Murine RMS Cell Cultures
Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S)and 5 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). At confluency, cells were washed with Phosphate Buffered Saline (PBS) and further incubated in the medium without FBS. Freshly-made ascorbate was added daily at 50 μg/mL concentration to the medium.
Immunoblotting
For the analysis of secreted proteins, the medium was collected and proteins were precipitated with 0.2 μg/mL ammonium sulfate, centrifuged, dissolved inTris Buffered Saline(TBS) and run on the SDS-PAGE. Western blotting with antibodies against COL18A1 (anti-NC1, 1087+; anti-NC11, 1112+ from Dr.Takako Sasaki) was performed after transfer to a nitrocellulose membrane. Cells were also fixed in methanol and stained withrabbit polyclonal antibody against murine type IV collagen from PF-HR9 cells (15),rabbit polyclonal against murine NC2 domain of collagen XVI (trimeric form, native conformation) purified using antigen-coupled column from serum (Hans Peter Bächinger’s Laboratory, unpublished), antibodies against murineCOL18A1 (anti-NC1, 1087+; anti-NC11, 1112+ from Dr.Takako Sasaki) and antibody against murinefibrillin-1 (9543 from Dr. Lynn Sakai, Oregon Health & Science University).
Tissue Microarrays
Four samples of formalin-fixed paraffin embedded (FFPE) human RMS were available to include in a custom mouse model tissue microarray (TMA) (1 x aRMS, 3 x eRMS). Forty-eight murine model sarcomas were also included, representing developmental stages and genotypes including early myoblast (origin), postnatal stem cell (origin), maturing myofiber (origin), Pax3:Foxo1-expressing, Trp53 wild type or mutated and Rb1wildtype or mutated(5, 7, 10).
The TMA was stained with a standard H&E stain for histologic verification. Co-author AM classified each tumor as non-rhabdomyosarcoma and rhabdomyosarcoma. The latter was further divided into aRMS, eRMS, pleomorphic RMS and RMS not otherwise specified(RMS NOS).
Immunohistochemical (IHC) staining
All 7 IHC stains were performed on the TMA by an immunoperoxidase technique using the following commercial antibodies: anti-COL18A1 (rabbit polyclonal, 1:50), anti-PLOD1 (rabbit polyclonal, 1:100), anti-PLOD2 (rabbit polyclonal, 1:100), anti-FBN1 (mouse monoclonal, 1:400), anti-FBN2 (rabbit polyclonal, 1:400), anti-COL4A1 (rabbit polyclonal, 1:50) and anti-COL4A2 (rabbit polyclonal, 1:50).All antibodies were purchased from LifesSpan Biosciences (Seattle, WA).
Each IHC stain was scored by co-author SHG for intensity and percentage of positive cells in each individual tumor sample (some tumors were representedmore than once in the TMA). The intensity of each stain was scored accordingly: 0-negative; 1-indeterminate; 2-weak positive; 3-strong positive. The intensity and percentages were averaged for all cases containing more than one TMA fragment. Scoring of intensity was further simplified in a binary scheme where average intensity of greater than 1.5 was considered positive, and 1.5 or less was considered negative.
Statistical Analysis
Binary immunohistochemistry expression values were compared between histologic groups using the Fisher exact test. RNA expression comparisons between histologic groups were analyzed using one-way analysis of variance (ANOVA) with Tukey contrasts for multiple pairwise comparison of means andthese analyses werecarried out in log2 units.Statistical significance was set at *P <0.05, **P < 0.01, and ***P < 0.001.Error bars indicate mean ± SD or SEM. Comparisons with a p-value less than 0.05 were considered statistically significant. All statistical analyses were performed using R statistical software (version 3.3.1; R Foundation, Vienna, Austria) with the R commander graphical user interface.