Department of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039, USA.
J Surg Res. 2012 Mar;173(1):1-9. doi: 10.1016/j.jss.2011.04.041. Epub 2011 May 17.
Expression of epidermal growth factor receptor (EGFR), a potent regulator of cellular homeostasis, is associated with aggressive tumor behavior. The mechanism by which EGFR inhibition functions is unclear, with controversial results demonstrating an effect on the tumor cells, endothelial cells, or pericytes. EGFR activation has been linked to the expression of vascular endothelial growth factor (VEGF), a known mitogen of angiogenesis, but the relationship between these factors and their effect on tumor vessel development is vague. We hypothesized that using an EGFR inhibitor on a human Ewing's sarcoma model would inhibit tumor growth by suppressing vessel proliferation.
A cell proliferation assay was performed on the Ewing's sarcoma (SK-NEP-1) cell line. Tumor cells were implanted intrarenally in athymic mice. Animals received daily gavage with vehicle or gefitinib 1 wk following implantation. Mice (n = 12/cohort) were euthanized 6 wk following implantation. Remaining mice were maintained without treatment for 2 wk. Vascular changes were assessed by angiography and immunohistochemically. EGFR and vascular endothelial growth factor (VEGF) expression were quantified using quantitative polymerase chain reaction (qPCR).
Gefitinib suppressed in vitro cell growth with an IC(50) = 1.36 μM. Minimal tumor growth suppression was noted at 6 wk (6.01 ± 1.2 g in control versus 4.61 ± 0.9 g treated, P = 0.36). After cessation of gefitinib, tumor growth was increased in both groups (7.37 ± 1.62 g versus 6.77 ± 1.53 g, P = 0.79). Microvessel density was unchanged despite EGFR inhibition (161,000 ± 16,000 pixels versus 135,000 ± 18,000 pixels, P = 0.31). At 6 wk, the vascular maturity index was similar in both groups (3.63 ± 1.12 versus 4.09 ± 1.71, P = 0.83). A downward trend in EGFR expression (49% of control) and an upward trend in VEGF levels (50% of control) occurred in the treated group.
EGFR expression was suppressed in cultured cells and xenograft tumors. Despite a cytotoxic effect on cell lines, gefitinib had little effect on tumor growth. No effects on the tumor vasculature were noted in the setting of EGFR suppression, suggesting that angiogenesis induced by SK-NEP-1 cells is refractory to EGFR inhibition. Interestingly, the resulting increase in VEGF expression following EGFR blockade, provides an alternative pro-angiogenic pathway promoting tumor survival.
表皮生长因子受体（EGFR）的表达是细胞内稳态的一个强有力的调节剂，与侵袭性肿瘤行为有关。EGFR 抑制作用的机制尚不清楚，有争议的结果表明其对肿瘤细胞、内皮细胞或周细胞有影响。EGFR 的激活与血管内皮生长因子（VEGF）的表达有关，VEGF 是已知的血管生成有丝分裂原，但这些因子之间的关系及其对肿瘤血管发育的影响尚不清楚。我们假设，在人尤文肉瘤模型中使用 EGFR 抑制剂，通过抑制血管增殖来抑制肿瘤生长。
对尤文肉瘤（SK-NEP-1）细胞系进行细胞增殖测定。将肿瘤细胞植入无胸腺小鼠的肾脏内。在植入后 1 周，动物接受每日灌胃治疗，给予载体或吉非替尼。植入后 6 周处死小鼠（n = 12/组）。其余小鼠继续不治疗 2 周。通过血管造影和免疫组织化学评估血管变化。使用定量聚合酶链反应（qPCR）定量 EGFR 和血管内皮生长因子（VEGF）的表达。
吉非替尼在体外抑制细胞生长的 IC50 为 1.36 μM。6 周时肿瘤生长抑制作用较小（对照组为 6.01 ± 1.2 g，治疗组为 4.61 ± 0.9 g，P = 0.36）。停止吉非替尼治疗后，两组肿瘤生长均增加（对照组为 7.37 ± 1.62 g，治疗组为 6.77 ± 1.53 g，P = 0.79）。尽管 EGFR 被抑制，但微血管密度仍无变化（161000 ± 16000 像素与 135000 ± 18000 像素，P = 0.31）。两组血管成熟指数相似（对照组为 3.63 ± 1.12，治疗组为 4.09 ± 1.71，P = 0.83）。在治疗组中，EGFR 表达下降（对照组的 49%），VEGF 水平升高（对照组的 50%）。
EGFR 在培养细胞和异种移植瘤中表达受到抑制。尽管对细胞系有细胞毒性作用，但吉非替尼对肿瘤生长几乎没有影响。在 EGFR 抑制的情况下，肿瘤血管未见变化，这表明 SK-NEP-1 细胞诱导的血管生成对 EGFR 抑制有抗性。有趣的是，EGFR 阻断后 VEGF 表达的增加提供了促进肿瘤存活的替代促血管生成途径。
