Article Figures & Data
Figures
- Fig. 1.
In vivo growth, histologic, and immunohistochemical characteristics of orthotopic medulloblastoma xenograft models in SCID mice. A, representative images showing the time course of in vivo growth of ICb-D283 xenografts. Mice injected with 105 tumor cells in the right cerebellar were euthanized (five mice each) at 2, 4, and 6 weeks postinjection and at the end of experiment when they developed neurologic deficits. The mouse brains were then paraffin embedded, serially sectioned, and stained with H&E (a-d). B, histologic features of intracerebellar xenografts at 2 and 4 weeks postinjection. For ICb-D283 tumors, early invasion (e, inset), the small round blue cells (*), larger, more aggressive-appearing cells (**; f), and cell wrapping (arrow; g) were shown. For ICb-DAOY tumors, early subarachnoidal spread (arrow head; h), subsequent deep invasion into the surrounding normal folium (white arrows; i), and striking cellular polymorphism were presented. For ICb-MED-1, early invasion into normal tissues (k) and more uniform morphology (l and m) could be seen. Magnification, 4 × 10 (e, h, and k), 10 × 10 (f, I, and l), and 10 × 40 (g, j, and m). C, immunohistochemical analysis of AcH3 and neuronal marker MAP-2 expression induced by valproic acid. Five mice from each model were euthanized at the end of valproic acid treatment (4 weeks post tumor injection; VPA-Treated) and compared with the untreated mice (Control). Magnification, 10 × 40.
- Fig. 2.
Log-rank survival analysis of mice bearing intracerebellar medulloblastoma xenografts from D283, DAOY, and MED-1 cell lines. A, effect of cell number on animal survival times. For each cell line, three different numbers of tumor cells (103, 104, and 105) were suspended in 2 μL of growth medium and injected into the right cerebellum of five Rag2 SCID mice, respectively. P < 0.05, for all group comparisons (103/104, 104/105, 103/105) except for comparison between mice injected with 104 and 105 of D283 cells. B, effect of valproic acid treatment on animal survival times. Each mouse received injection of 105 tumor cells in the right cerebellum. Two weeks later, when solid tumors are formed, systemic treatment with valproic acid was started for a duration of 14 days (blue bar) using two s.c. osmotic minipumps that release valproic acid (600 mg/mL) at a constant rate of 1 μL/h (600 μg/h). The control group received no treatment.
- Fig. 3.
In vivo antitumor activities of valproic acid. Mice bearing intracerebellar xenografts were sacrificed at the end of valproic acid treatment (4 weeks post tumor injection) and compared with the untreated group. A, suppression of tumor growth. Tumor size was determined by measuring the maximum cross-sectional areas in at least three consecutive sections for each of the tumors (n = 5 per group). Tumor sizes in the treated groups were normalized with the corresponding control groups. B and D, inhibition of cell proliferation. The proliferative cells identified by immunohistochemical staining with Ki-67 (B) were counted in digitally captured images under high-power (10 × 40) magnification and graphed as the percentage of total cells (D). C and E, induction of apoptosis. Apoptotic cells were detected with terminal deoxynuleotidyl transferase–mediated dUTP nick end labeling assay and counted in digitally captured images under high-power (10 × 20) magnifications. F to I, inhibition of angiogenesis. Microvessel density was determined by counting blood vessels (arrow) identified by immunostaining with von Willebrand factor (vWF; F) in digitally captured images under high power (10 × 40) magnifications (F and H). Changes of VEGF expression were evaluated by counting the immunopositive cells (G and I). The VEGF-positive cells in ICb-DAOY (arrowhead) were much larger than those in ICb-D283 and ICb-MED-1 tumors. Columns, mean; bars, SE. *, P < 0.05; **, P < 0.01.
Tables
- Table 1.
Summary of immunohistochemical examination of histone acetylation (AcH3 and AcH4), markers associated with neuronal (MAP-2 and synaptophysin) and glial (GFAP) differentiation, and intermediate filaments (VMT) in xenografts treated with or without valproic acid
Markers ICb-D283 ICb-DAOY ICb-MED-1 Untreated Treated Untreated Treated Untreated Treated AcH3 +/−, 30% +, 90% +/−, 10% +, 50% +, 15% +, 70% AcH4 +, 25% +, 80% (−) +, 5% +, 20% +, 85% MAP-2 ++, 5% +++, 30% +, 20% ++, 70% +++, 50% +++, 75% Synaptophysin ++, 50% ++, 60% +, 5% +, 5% +, 10% +, 10% GFAP (−) (−) (−) (−) (−) (−) VMT +++, 85% +++, 80% +++, 100% +++, 100% +++, 90% +++, 70%
Volume 12, Issue 15
