Animal experiments

All animal experiments were performed after approval of the Lower Saxony State Office for Consumer Protection and Food Safety and in accordance with all relevant ethical and legal requirements. Housing and breeding of animals was performed within the animal facility at the Institute of Human Genetics, University Medical Center Göttingen. In general, mice were housed at a 12 h light-dark cycle at 20 ± 2 °C and a relative humidity of 55 ± 10 % in individually ventilated Makrolon type 2 cages located in air-flow racks. Health checks were performed daily by animal caretakers. Mice received tap water and food pellets ad libitum. All used mouse lines are listed in Tab. 15.

5.5.1 Tumor xenografts

Immunodeficient Nu/Nu mice were used for transplantation studies and were purchased from Charles River Laboratories (Wilmington, USA). These mice were housed in autoclaved and individually ventilated type 2 cages and received sterilized food pellets as well as autoclaved water ad libitum. Genotypes of Nu/Nu mice were determined by the presence (Nu/+) or absence (Nu/Nu) of hairs.

For analyses of the in vivo growth behavior of oncRAS-expressing ERMS cell lines, cells harboring the control vector or cells expressing oncRAS were transplanted subcutaneously into the left and right flank of one Nu/Nu mouse, respectively. For the transplantation experiments animals of both genders at an age between 8 and 11 weeks were used.

Ahead of transplantation ERMS cell lines were grown in cell culture to 90 % confluence. One day before transplantation the cells were split 1:3 to ensure exponential growth. On the day of transplantation cells were collected and counted (compare sections 5.1.1 and 5.1.3).

Then, 2 × 10⁶ TE617.T cells in PBS or 9 × 10⁶ RUCH-2 cells in 1:1 Matrigel:PBS in a total volume of 200 µl were transferred into pre-cooled Sub-Q syringes and kept on ice. Mice were anesthetized with 1-3 % isoflurane in a 1:1 air/oxygen mix. Then the cells were injected subcutaneously. After transplantation mice were carefully monitored and tumor size was measured every second or every day by a caliper. The tumor volume was calculated by assuming an ellipsoid shape using de formula:

Tumor volume =tumor width x height x length 2

Mice were sacrificed when a tumor reached a diameter of 1.5 cm or latest 55 days after transplantation. The tumors were isolated, their exact size and weight was documented and then they were fixed in 4 % PFA in PBS. If possible, parts of the tumor tissue were frozen on dry ice and stored at -80 °C for further experiments.

Transplantation and tumor monitoring of TE617.T KRAS xenografts was part of the doctoral thesis of N. Cuvelier ²⁹⁹.

5.5.2 Breeding of mice

For ERMS monitoring studies Ptch^del/+ Balb mice were used. These mice harbor a heterozygous deletion of exon 8 and 9 within the Ptch gene and spontaneously develop RMS, which resembles human ERMS ^137,143. These mice were bred to conditional HRasflox_B6 ³⁰³, KRasflox_B6 ³¹⁷ or NRasflox_B6 ²²⁰ mice. The latter mice are heterozygous for a floxed oncogenic Ras allele that is expressed upon Cre-mediated recombination. The resulting Ptch^del/+oncRas^fl/+ offspring were crossed to Myf5^CreER/+ mice ³⁰⁵. Since, as in humans, RMS of Ptch^del/+ Balb mice express high levels of Myf5 ^143,323, activation of the Cre recombinase in this model should result in expression of oncogenic Ras isoforms in the tumors. An overview of the breeding is displayed below (Fig. 6).

Figure 6: Breeding scheme for tumor monitoring studies

Overview of breeding strategies for generating and maintaining colonies of Ptch^del/+oncRas^fl/+Myf5^CreER mice. For details, see text.

When analyzing potential oncRas-dependent effects on ERMS precursor lesions the parental Myf5^CreER/+ mice were on a mixed C57BL/6 x BALB/c background, whereas those used for the analyses of oncRas-dependent effects in established ERMS were incipient-congenic and had been backcrossed 8 generations to BALB/c. Backcrossing had been done because the BALB/c background confers high susceptibility to ERMS ^324,325.

5.5.3 Identification of mice

Mice were weaned at the age of 3 weeks and were numbered by ear clipping. Younger mice, which still were not weaned, were labeled by a tattoo code on their paws. At the same time tail tip biopsies were taken and were used for genotyping of the respective mice (see section 5.2.3.1).

5.5.4 Tamoxifen application

To induce recombination of the floxed Ras loci in Ptch^del/+oncRas^fl/+Myf5^CreER/+ mice 1 mg of tamoxifen in 100 µl solvent was injected intraperitoneally (i.p.) for 5 consecutive days resulting in a total dose of 5 mg tamoxifen per mouse. Tamoxifen was prepared by mixing 200 mg tamoxifen with 20 ml Sunflower seed oil and 800 µl pure EtOH. The solution without tamoxifen served as solvent control. The solutions were aliquoted into syringes and frozen at -20 °C until use.

5.5.5 Tissue isolation

Mice were euthanized by CO₂ or isoflurane narcosis and subsequent cervical dislocation. All tumors, skeletal muscles from the leg and back, cysts or suspicious tissues were taken and washed with PBS. If possible, tumors were weighed and measured using a caliper. Tumor tissue, cysts and suspicious tissues were fixed in 4 % PFA in PBS and were embedded in paraffin for immunohistochemical stainings (see section 5.4). The skeletal muscles and parts of the tumor samples were frozen on dry ice or snap frozen in liquid nitrogen and stored at -80 °C for protein and gene expression analyses.

5.5.6 Testing of the Myf5^CreER/+ deleter

To check the activity of the Myf5^CreER/+ deleter in ERMS, Ptch^del/+Myf5^CreER/+R26R^+/- reporter mice were generated ²⁹⁹. Tumor bearing mice were injected with 1 mg tamoxifen on 5 consecutive days and sacrificed 1 or 5 weeks thereafter. X-Gal stainings were performed on frozen sections of brain, heart, intestine, skeletal muscle and ERMS as described in section 2.4.3.

5.5.7 Tumor monitoring: induction of oncRas in ERMS precursor lesions

At the age of 4 weeks mice were injected with 1 mg. of tamoxifen i.p for 5 consecutive days to induce cre-mediated recombination at the respective Ras loci. Mice were monitored once a week for palpable tumors and were visually controlled for other abnormalities until the age of 200 days. The appearance of palpable tumors was recorded. Animals with poor general condition or those carrying tumors exceeding a size of 1.5 cm were sacrificed. Tissue was isolated as described in section 5.5.5. An overview of the study design is shown in Fig. 7.

Figure 7: Study design focusing on induction of Ras mutations in ERMS precursor lesions

Ptch^del/+ or Ptch^del/+oncRas^fl/+Myf5^CreER/+ mice at the age of 4 weeks were injected with tamoxifen for 5 consecutive days or left untreated. Afterwards mice were monitored weekly until the age of 200 days. For details, see text.

The studies were performed using tamoxifen-treated Ptch^del/+HRas^fl/+Myf5^CreER/+, Ptch^del/+KRas^fl/+Myf5^CreER/+ and Ptch^del/+NRas^fl/+Myf5^CreER/+ mice. Untreated siblings of the same genotype served as control. In order to analyze the effect of tamoxifen itself on tumor growth, ERMS-growth of tamoxifen- and untreated Ptch^del/+ mice from the same breeding were used.

Part of these experiments have been done by N. Cuvelier and are described in her doctoral thesis ²⁹⁹. Here, the already generated data were re-analyzed and the collected material was used for further analyses. Thus, the genotypes of each mouse were verified by a second round of genotyping PCR (see section 5.2.3.1). For this purpose, frozen and embedded tissue samples were used. In addition, recombination assays were performed using frozen or embedded samples from skeletal muscle and ERMS (see section 5.2.2). Finally, the study using Ptch^del/+NRas^fl/+Myf5^CreER/+ mice was completely repeated.

5.5.8 Tumor monitoring: induction of oncRas in already established ERMS After weaning, mice were monitored once a week for palpable ERMS and were visually controlled for other abnormalities. Tumors occurred mainly at the extremities and the lower back and were observed until they reached a diameter of approximately 0.5 cm. Then the mice were subjected to micro computed tomography (µCT) measurement to determine the exact tumor volume (see section 5.5.9). Afterwards the mice were injected with 1 mg of tamoxifen for 5 consecutive days to induce the cre-mediated recombination at the Ras loci.

After tamoxifen induction the mice were further monitored for changes in ERMS growth and general health conditions. Seven weeks after onset of the tamoxifen treatment, the mice underwent a second µCT measurement, which was defined as the end point of the study.

Animals were euthanized, and tissue samples were taken as described in section 5.5.5. An overview of the study design is shown in Fig. 8.

Figure 8: Study design focusing on induction of Ras mutations in established ERMS

After weaning, Ptch^del/+ or Ptch^del/+oncRas^fl/+Myf5^CreER/+ mice were monitored for palpable tumors. After tumor detection, mice were subjected to micro computed tomography (µCT) measurement and were injected with tamoxifen or solvent for 5 consecutive days. The mice were monitored for further 7 weeks and then subjected to a second µCT measurement. For more details, see text.

The study was performed with Ptch^del/+HRas^fl/+Myf5^CreER/+, Ptch^del/+KRas^fl/+Myf5^CreER/+ and Ptch^del/+NRas^fl/ Myf5^CreER/+ mice. Mice were injected with 1 mg of tamoxifen i.p. for 5 consecutive days to induce cre-mediated recombination or with solvent as a control. In addition, tamoxifen-treated or solvent-treated Ptch^del/+ siblings from each breeding cohort served as controls.

5.5.9 Tumor volume measurement by µCT

A low dose in vivo μCT (QuantumFX) was used to measure the tumor volume in living animals. The data acquisition was performed at 90 kVp tube voltage, 200 μA tube current and with 2 min total acquisition time. Mice were anesthetized with 1-3 % isoflurane in a 1:1 air/oxygen mix. Then the contrast agent Imeron 300 (5 ml/kg) was injected intravenously (i.v.) into the tail vain or into the retro-bulbar venous plexus. Approximately 30 sec later, the imaging was performed. Data sets were reconstructed with a voxel size of 80 μm and analyzed. Size of each tumor (precisely tumor width, height and length) at onset and end of the study was determined using simple viewer software.

The tumor volume was calculated by assuming an ellipsoid shape using the formula:

Tumor volume =tumor width x height x length 2

For data evaluation logarithmic values of the calculated tumor volume were plotted.