Immunohistochemistry (IHC) and immunoreactivity scoring

For IHC, 4-μm FFPE sections were cut and antigen retrieval was carried out by heat treatment with Target Retrieval Solution (S1699, Agilent Technologies). The slides were stained with either polyclonal anti-PRC1 antibody raised in rabbit (1:200, 15617-1-AP, Proteintech), monoclonal anti-γ-H2AX raised in rabbit (1:8000, ab81299, Abcam), or with monoclonal anti-Ki-67 raised in rabbit (1:200, 275R-15, Cell Marque) for 60 min at RT, followed by a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody (ImmPRESS Reagent Kit, MP-7401, Vector Laboratories) or for 120 min at RT followed by Dako REAL™ Detection kit, Alkaline Phosphatase/RED Rabbit/Mouse (K5005, Dako). AEC-Plus (K3461, Agilent Technologies) or Substrate RED (K5005, Dako) was used as chromogen in the corresponding detection system. Samples were counterstained with hematoxylin (H-3401, Vector Laboratories and T865.1 Roth). For cleaved caspase-3 (CC3) staining, antigen retrieval was carried out by heat treatment with Target Retrieval Solution Citrate pH6 (S2369, Agilent Technologies). Slides were incubated with the polyclonal CC3 primary antibody raised in rabbit (1:100; 9661, Cell Signaling) for 60 min at RT followed by ImmPRESS Reagent Kit (MP-7401, Vector Laboratories) or for 120 min at RT followed by Dako REAL™ Detection kit, Alkaline Phosphatase/RED Rabbit/Mouse (K5005, Dako). DAB+(K3468, Agilent Technologies) or Substrate RED (K5005, Dako) was used as chromogen in the corresponding detection system and hematoxylin for counterstaining. For human mitochondrial staining, antigen retrieval was carried out by heat treatment with Pro Taqs II Antigen Enhancer (401602192, Quartett). Slides were incubated with the monoclonal anti-human mitochondria antibody raised in mouse (1:1,000; ab92824, Abcam) for 60 min at RT followed by ImmPRESS Reagent Kit (MP-7402, Vector Laboratories). AEC-Plus (K3461, Agilent Technologies) was used as the chromogen. For CD99 staining, slides were stained with monoclonal anti-human CD99 antibody raised in mouse (1:40, ab8855, Abcam) for 32 min using the ultraView detection kit in a VENTANA BenchMark system (Roche, Basel, Switzerland), and counterstained with hematoxylin.

To assess tissue integrity and for detection of mitotic defects, e.g., monster’ cells, FFPE blocks of EwS xenografts were stained with hematoxylin and eosin (H&E). Evaluation of PRC1 immunoreactivity was carried out in analogy to the scoring of hormone receptor Immune Reactive Score (IRS) ranging from 0–12. This modified IRS scoring scheme has been adapted to EwS and has been described and validated previously^{12,18,45–47}. The percentage of cells with expression of the given antigen was scored and classified in five grades (grade 0 = 0–19%, grade 1 = 20–39%, grade 2 = 40–59%, grade 3 = 60–79%, and grade 4 = 80–100%). In addition, the intensity of marker immunoreactivity was determined (grade 0 = none, grade 1 = low, grade 2 = moderate, and grade 3 = strong). The product of these two grades defined the final IRS. Evaluation of Ki-67, CC3, and γ-H2AX immunoreactivity was quantified based on their positive staining percentage of cells per high-power field (HPF). Final scores were determined by examination of 5–15 HPFs of at least one section per sample.