 10 min. The obtained cells were seeded in a tissue culture plate (Thermo Fisher Scientific, Waltham, MA, USA). They were maintained in Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 100 U penicillin G, and 100 μg/ml streptomycin (Gibco) at 37ºC in a humidified atmosphere of 5% CO₂. The culture medium was changed once or twice a week. When the cells reached sub-confluence, they were dispersed with 0.1% trypsin-EDTA (Gibco) and seeded in a new culture plate.

Authentication and quality control of established cell lines

Genomic DNA was extracted from tumor tissues or cell lines using AllPrep DNA/RNA Mini Kits (Qiagen, Hilden, Germany), quantified with a NanoDrop 8000 (Thermo Fisher Scientific), and stored at –80ºC until use. To authenticate cells, we amplified short tandem repeats (STRs) at 10 loci using 500 pg genomic DNA and the GenePrint 10 STR multiplex assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The amplicons were analyzed with a 3500xL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and GeneMapper 5 software (Applied Biosystems). The resulting STR profiles were compared with those of cells in public cell banks, including the American Type Culture Collection (ATCC), the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), and the Japanese Collection of Research Bioresources Cell Bank (JCRB).

DNA in the culture medium was tested to rule out the possibility of mycoplasma contamination according to the guidelines of the International Cell Line Authentication Committee²⁷⁾. Briefly, DNA was isolated from the medium of cultures at 70–90% confluency, heated at 95ºC for 10 min, and amplified using an e-Myco Mycoplasma PCR Detection Kit (iNtRON Biotechnology, Gyeonggi-do, South Korea). The amplified DNA fragments were separated by electrophoresis on a 1.5% agarose gel, stained with Midori Green Advance (Nippon Genetics, Tokyo, Japan), and detected using an Amersham Imager 600 (GE Healthcare Life Sciences, Little Chalfont, UK).

Immunocytochemical analysis of established cell lines

Suspensions of cells were solidified using iPGell (Genostaff, Tokyo, Japan), and cell blocks were created. The cell blocks were fixed with 10% neutral buffered formalin solution, embedded in paraffin, and cut into 4-μm-thick sections. The sections were then stained with HE or antibodies against desmin (D33, 1:100) and myogenin (F5D, 1:100) (both Dako/Agilent Technologies, Santa Clara, CA, USA). Bound antibodies were visualized using the EnVision system (Dako). Nuclear staining was performed using 3,3′-diaminobenzidine (DAB).

Characterization of cell growth

Cells were seeded in 96-well culture plates at 8,000 or 16,000 cells/well in triplicate and counted using a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) at several time points over 96 h of incubation at 37ºC. Cells in each well were incubated for 2 h with CCK-8 reagent at 24, 48, 72, and 96 h after seeding. Absorbance values at a wavelength of 450 nm were measured using a microplate reader (Bio-Rad, Hercules, CA, USA). Growth curves were then generated by plotting time after seeding day on the horizontal axis against absorbance value at 450 nm on the vertical axis.

Spheroid formation assay

To test their ability to form spheroids, 100,000 cells were seeded in a 6-cm ultra-low-attachment culture dish (Thermo Fisher Scientific) containing 10% FBS in DMEM. After 3 weeks of incubation at 37ºC in a humidified atmosphere containing 5% CO₂, the presence of spheroids was assessed under a microscope (Keyence, Osaka, Japan). All assays were performed in duplicate. The size distribution of spheroids was examined and summarized in a histogram (Supplementary Figure 1)^28,29,30).

Cell invasion assay

The invasive capacity of the cultured tumor cells was assessed using BD BioCoat Matrigel Invasion Chambers (BD Biosciences, Bedford, MA, USA) according to the manufacturer’s instructions. In brief, 50,000 or 100,000 cells in serum-free DMEM were transferred to the upper chambers, and medium containing 10% FBS was placed in the lower chambers. Forty-eight hours later, the tumor cells on the bottom surface of the upper chambers were stained and counted. Cells in nine separate areas were counted at 200 × magnification.

Screening anti-cancer drugs for cell growth inhibition

Cells were seeded in a 96-well culture plate with DMEM containing 10% FBS at 5,000 cells/well in duplicate and grown overnight at 37ºC under 5% CO₂. One hundred sixty-four low-molecular-weight chemical compounds, which included U.S. Food and Drug Administration (FDA)-approved drugs (Selleck Chemicals, Houston, TX, USA), were added using the Bravo Automated Liquid Handling Platform (Agilent Technologies). After treatment for 72 h, living cells were quantified by CCK-8 assay. The experiment was performed in duplicate. Anti-cancer drugs used in this study are listed in Supplementary Table 1.

Results

A schematic of the workflow used to establish the patient-derived cancer models in this study is given in Figure 2. We used the surgically resected tumor tissue to generate both PDXs and patient-derived cell (PDC) lines. The cell line directly established from the patient’s tumor was named NCC-pRMS1-C1, and that derived from fourth-generation PDX tissue was named NCC-pRMS1-X4-C1. Third-generation tumor tissue was frozen in liquid nitrogen and inoculated into the skin of an additional mouse. We confirmed that the tumor grew in subcutaneous tissue after being stored in liquid nitrogen.

Figure 2.

Workflow schema.

The xenografts were generated using surgically resected tumor tissue. Cell lines, the names of which are shown below the corresponding images, were established from both xenograft and surgically resected tumor tissue.

PDX growth characteristics and morphology

The surgically resected tumor tissue was subcutaneously inoculated into immune-deficient mice, and after propagation in vivo, these PDXs were serially transplanted into other mice. Third-2 PDX tissue was frozen and later inoculated into mice, which confirmed that the tumor tissue could be successfully propagated after prolonged storage. The histological features of PDX tumors of different generations were found to be quite similar, as shown in Figure 3A. In contrast, growth rates varied between passages. The second-generation PDX gradually increased in size soon after transplantation, whereas PDXs of other generations started to grow between 20 and 40 days after being transplanted (Figure 3B). We established a cell line from a PDX tumor of the fourth generation (Figure 2 and Figure 3B).

Figure 3.

Histological analysis and growth curves of xenograft tumors derived from surgical specimens.

H&E staining of xenograft tumor tissue of different generations is shown (A). Tumors of the third generation were frozen and later re-implanted (fourth generation). Original magnification: 400×. Tumor growth rates were measured (B). Scale bars indicate 200 μm.

Authentication of established cell lines

We subjected the original tumor tissue, a PDX, and the PDC lines to STR typing of 10 loci (Table 1). All samples had the same STR profile, with the exception of the D16S539 locus. We therefore concluded that all of the established PDXs and PDC lines were derived from the patient’s tumor. Evaluations higher than 0.75 were not found in the STR profile databases of public cell banks; therefore, we judged the established PDXs and cell lines to be novel. PCR tests indicated that the cell lines were not contaminated with mycoplasma (data not shown).

Table 1. Results of STR analysis

Microsatellite (Chromosome)	NCC-pRMS1-X4-C1 (P3)	NCC-pRMS1-C1 (P21)	NCC-pRMS1-X3	Tumor tissue
Amelogenin (X Y)	X,Y	X,Y	X,Y	X,Y
TH01 (3)	9,10	9,10	9,10	9,10
D21S11 (21)	28,32.2	28,32.2	28,32.2	28,32.2
D5S818 (5)	11,12	11,12	11,12	11,12
D13S317 (13)	12	12	12	12
D7S820 (7)	12	12	12	12
D16S539 (16)	13	13	10,13	10,13
CSF1PO (5)	11,12	11,12	11,12	11,12
vWA (12)	16	16	16	16
TPOX (2)	8,9	8,9	8,9	8,9

Cells were compared to a normal and tumor tissue from the patient. P indicates a passage number of a cell line.

Phenotypic characterization of established cell lines

NCC-pRMS1-C1 cells from the original tumor tissue were maintained for over 24 months and 60 passages, and NCC-pRMS1-X4-C1 cells from the PDX tumor tissues were maintained for 17 months and 55 passages. The cells exhibited a spindle shape (Figure 4A and B), consistent with the morphology of those of the original tumor tissue. Immunocytochemistry revealed that NCC-pRMS1-C1 cells did not express myogenin (Figure 4C) or desmin (Figure 4D). Myogenin was expressed (Figure 4E) by a small portion of NCC-pRMS1-X4-C1 cells. A population of NCC-pRMS1-X4-C1 cells also expressed desmin (Figure 4F). When seeded on ultra-low-attachment tissue culture dishes, cells of both lines formed spheroids (Figure 4G and H). The size distribution of the spheroids was evaluated, with no substantial difference between the two cell lines (Supplementary Figure 1).

Figure 4.

Morphological and immunohistochemical analysis of established cell lines.

Phase-contrast images of NCC-pRMS1-C1 (A) and NCC-pRMS-X4-C1 (B) cells cultured in serum-containing medium are shown. Immunocytochemical staining of NCC-pRMS1-C1 (C, D) and NCC-pRMS-X4-C1 (E, F) cells for myogenin (C, E) and desmin (D, F) was performed. Myogenin and desmin were not expressed in NCC-pRMS-C1 cells (C and D, respectively). Myogenin was expressed in a small portion of NCC-pRMS1-X4-C1 cells (E). A portion of NCC-pRMS1-X4-C1 cells expressed desmin (F). Arrow heads indicate the cells expressing myogenin (E) or desmin (F). Scale bars indicate 100 μm. Phase-contrast images of NCC-pRMS1-C1 (G) and NCC-pRMS1-X4-C1 (H) cells cultured in ultra-low-attachment culture dishes are shown. Bar: 100 μm. Both cell lines exhibited spheroid formation.

NCC-pRMS1-C1 and NCC-pRMS1-X4-C1 cells exhibited constant growth at different seeding densities (Figure 5A and B), with doubling times of 61 and 40 h, respectively, when seeded at 8,000 cells per well. In addition, the invasive capacity of these cell lines differed. The numbers of invading NCC-pRMS1-C1 cells were obviously smaller than those of invading NCC-pRMS1-X4-C1 cells at both densities tested (Figure 5C–F). The results of this assay are summarized in Figure 5G, which demonstrates that NCC-pRMS1-X4-C1 cells were considerably more invasive than NCC-pRMS1-C1 cells.

Figure 5.

Characterization of established cell lines.

NCC-pRMS-C1 (A) and NCC-pRMS-X4-C1 (B) cell proliferation was evaluated with the Cell Counting Kit-8 assay. Representative images of the invasion chamber assay of NCC-pRMS-C1 (C, D) and NCC-pRMS-X4-C1 (E, F) cells, 50,000 (C, E) or 100,000 (D, F) of which were seeded in each invasion chamber, are shown. Scale bars indicate 50 μm. Columns in the graph represent cell counts (G). Anti-cancer drug screening in NCC-pRMS1-C1 and NCC-pRMS1-X4-C1 cells. NCC-pRMS1-C1 and NCC-pRMS1-X4-C1 cells were treated with 164 anti-cancer compounds (10 μM) for 72 h (H). The cell viability following anti-cancer drug treatments is plotted.

Screening of anti-cancer drugs in NCC-pRMS1-X4-C1 and NCC-pRMS1-C1 cells

The sensitivity to anti-cancer drugs that are clinically available for cancer therapy was monitored in NCC-pRMS1-C1 and NCC-pRMS1-X4-C1 cells. The cells were treated with all 164 anti-cancer drugs at a fixed concentration of 10 μM. As shown in Figure 5 and Supplementary Table 1, we identified seven drugs with a high inhibition effect (less than 10% of viable cells remaining) on both NCC-pRMS1-C1 and NCC-pRMS1-X4-C1 cells: mitoxantrone, ponatinib, romidepsin, vandetanib, belinostat, bortezomib, and vorinostat. Although the two cell lines originated from the same patient, they showed different responses to the drug treatments (Figure 5H).

Discussion

Numerous RMS cell lines have been established for basic and translational research and preclinical studies²³⁾. Among them, only two pRMS lines, HS-RMS-1²⁴⁾ and HS-RMS-2²⁵⁾, have been generated, and a PDX model of this disease has not yet been described, likely because pRMS is the rarest of the three RMS subtypes. The HS-RMS-1 line was established from a 26-year-old patient²⁴⁾, and although acknowledged as the first pRMS cell line, the donor from which it was derived may not represent a typical case of this disease, considering that pRMS patients are on average (both median and mean) in their 50s¹⁹⁾. Using a primary tumor from a 73-year-old pRMS patient, the present work resulted in the third and fourth pRMS cell lines and the first pRMS PDX to be described.

Our models are unique for two reasons. Firstly, they were established from an untreated primary tumor. The majority of RMS cell lines are derived from tumor relapses or distant metastases²³⁾; thus, their characteristics may differ from those of the original untreated tumor cells, owing to the selective pressure exerted by treatments and genetic and epigenetic changes. These cell lines may therefore exhibit different responses to anti-cancer drugs and greater metastatic potential. Although potentially easier to establish, they may not be suitable for studying primary tumors.

Secondly, our models comprise xenografts and cell lines from the same tumor tissue. There has been much discussion and debate concerning the utility of patient-derived cancer models. Xenografts may faithfully reflect the characteristics of tumors in the human body, and it is thought that patient outcomes might be predicted by examining corresponding PDXs^31,32,33,34). For instance, in a recent study of 92 patients with various solid cancers, Izumchenko et al. reported an 87% association between the responses of patients and corresponding PDXs to 129 treatments³⁵⁾. However, the question of how accurately xenografts reproduce the behaviors of tumors in humans remains. Notably, the genomic instability of xenografts of various types of malignancies has been repeatedly reported^36,37,38). Kresse et al. found that sarcoma xenografts, especially those of osteosarcomas, acquire additional mutations³⁹⁾, and Ben-David et al. reported the development of mouse-specific genetic aberrations in PDXs that alter responses to anti-cancer drugs³⁸⁾. Consistent with a previous report⁴⁰⁾, we observed in the present study that PDX tumor growth rate increased over time (Figure 3), suggesting changes in the behavior of tumor cells inside mice. Alterations of the features of PDX tumors in mice may stem from the selection pressure on tumor cells and clonal expansion of specific tumor cell populations.

Through the screening of anti-proliferative effects of anti-cancer drugs, we identified candidate drugs for pRMS treatment with different modes of action. Mitoxantrone is a derivative of anthracene and a type II topoisomerase inhibitor. It interacts with DNA, interfering with the strand-reunion reaction of topoisomerase II and producing DNA breaks⁴¹⁾. Ponatinib⁴²⁾ and vandetanib^43,44,45) are multikinase inhibitors. Romidepsin⁴⁶⁾, belinostat⁴⁷⁾, and vorinostat^48,49) are inhibitors of histone deacetylase. Their possible clinical utility should be further evaluated using additional cell lines and PDXs of pRMS.

Cell lines are also invaluable tools for experimental preclinical studies, and examples of discoveries that could not have been made without their use are numerous⁵⁰⁾. We found that the two established cell lines exhibited different sensitivity to anti-cancer drugs; the cell line established from the original tumor showed more resistance than the cell line established from the xenograft tissue. From a practical perspective, cell lines have several advantages over xenografts, given their low cost, suitability to high-throughput applications⁵¹⁾, and compatibility with informatics approaches to drug development such as Connectivity Map⁵²⁾. However, there is much evidence to suggest that cultured cells lose the traits of the tumor from which they are derived. Specific mutations and irreversible modifications of gene expression patterns are induced during in vitro culture, changing treatment response^53,54,55). Nevertheless, the utility of cell lines in predicting the response of patients to treatments should not be overlooked, as emphasized by a study in which the Cancer Cell Line Encyclopedia was employed to identify relationships between gene expression-based signatures and drug sensitivity⁵⁶⁾. Rees et al. also revealed that differential basal gene expression correlates with patterns of small-molecule sensitivity among many cell lines using a method made available via the Cancer Therapeutics Response Portal⁵⁷⁾. Controversies surrounding patient-derived cancer models should be resolved by clarifying the advantages and limitations of each. Our matched set of xenografts and cell lines from the same pRMS patient will be useful research tools to address these issues and should pave the way for basic, translational, and preclinical studies of this disease.

Our study has one critical limitation, in that we generated PDXs and PDC lines from only one case of pRMS, a clinically diverse malignancy. To more firmly link in vitro data to clinical observations, we need to establish PDXs and PDC lines from a greater number of patients. Moreover, multiple PDXs and PDC lines should be produced from each tumor to understand in more detail the features of tumor tissues, which are composed of several cell populations. A continuous effort will be required to obtain these patient-derived cancer models.

Conclusions

We established the third and fourth pRMS cell lines and the first xenograft model of this disease using tumor tissue from a typical pRMS patient. Patient-derived models of rare cancers can only be generated in a limited subset of research institutes and should therefore be made available to the community. We hope to share our cell lines with researchers interested in basic science with clinical applications and translational research. The accumulation of data from different researchers using common models will facilitate cancer research, providing us novel biological insights.

Conflict of interest

The authors declare that they have no conflicts of interest.

Sources of support

This work was supported by the National Cancer Center Research Core Facility and National Cancer Center Research and Development Fund [26-A-3, 26-A-9, 29-A-2].

Acknowledgments

We thank Drs. Y. Minami, K. Shimizu, T. Mori, T. Uehara, M. Sugawara, Y. Araki, and Ms. R. Nakano of the Division of Musculoskeletal Oncology, National Cancer Center Hospital, for sampling tumor tissue specimens from surgically resected materials. We would like to thank Editage (www.editage.jp) for English language editing and constructive comments on the manuscript.

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