Fig 1.
DIPG xenograft tumors are produced in the CAM model following inoculation with patient-derived cell lines.
(A) Experimental procedures follow the corresponding timeline where 1x106 DIPG cells are inoculated onto the CAM on embryonic development day 9. DIPGIV (B) and DIPGXIIIp* (C) tumors (dashed perimeter and black arrow) are visible within 48 hours of inoculation. Ultrasound visualization using RF Power Doppler Mode of DIPGIV (D) and DIPGXIIIp* (E) tumors reveals that the tumors grow below the CAM surface with blood flow visible around the edges of tumor and within CAM (red fluorescence: active blood flow). DIPGIV tumor growth can be appreciated through visualization of increased GFP signal of tagged tumor cells from day 11 (F) to day 13 (G) to day 15 (H).
Fig 2.
DIPG CAM tumors maintain molecular features of native tumor and rodent xenografts.
(A) Hemoxylin and Eosin (H&E) staining of DIPGXIIIp* rodent, DIPGXIIIp* CAM and DIPGIV CAM tumors show tumor cell growth and invasion. Immunohistochemistry (IHC) staining shows positive H3K27M mutation status (B) and reduction of H3K27 trimethylation (C) for all tumors. All images were taken at 20X magnification.
Fig 3.
Volumetric analysis of DIPGXIIIp* and DIPGIV CAM tumors following drug treatment.
DIPGXIIIp* (A) and DIPGIV (B) CAM tumors were treated with alisertib, bortezomib, MSN1-Leu, panobinostat, ponatinib and bevacizumab on embryonic development days 11, 13 and 15. Tumor volume was determined following 3-D ultrasound on embryonic development day 16. Vehicle is 0.5% DMSO in sterile PBS. PBS control is 100% sterile PBS. Differences were determined with one-way ANOVA and Dunnett’s test for multiple comparisons where P<0.05 is considered significant. P<0.05 = *, P<0.01 = **, P<0.001 = ***, P<0.0001 = ****.
Fig 4.
Analysis of tumor vascularity of DIPGXIIIp* and DIPGIV CAM tumors following drug treatment.
DIPGXIIIp* (A) and DIPGIV (B) CAM tumors were treated with alisertib, bortezomib, MSN1-Leu, panobinostat, ponatinib and bevacizumab on embryonic development days 11, 13 and 15. Tumor vascularity was assessed following 3-D ultrasound on embryonic development day 16. Vehicle is 0.5% DMSO in sterile PBS. PBS control is 100% sterile PBS. Differences were determined with one-way ANOVA and Dunnett’s test for multiple comparisons where P<0.05 is considered significant. P<0.05 = *, P<0.01 = **, P<0.001 = ***, P<0.0001 = ****.
Fig 5.
Volumetric and vasculature analysis of DIPG CAM tumors.
DIPGXIIIp* and DIPGIV CAM tumors underwent a single dose of 2Grey radiation treatment on embryonic development day 13 and were analyzed by 3-D ultrasound on embryonic development 16. DIPGXIIIp* and DIPGIV CAM tumors were analyzed for differences in volume (A, B, respectively) and vascularity (C, D, respectively). Differences were analyzed by Student’s T-Test where P< 0.05. * = P<0.05, ** = P<0.001, *** = P<0.0001.