Fig 1.
Cleaning, albumen removal and windowing of the eggs.
A) Upon delivery eggs were cleaned either with ethanol or water (n = 20 each). Viability of the embryos was checked at the indicated time points (EDD = embryonic development day). B) Removal of albumen using diaphanoscopy, windowing of the eggs and resealing for further incubation. C) Viability before (n = 50) and after (n = 78) implementation of the described optimization steps (** p<0.01).
Fig 2.
Effects of the cell matrix, the type of CAM pretreatment and the cell number on tumor take rates and tumor volumes.
A) Tumor volumes achieved after transplantation of 1x106 MNNG-HOS cells suspended in different matrices (n = 20 each). B) Calculated tumor take rates of MNNG-HOS cells suspended in different matrices. C) Influence of CAM pretreatment on the tumor volumes (n = 10 each). D) Calculated tumor take rates after different pretreatments. E) Influence of the number of transplanted MNNG-HOS cells on tumor volumes. F) Overall tumor take rates before (n = 25) and after (n = 78) implementation of the described optimization steps (** p<0.01).
Fig 3.
Increased tumor volumes after inoculation of cell suspensions compared to premade cell pellets.
A) Calculated tumor volumes after transplantation of MNNG-HOS cells as cell pellets or cell suspension (n = 20 each). B) Photographs of resected xenografts C) Representative hematoxilin stainings of xenografts derived by transplantation of cell pellets or cell suspensions (** p<0.01).
Fig 4.
Determination of the optimal time points for cell transplantation and tumor resection.
A) Tumor take rates achieved at different transplantation (T) and resection (R) time points. T7R16 (n = 14), T7R17 (n = 15), T8R16 (n = 12), T8R17 (n = 12), T9R16 (n = 13) and T9R17 (n = 12). B) Influence of these transplantation and resection time points on the viability of the embryos. C) Tumor volumes achieved at different transplantation and resection time points. (T = transplantation, R = resection).
Fig 5.
Suitability of the established CAM assay protocol for the analysis of osteosarcoma cell lines.
A) Tumor volumes of xenografts derived after transplantation of the following osteosarcoma cell lines: HOS143B (n = 14). MNNG HOS (n = 16), MG 63 (n = 14), Cal72 (n = 14), HOS (n = 16), SaOS (n = 16) and U2OS (n = 14). B) Tumor take rates after transplantation of these osteosarcoma cell lines.
Fig 6.
Characterization of CAM assay derived xenografts.
A) and C) ALU in situ hybridization and B) and D) CR1 in situ hybridization of CAM xenografts. Nuclei of human (ALU) and chicken (CR1) cells are stained dark purple. Sections were counterstained with methyl green (Magnification in A and B is 50-fold, in C and D 200-fold). E-L) Immunohistochemical stainings of RUNX2, PRIM1, SPARC and BMP4 in CAM xenografts and osteosarcoma tissue (Magnification 100-fold). M) and N) CD34 staining of CAM xenografts. Vessels are indicated by arrows. O) and P) Lens culinaris agglutinin staining of CAM xenografts. Vessels are indicated by arrows (Magnification in M and O is 100-fold, in N and P 200-fold).
Fig 7.
Comparison of the established CAM assay with a rat animal xenograft model.
Rat osteosarcoma cells UMR-106 were stable transfected with sFLT1 and ANG2, respectively, and transplanted to chicken eggs (wild-type n = 24, sFLT1 n = 23, ANG2 n = 23) or subcutaneously injected into the lower leg of rats (n = 6 each group). A) Tumor take rates observed after transplantation of wild-type and transfected cells. B) Cumulative tumor volumes of wild-type and transfected cells (** p<0.01 compared to wild-type cells).