1. EGFR Inhibition Fails to Suppress Vascular Proliferation and Tumor Growth in a Ewing's Sarcoma Model 
Artur Chernoguz, M.D., Kelly Crawford, B.S., Eileen Donovan, Abbey Vandersall, Caroline Berglund, B.S., Timothy P. Cripe, M.D., Ph.D., Jason S. Frischer, M.D. 
Journal of Surgical Research 
Volume 173, Issue 1, Pages 1-9 (March 2012)
DOI: /j.jss
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2. Fig. 1
Cell viability assay was performed by exposing cultured SK-NEP-1 cells to gradient concentrations of gefitinib (0, 0.01, 0.1, 1, 10, and 100 μM) for 2, 4, and 6 d. IC50 = 1.36.
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3. Fig. 2
(A) Western blot analysis of the SK-NEP1 cell lysates stimulated with EGF in the presence of gradient gefitinib concentrations (0, 0.01, 0.1, 1, 10, and 100 μM). Eighty-eight percent reduction in MAPK activation was noted in the cells exposed to 0.1 μM gefitinib (62.09% ± 0.36% in control versus 13.39% ± 2.0% in treated, P < 0.01). Treatment with ≥ 10 μM gefitinib virtually abolished EGF-induced MAPK phosphorylation in cultured SK-NEP-1 cells. (B) MAPK activation was reliably reduced in tumor xenografts following a 5-wk treatment regimen with gefitinib (7.29% ± 1.3% in control versus 2.08% ± 0.37%, P < 0.01). Levels of phosphorylated MAPK returned to control levels following a 2-wk gefitinib withdrawal (6.11% ± 1.39% in controls versus 6.86 ± 1.25 in treated, P = 0.29).
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4. Fig. 3
Five-week regimen of gefitinib failed to significantly reduce the weight of orthotopic SK-NEP-1 Ewing's sarcoma tumors (control: 6.01 ± 1.2 g versus treated: 4.61 ± 0.9 g, P = 0.36). After a 2-wk gefitinib withdrawal period, tumor weights increased in both, control and treated groups (7.37 ± 1.62 g versus 6.77 ± 1.53 g, P = 0.79).
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5. Fig. 4
(A) Fluorescent immunohistochemical staining with anti-CD34 antibody reliably detected microvasculature in SK-NEP-1 Ewing's sarcoma tumor tissues. (B) Microvessel density was calculated using microangiography images obtained from an orthotopic model of Ewing's sarcoma. No statistical significant difference was observed in the gefitinib treated group compared to controls (n = 5/group) immediately at completion of the treatment regimen (wk 6: control: 161,000 ± 16,000 pixels versus treated: 135,000 ± 18,000 pixels, P = 0.31), or after a 2 wk cessation of treatment (wk 8: 169,000 ± 16,000 pixels versus 159,000 ± 8500 pixels, P = 0.59). (Color version of figure is available online.)
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6. Fig. 5
(A) The degree of pericyte coverage in SK-NEP-1 tumor vessels, reflected by the CD34/ASMA ratio (Vascular Maturity Index) remained unchanged despite treatment with gefitinib. (B) Double immunofluorescent immunohistochemical staining with endothelial cells marked by anti-CD34 (green) and anti-α smooth muscle cell, ASMA (red), antibody revealed the distribution of tumor microvessels, and associated mural cells. (Color version of figure is available online.)
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7. Fig. 6
Quantitative PCR was performed in SK-NEP-1 Ewing's sarcoma xenograft tumors in order to assess VEGF and EGFR expression following treatment with the EGFR inhibitor, gefitinib. (A) A 50% increase in VEGF levels was noted following the five weeks of gefitinib treatment, (B) with an associated downward trend in EGFR levels (49%).
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