Establishment of a patient-derived orthotopic osteosarcoma mouse model

Tumor tissue samples were obtained from a patient with relapsed high-grade OS [11]. The study was approved by the ethics committee of the Medical Faculty of the University of Heidelberg . After surgical resection and histopathological confirmation of the OS, the samples are stored in NaCl 0.9% on ice. A portion of the sample was xenotransplanted immediately (fraction 1, see below). The remaining part of the samples was split into three further fractions (fraction 2 to 4) as follows: fraction 2 was put into DMEM medium, 10% DMSO (Dimethylsulfoxide), 20% FCS (fetal calf serum) and 1% NEAA (Non Essential Amino Acid) under sterile conditions, was frozen stepwise to −80°C and then transferred to liquid nitrogen. Fraction 3 was snap frozen and stored in liquid nitrogen. The fourth fraction was brought into cell culture in DMEM medium, 10% FCS and 1% NEAA (Non Essential Amino Acid) under sterile conditions and was cultured under standard conditions.

Fraction 1 of the human OS tissue was inserted into ten week old athymic BALB/c Nu/Nu mice (Charles River, Wilmington, Mass.) as follows:

Tumor samples were cut into 1 × 1 × 1 mm³ pieces. The right tibia of 6 mice was opened in the central (medial) part by drilling with a dental drill with a diameter of 0.5 mm to insert one tumor fragment in contact with the bone marrow. The anesthesia of mice was performed with isoflurane inhalation (1,5-3,5 Vol% per liter oxygen). After, the surgical wound was sutured. The whole procedure took about 10 min per mouse. The percentage of success was 92%. The mice were maintained under specific pathogen-free conditions, food and water were supplied ad libitum. Housing and all procedures involving the mice were performed according to the protocols approved by the German Cancer Research Center institutional animal care and use committee and by the local responsible government department (Regierungspräsidium Karlsruhe). Mice were observed daily for tumor growth by palpation and inspection.

MicroCT scans were acquired using an Inveon PET/SPECT/CT system (Siemens Medical Solutions, Knoxville, TN, USA). A 12 minute and 30 seconds CT scan was performed with parameter settings: 360 rotation steps, tube voltage 50 kV, tube current 500 μA, binning 1 and exposure time 400 ms. The pixel size was 0.0143 × 0.0143 × 0.0143 mm. Image reconstruction was performed using the conventional Inveon Research Workplace software 4.0. The reconstruction filter was Shepp-Logan with a downsample factor of 2. MRI was performed using a small animal MRI scanner (Bruker Icon, 1 Tesla, Ettlingen, Germany). Because sufficient contrast for tumor discrimination could not be achieved using i.p. injection of Magnevist (Bayer Schering Pharma) at a concentration of 0.5 mmol/ml for contrast enhanced T1w mri, tumor growth was visualized using T2w images (TE = 100 ms, TR = 2842 ms, FA = 180, voxel size = 0.16x0,16x1 mm³, FOV 30x30 mm, matrix size = 192x192, averages = 4). Lesion sizes were measured using T2 weighed (T2w) images and were found to match with tumor sizes obtained via μCT (Figure 1) and with weighed resected tumor tissues (data not shown). Measurements were performed by manual segmentation of the mass using Bruker ParaVision software. Mice were immobilized during the imaging procedures via Sevofluran inhalation narcosis at a dosage of 4% in air.

“Sufficient tumor growth” was defined as tumors in 3 of 6 mice attaining 1500 mm³. Each tumor was then removed and processed for the following analyses: (1) histopathological examination (2) array-based comparative genomic hybridization (aCGH) (see below), (3) orthotopic re-transplantation into 6 further mice, (4) re-cultivation in cell culture. One of the six tumors was cut into 1 × 1 × 1 mm³ fragments and transferred twice into 6 mice per passage (designated as passage P1 to P3). In the first passage (P1) three of six mice, in the second passage (P2) four of six mice and in the third passage (P3) six of six mice developed an orthotopic OS, which were frozen as outlined above for further analysis.

The original tumor tissue was collected and directly cultivated into medium (P1*). After 26 days, cells were split and 3.75 × 10⁶ cells were injected subcutaneously into the left flank of six athymic BALB/c Nu/Nu mice (Charles River, Wilmington, Mass.). After three weeks, four of six mice developed a tumor mass which was removed when reaching a volume of 1500 mm³. Tumor fragments were disaggregated and passaged again (P2*), split after 20 days and injected into the flank of six further mice. After two weeks, all mice developed a tumor mass, which was removed, disaggregated and re-passaged (P3*). Each resected xenograft was first disaggregated into a cell line and then cells were re-injected. The cell line “passages” means sequentially passaged cells subcutaneously through the mouse and not just in culture. The third passage was used for further analysis. Cells were tested and proved to be free of mycoplasma, viral as well as cell contamination using in-house Multiplex cell Contamination testing (McCT) service [12]. Genetic stability of the cells was compared to the original human tumor sample at each passage by aCGH (see below).

Genomic DNA from fresh frozen tissue was isolated using standard phenol-chloroform extraction. Genomic DNA from cultured cells was isolated using the Blood and Cell Culture Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Selection of genomic clones, isolation of BAC DNA, performance of degenerate oligonucleotide primer-PCR, and preparation of microarrays were performed as previously described [13]. Labeling, hybridization, and washing procedure were performed as reported previously [14]. Array- (or matrix-) CGH was carried out as described, gains were defined as copy number imbalances log2 ratio > 0,25 and losses log2 ratio < −0,25 [15,16].

Histology was performed nine weeks after tumor implantation into the mice. 3 μm thick whole tumor sections were cut from formalin-fixed, paraffin-embedded (FFPE) tissue blocks of all tumors. Sections were stained with hematoxylin-eosin (HE; Sigma-Aldrich, St Louis, USA). Histological comparisons were performed by two pathologists (AS, WW) using conventional HE-stainings as well as PAS and Masson Trichrom staining.