Additional file 8 of Capmatinib is an effective treatment for MET-fusion driven pediatric high-grade glioma and synergizes with radiotherapy

Additional file 8: Supplementary Fig.4. Characterization of human tumor samples and derived cell cultures.a, H&E staining of the representative sections from the initial tumor (TRIM24-MET-i). The upper panel shows areas in the parietal region, the lower panel in the occipital region, both displaying variable histologies, compact (left) and infiltrative (right) tumor cells. Scale bar=50 µm. b, H&E staining of four representative sections from the recurrent tumor (TRIM24-MET-r), showing diverse cytomorphology and growth patterns. Scale bar=50µm. c, Initial (TRIM24-MET-i) and recurrent tumor (TRIM24-MET-r), showing punctuated GFAP expression (upper panel). Ki-67 staining indicates that most tumor cells are actively proliferating (the lower panel). Scale=50µm. d, Sanger sequencing results of RT-PCR amplicons, demonstrating the TRIM24-MET fusion junction in the initial (TRIM24-MET-i) and recurrent (TRIM24-MET-r) tumor samples. e, RT-QPCR data demonstrating the relative expression levels (normalized to GAPDH) of the TRIM24 N-terminal region, c-MET N-terminal region, MET-kinase domain and the TRIM24-MET fusion in TRIM24-MET-i and TRIM24-MET-r cells as well as in control pHGG tumor cells without TRIM24-MET fusions (SJHGGx6c, SJDIPGx37c). f, Immunoprecipitation (IP)-Western blots confirming the existence of TRIM24-MET protein in initial (TRIM24-MET-i) and recurrent tumor (TRIM24-MET-r) cells. Pro-MET (pM=170kD) and the mature c-MET protein (M=140kD) were identified in SJHGGx6c cells (c-MET-expressing tumor cells), and the TRIM24-MET fusion (TM=117kD) in TRIM24-MET-i and TRIM24-MET-r cells. SJDIPGx37c cells were used as a negative control of endogenous c-MET expression. g, IP-Western blot showing existence of TRIM24-MET. The same protein samples in “D” were blotted with a rabbit poly clonal antibody recognizing the N-terminus of TRIM24. The Western blot identifies the TRIM24-MET protein in TRIM24-MET-i and TRIM24-MET-r cells but not in control cells (SJHGGx6c and SJDIPGx37c). h, In vitro proliferation rate of initial tumor cells (TRIM24-MET-i) and recurrent tumor cells (TRIM24-MET-r), based on luminescent cell viability assay. Values were normalized to day 0.