Fig 2: PDGFRA-induced zebrafish MPNST tumors exhibit similar histology to that of human MPNSTs. (a–c) The histopathology after H&E staining of the zebrafish MPNSTs was identical in the nf1a+/−; nf1b−/−; p53m/m zebrafish when either PDGFRA wild-type or mutant proteins were overexpressed. In each genotype, the zebrafish MPNST histopathology was very similar to that of human MPNSTs. The zebrafish MPNSTs comprise spindle cells that stack into short fascicles, typically with a whirling organization pattern. MPNST cells have long serpentine-like nuclei and are spindle-shaped. (d–i) Markers of cells of neural crest origin were expressed by the zebrafish MPNST tumors, such as S100 (d–f) and Sox10 (g–i). S100 was detected in the cell membrane and nucleus, whereas Sox10 was detected in the nucleus. Scale bar=200 μm.

Fig 3: Overexpression of the PDGFRA wild-type gene activates AKT and ERK at levels optimal for tumorigenicity. (a) Western blot analysis for AKT and ERK1/2 activation in protein lysates prepared from tumors of mCherry control, PDGFRA wild-type, and PDGFRA mutant fish. Similar levels of p-AKT and p-ERK1/2 were detected in PDGFRA wild-type tumors as compared with mCherry control tumors; however, tumors induced by the PDGFRA mutant showed increased levels of p-AKT and p-ERK1/2. Proteins were detected by stripping the membrane and reprobing. Arrows denote protein size (kDa). (b and c) Statistical analysis of mean±s.d. of p-AKT/total AKT and p-ERK/total AKT using ImageJ. Asterisks indicate statistical significance (**P<0.005). (d, g and j) Representative images of SA-β galactosidase-stained transgenic fish embryos of mCherry control (n=12), PDGFRA wild-type (n=36) and PDGFRA mutant (n=32) in the nf1a+/−; nf1b−/−; p53m/m background at 4 d.p.f. The boxed areas were magnified at the right bottom corner of each panel. (e, h and k) Transverse cryosections through the spinal cord of embryos at 4 d.p.f., with mCherry and DAPI (4',6-diamidino-2-phenylindole; f, i and l).

Fig 4: Sunitinib promotes apoptosis of primary MPNST progression in transgenic fish expressing wild-type PDGFRA. (a–d) Histopathology after H&E staining of the DMSO- (a and b) or sunitinib- (c and d) treated MPNSTs. Black arrowheads show areas of MPNSTs (black scale bar=5 mm). Proliferating cell nuclear antigen (e–h) and cleaved caspase-3 (i–l) stains of each condition for proliferation and apoptosis analysis, respectively. The right upper corner of each panel shows magnified stained areas.

Fig 5: (A) Immunohistochemistry for α-SMA, PDPN, vimentin, PDGFRA, CD73, and CD31 in metastatic lymph node (400×). α-SMA, PDPN, vimentin, PDGFRA, and CD73 stained stromal cells in metastatic lymph node. CD31 was not stained in stromal cells. (B) Immunofluorescence images of the stroma of a metastatic lymph node (400×), stained for α-SMA (green), PDPN (red), and DAPI (blue). Dual staining for PDPN and α-SMA confirms the co-expression of the two proteins in the FRCs of metastatic lymph node. α-SMA: α-smooth muscle actin; PDPN: podoplanin; PDGFRA: platelet-derived growth factor receptor α; DAPI: 4’,6-Diamidino-2-phenylindole; FRCs: fibroblastic reticular cells.