The present study was approved by the Institutional Gene Recombination Experimentation Committee of the Kyoto Prefectural University of Medicine (approval no. #2019-35; Kyoto, Japan). The human ERMS cell line RD was purchased from the Japanese Collection of Research Bioresources Cell Bank, and the human ARMS cell line SJ-Rh30 was kindly provided by Dr Peter J. Houghton (Greehey Children's Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX, USA) (30). Firefly luciferase-expressing RD and SJ-Rh30 were established by transducing RediFect Red-Luc-Puromycin lentiviral particles (PerkinElmer, Inc.) into the cells. Briefly, RD and SJ-Rh30 cells were plated at 5.0×10⁵ cells/100-mm dish. After 24 h, hexadimethrine bromide was added (8 mg/ml; cat. no, H9268; MilliporeSigma), followed by each particle solution (MOI, 0.5). After another 24 h, media were removed and fresh media were added. The following day, puromycin was added (5 mg/ml; cat. no. P8833; MilliporeSigma). Puromycin-resistant clones were selected with cloning rings at day 14, with continuous puromycin selection at all times. Cell lines were cultured in Dulbecco's modified Eagle's medium (4.5 g/l glucose) (Nacalai Tesque, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1 µg/ml puromycin (Thermo Fisher Scientific, Inc.) at 37°C under a 5% CO₂ atmosphere in an incubator. Vincristine (VCR) (cat. no. S1241; Selleck Chemicals) was dissolved in dimethyl sulfoxide (cat. no. D2950; MilliporeSigma) and stored as a 1 mM stock solution at −20°C. D-Luciferin potassium salt (D-luciferin; cat. no. 12507; AAT Bioquest, Inc.) was dissolved in phosphate-buffered saline (PBS; Nacalai Tesque, Inc.) and stored as a 15 mg/ml stock solution at −20°C. The cells were screened periodically for mycoplasma contamination using a Mycoplasma Detection kit (MycoStrip™; Invivogen).

A total of 350 fertilized eggs from a local commercial hatchery were incubated in an upright position at 37°C and 60% humidity. The day the eggs were kept in the incubator was designated as day 0 (Fig. 1A). On day 9, a small hole was made in the air chamber side of the eggshell with an egg piercer and surgical scissors after tracing the edges of the air chamber with a pencil under illumination in a darkroom. The eggshell membrane was removed with tweezers and a sterile silicon ring (inner diameter, 5 mm) was placed at the center of the CAM (Fig. 1B-F). Tumor cells were detached from culture dishes using trypsin/ethylenediaminetetraacetic acid and were counted. Cell line suspensions were resuspended in Matrigel and PBS (3:1 ratio) at 2.0×10⁶ cells/25 µl Matrigel solution and the mixture was directly pipetted into the center of the ring (Fig. 1G and I). The hole was resealed with parafilm and the eggs were placed in the incubator (Fig. 1H). Successful development of cell xenografts was confirmed by visual observation and chemiluminescent imaging with the G:BOX Chemi XRQ gel doc system (Syngene International, Ltd.). On days 12, 14, 16 and 18,50 µl D-luciferin was added to cell xenografts before chemiluminescent imaging (D-luciferin was thawed and diluted to 1:100 with Dulbecco's modified Eagle's medium before use). On days 14, 16 and 18, the xenograft tumors were resected, and the length of three sides was measured with a vernier caliper and the product of the lengths of three sides was taken as the volume (length × width × height). For the control immunostaining samples, the leg muscular tissues from one chick embryo were also resected on day 16. Chick embryos were euthanized by an overdose of pentobarbital (100 mg/kg) before resection of the xenograft tumors. The excised tumors were fixed with 100% formalin overnight at room temperature and paraffin-embedded for hematoxylin and eosin (H&E) staining. Paraffin-embedded sections (4 µm) were deparaffinized in xylene and rehydrated in ethanol after incubation on a paraffin spreading unit at 65°C for 15 min. The sections were stained with hematoxylin (6.5 min) and eosin (1 min) at room temperature, and the slides were observed under a light microscope.

Formalin-fixed paraffin-embedded chick embryo tissues and CAM assay xenografts were deparaffinized in xylene and rehydrated in ethanol. IHC was performed using an anti-human vimentin antibody (1:200; cat. no. ab16700; Abcam). Blocking, HRP micro-polymer secondary antibody incubation and DAB detection were performed using a rabbit-specific HRP/DAB Detection IHC Kit (cat. no. ab236469; Abcam). The sections were blocked with Protein Block for 10 min at room temperature and incubated with primary antibody overnight at 4°C. After blocking with 3% hydrogen peroxide for 10 min at room temperature, incubation with secondary antibody was performed for 20 min at room temperature. DAB detection was performed for 3 min at room temperature and nuclei were counterstained with 3% methyl green for 20 min at room temperature (cat. no. 12001; Muto Pure Chemicals Co. Ltd.). The slides were observed under a light microscope.

WST-8 colorimetric assays were performed using a Cell Counting Kit-8 (Dojindo Laboratories, Inc.) according to the manufacturer's instructions. RD cells were plated in a 96-well plate at a density of 5.0×10³/well in 80 µl culture media. After 24 h, dimethyl sulfoxide or VCR (1 pM-1 µM) was added to each well. Cell viability was determined every 24 h after treatment with VCR by measuring the absorbance at 450 nm using a microplate reader (Multiscan JX; Sumitomo Pharma Co., Ltd.). The dose-response curve was generated using ImageJ (National Institutes of Health; version 1.52a). The mean half-maximal inhibitory concentration (IC₅₀) was calculated based on the dose-response curve on day 3.

All data are presented by the mean ± standard error. The statistical significance of differences between samples was determined using one-way ANOVA and Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference. R² values were calculated using the least-squares method. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing; version 1.40) (31).

As shown in Fig. 1, day 0 was defined as the day when the commercial fertilized eggs were kept in the incubator and a hole was made in the eggshell on day 9. A total of 7 days after transplantation with firefly-expressing RD and Rh30 cells onto the CAM, both tumors formed a mass that could be visualized by adding D-luciferin (Fig. 2A). The xenograft tumors were resected and their volume was calculated using measurements obtained with a Vernier caliper on days 14, 16 and 18 (i.e., 5, 7 and 9 days after xenograft generation on the CAM). The RD and Rh30 cell-derived tumors increased temporally and three-dimensionally over time (Fig. 2B). In addition, pathological analysis of the tumor tissue on day 16 was performed, which confirmed that tumor cells were densely aggregated along the CAM (Fig. 2C, black arrows), with the invasion of chick blood cells with prominent nuclei (32) (Fig. 2C, white dotted line) suggesting the formation of feeding vessels from the CAM. Tumor cells were positive for human-specific vimentin and CAM was negative as confirmed by IHC (Fig. 2D), which indicated the RMS characteristics of these tumor masses. Notably, human vimentin antibodies did not cross-react with chicken tissues (Fig. S1; Data S1), indicating that IHC for human vimentin could discriminate between the human RMS-derived tissue and chicken muscular tissues.

VCR is one of the key chemotherapeutic agents for RMS and is widely used to treat RMS. For the anticancer drug screening using the RD-derived tumor established on the CAM, the present study first evaluated the sensitivity of VCR against RD cells using the WST-8 cell viability assay (Fig. 3A). The IC₅₀ of VCR was 0.114 nM and a clear decrease in the viability of RD cells was detected when they were treated with >1 nM VCR (Fig. 3B). Based on these results, the concentrations of VCR for the treatment of RD-derived tumors on the CAM were selected. Briefly, the RD cell suspension was grafted on the CAM on day 9. The implanted eggs were then divided into three groups, and 1 nM, 10 nM or 1 µM VCR was placed directly on the RD-derived tumors on day 12 (3 days after xenograft generation). Tumors were resected on day 16 for further evaluation. It was observed that the volume of resected tumors decreased in a concentration-dependent manner (Fig. 3C). Furthermore, it was observed that the range of necrotic tissue spread in a concentration-dependent manner (Fig. 3D); suggesting that the RD-derived tissue on the CAM was sensitive to VCR and the findings were similar to those obtained from the in vitro drug-sensitivity assay.