Rhabdomyosarcoma (RMS) is the most common highly malignant pediatric soft tissue sarcoma. While recent multidisciplinary treatments have improved the 5-year survival rate of low/intermediate-risk patients to 70–90%, there are various complications that arise due to treatment-related toxicities. Immunodeficient mice-derived xenograft models have been widely used in cancer drug research; however, these models have some limitations, including i) they are time-consuming and expensive, ii) their use needs to be approved by animal experimental ethics committees, and iii) the inability to visualize where tumor cells or tissues were engrafted. The present study performed a chorioallantoic membrane (CAM) assay in fertilized chicken eggs, which is time-saving, simple, and easy to standardize and handle because of the high vascularization and the immature immune system of the fertilized eggs. The present study aimed to examine the usability of the CAM assay as a novel therapeutic model for the development of precision medicine for pediatric cancer. A protocol was developed for constructing cell line-derived xenograft (CDX) models using a CAM assay by transplanting RMS cells on the CAM. It was then examined as to whether these CDX models could be used as therapeutic drug evaluation models using vincristine (VCR) and human RMS cell lines. After grafting and culturing the RMS cell suspension on the CAM, three-dimensional proliferation over time was observed visually and by comparing volumes. VCR reduced the size of the RMS tumor on the CAM in a dose-dependent manner. Currently, treatment strategies based on patient-specific oncogenic backgrounds have not been adequately developed in the field of pediatric cancer. The establishment of a CDX model with the CAM assay may lead to the advancement of precision medicine and help formulate novel therapeutic strategies for intractable pediatric cancer.
Drug sensitivity and the severity of side effects vary from patient to patient; therefore, it is necessary to develop precision medicine designed to provide the optimal type and amount of treatment for each patient. The development of precision medicine based on patient genetic information has markedly improved therapeutic methods for some types of adult cancer, such as chronic myelocytic leukemia and breast cancer; however, precision medicine for pediatric cancer has not been fully developed because of its rarity and diversity (
Immunodeficient mouse models, cell-derived xenograft (CDX) models and patient-derived xenograft (PDX) models have been commonly used for cancer drug research; however, these murine models have some weaknesses: i) Establishment and maintenance of a PDX model is time-consuming and a high cost is incurred to manage its quality, ii) experimental procedures should be undertaken to reduce the number of animals used per study or to refine procedures to improve animal welfare, and iii) it is impossible to visualize the site of xenografts (
The chorioallantoic membrane (CAM) is an extraembryonic membrane consisting of chorion and allantois in fertilized chicken eggs. The CAM assay has been widely used to study angiogenesis, infiltration and metastasis using human-, rodent- and bird-derived xenografts (
Rhabdomyosarcoma (RMS) is the most common type of highly malignant pediatric soft tissue sarcoma in the United States (≥50% of pediatric soft tissue sarcomas) (
Progress in multidisciplinary treatment has improved the 5-year survival rate of low/intermediate-risk patients to 70–90%, but that of high-risk patients remains <30% in the United States (
The present study aimed to explore whether the CAM assay is a novel treatment model that could contribute to precision medicine for pediatric cancer. As a first step, this study aimed to establish a protocol for the CAM assay with transplantation of RMS cells on the CAM and examined how to evaluate the effect of anticancer drugs.
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) (
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 (
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) (
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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 (
The present study established a CDX model using a CAM assay with the human ERMS cell line, RD, and the ARMS cell line, Rh30. The formation of a three-dimensional tumor mass on the CAM was confirmed by visual observation and the temporal multiplication of grafted cells by calculating the volume using a Vernier caliper. Moreover, pathological assessments confirmed that transplanted cells gathered and formed tumor tissue along the CAM. Some chick red blood cells infiltrated the tissue, indicating that the transplanted cells were nourished by the chick host and that the CAM assay was helpful in the establishment of CDX models. Moreover, tumors on the CAM were sensitive to VCR in a concentration-dependent manner, confirming the utility of the CAM assay as a therapeutic model both three-dimensionally and histologically. Therefore, the CAM assay could be useful to determine the sensitivity of anticancer drugs.
The CAM assay has been widely used in the research field of oncological morphology; however, the protocol of tumor engraftment is not standardized. Some researchers have placed the fertilized eggs horizontally (
Although we initially tried to assess how grafted RMS cells multiplied on the CAM through fluorescence analysis using luciferase-transgenic cell lines, we shifted to calculating the volume of resected tumors with measurements using Vernier calipers because the results of this assessment were revealed to be consistent with those of H&E staining. Fluorescence analysis was limited to two dimensions in the range visible through the hole in the eggshell, and was easily influenced by tumor crookedness and movement under the eggshell related to embryo motion.
The CAM assay has a number of advantages over mouse models. First, it is time- and cost-effective, whereas mouse models require long observation periods (weeks to months) (
In the field of pediatric cancer, the use of precision medicine has made less progress than in adult cancer, and a remedy based on the oncogenic background of patients has not yet been established. Previous studies have applied the CAM assay as an alternative model for RMS studies (
The present study has some limitations. First, only one cell line was examined for each ERMS and ARMS; in the future, we aim to examine other RMS cell lines. Second, the less damaging method of administering VCR via intravenous injection could not adequately be examined.
In conclusion, the present study established a CAM assay protocol using human RMS cell lines and confirmed the formation of a three-dimensional tumor mass on the CAM. Moreover, the anticancer efficacy of VCR was demonstrated on established human RMS CDX models. The CAM assay may therefore be useful as both a CDX model and a therapeutic model. In the future, we aim to establish CDX models using other RMS cell lines and PDX models using tumor tissue resected from mouse CDX models or patients, and to examine the utility of the models.
The authors would like to thank Dr Peter J. Houghton, (Greehey Children's Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX, USA) for providing the Rh30 cell line. The authors are also grateful to Dr Satoshi Miyagaki, Dr Akihiro Nishida and Ms. Mami Kotoura (Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan) for teaching us the experimental techniques.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
CS performed the experiments. KK and TI designed this study and confirmed the authenticity of all the raw data. CS, KK, HY, MM, SY, KT and HH interpreted and processed the experimental data, and performed the analysis. TN contributed to the original technical methods using CAM. CS, KK, HY, MM, SY, KT, TN, HH and TI discussed the results and contributed to the final manuscript. All authors read and approved the final manuscript.
This study was approved by the Faculty of Science Ethics Committee of the Kyoto Prefectural University of Medicine (approval no. #2019-35).
Not applicable.
The authors declare that they have no competing interests.
alveolar RMS
chorioallantoic membrane
cell line-derived xenograft
D-luciferin potassium salt
embryonal RMS
hematoxylin and eosin
immunohistochemistry
phosphate-buffered saline
patient-derived xenograft
rhabdomyosarcoma
Diagram of cell-derived xenograft model generation using the CAM assay. (A) Day 0. Commercial fertilized eggs were placed upright in an incubator at 37°C and 60% humidity. (B) Day 9. Photographs of steps (C-H). Prior to step C, the edges of the air chamber were traced with a pencil while illuminating the eggs in the darkroom. (C) A hole was made on top of the egg with an egg piercer. (D) Scissors were used to cut the shell along the edge of the air chamber. (E) Eggshell membrane was removed with tweezers. (F) A silicon ring was placed on the CAM. (G) A cell suspension (25 µl) was grafted onto the CAM. (H) After cell inoculation, the window in the egg was tightly sealed using parafilm and the eggs were returned to the incubator. (I) Cell suspension was prepared by detaching tumor cells from culture dishes using trypsin/ethylenediaminetetraacetic acid and counting them. The cells were resuspended in Matrigel and PBS (3:1 ratio) at 2.0×106 cells/25 µl Matrigel solution. CAM, chorioallantoic membrane; PBS, phosphate-buffered saline.
Establishment of a cell-derived xenograft model on the CAM using the RMS cell lines, RD and Rh30. (A) Tumor formed on the CAM on day 16 (7 days after transplantation of RD or Rh30 cells). Images on the left were captured on a clean bench, whereas images on the right were observed using the G:BOX Chemi XRQ gel doc system following the addition of luciferin. (B) Temporal changes in tumor volume. Tumors were resected on days 14, 16 and 18, and the volume was calculated using Vernier caliper measurements. (C) Hematoxylin and eosin staining of the resected tumors on day 16 (left, ×40 magnification; right, ×200 magnification). Accumulation of cells and formation of RMS tissue along the CAM (black arrow), and infiltration of some chick red blood cells into the tissue (inside white dotted line) were observed. (D) Immunohistochemical staining of anti-human vimentin in the tumor tissue (left, ×40 magnification; right, ×200 magnification). Counterstaining of sections was performed with methyl green. These results indicated that the resected tumor consisted of human RMS cells transplanted on day 9. CAM, chorioallantoic membrane; RMS, rhabdomyosarcoma.
Administration of VCR resulted in a reduction in tumor volume on the CAM. (A and B) Results of the WST-8 assay. (A) Cell viability curve (VCR concentration, 0 pM-1 µM). (B) Viability inhibition curve on day 3. Half-maximal inhibitory concentration, 0.114 nM (dotted line). (C) Changes in tumor volume due to different concentrations of VCR administration. RD cells were grafted on the CAM on day 9 and 100 µl VCR was administered to each tumor on day 12. On day 16, tumors were resected and volumes were calculated. Data are presented as the mean ± standard error of three independent experiments. Data were analyzed using one-way ANOVA (P=0.0306), followed by Dunnett's post hoc test. *P<0.05 vs. control. (D) Hematoxylin and eosin staining of resected tumors. Necrotic area expansion was VCR concentration-dependent; necrotic areas are outlined with a white dotted line. Xenografts were treated with (a and b) 1 nM, (c and d) 10 nM and (e and f) 1 µM VCR. (a, c and e) ×40 magnification; (b, d and f) ×200 magnification. CAM, chorioallantoic membrane; VCR, vincristine.