Combination therapy with gefitinib and doxorubicin inhibits tumor growth in transgenic mice with adrenal neuroblastoma

Abstract

Highly relevant mouse models of human neuroblastoma (NB) are needed to evaluate new therapeutic strategies against NB. In this study, we characterized transgenic mice with bilateral adrenal tumors. On the basis of information from the tumoral gene expression profiles, we examined the antitumor effects of unencapsulated and liposomal doxorubicin (DXR), alone and in combination with gefitinib, on adrenal NB. We showed that intravenous injection of unencapsulated or liposomal DXR alone inhibited tumor growth in a dose-dependent manner, as assessed by magnetic resonance imaging (MRI). However, liposomal DXR did not exhibit greater antitumor effect than unencapsulated DXR. Immunohistochemical analysis revealed that the adrenal tumor vasculature with abundant pericyte coverage was a less leaky structure for liposomes. Combination therapy with unencapsulated or liposomal DXR plus gefitinib strongly suppressed tumor growth and delayed tumor regrowth than treatment with unencapsulated or liposomal DXR alone, even at a lower dose of DXR. Dynamic contrast-enhanced MRI analysis revealed that gefitinib treatment increased blood flow in the tumor, indicating that gefitinib treatment changes the tumor vascular environment in a manner that may increase the antitumor effect of DXR. In conclusion, the combination of gefitinib and DXR induces growth inhibition of adrenal NBs in transgenic mice. These findings will provide helpful insights into new treatments for NB.

Keywords: Adrenal neuroblastoma; doxorubicin; gefitinib; liposome; transgenic mouse.

Figure 1

Quantitative RT-PCR analysis of the mRNA expression levels of Topo IIα (A) and epidermal growth factor receptor (EGFR) (B) in the adrenal tumors from the transgenic mice. mRNA was purified from the adrenal tumors of transgenic mice aged 5–17 weeks and the adrenal glands of nontransgenic littermates aged 5 and 13 weeks. Each result represents the mean ± standard deviation (SD) (n = 3).

Figure 2

Antitumor effects of unencapsulated and liposomal DXR (A–D) and histological analysis after the treatment (E–G). Adrenal tumor size (A and B) and survival time (C and D) after treatment with unencapsulated doxorubicin (DXR) (A and C) or liposomal DXR (B and D). The arrows indicate the times of drug treatment. The tumor size represents the sum of the volumes of the left and right adrenal glands calculated from MR images. Each value represents the mean ± SD (n = 5–6). *P < 0.05, **P < 0.01 versus control mice at 19 weeks of age. (E–G) Histological analysis of the adrenal tumors 1 week after injection of saline (E), unencapsulated DXR (F), or liposomal DXR (G) at 5 mg/kg into 14-week-old transgenic mice. The sections were stained with H&E. N, necrotic area. Scale bar = 500 μm.

Figure 3

DXR levels in the serum (A), adrenal tumors (B), liver (C), spleen (D), and kidneys (E) after intravenous injection of unencapsulated DXR (•) or liposomal DXR (□) at 5 mg/kg into 14-week-old transgenic mice. Each value represents the mean ± SD (n = 3, serum and tissues; n = 6, tumor).

Figure 4

Immunostaining for endothelial cells and pericytes (A and C) and H&E staining (B and D) of mouse adrenal tumors (A and B) and human neuroblastoma (C and D). In (A) and (C), the green signals indicate endothelial cells; and the red signals, α-SMA-positive pericytes. The arrows in (B) and (D) indicate stromal cells. Scale bar = 200 μm.

Figure 5

Adrenal tumor size (A) and survival time (B) after treatment with unencapsulated or liposomal DXR in combination with gefitinib for 2 weeks. Transgenic mice were orally administered gefitinib at a dose of 100 mg/kg per day for five consecutive days with or without intravenous injection of unencapsulated or liposomal DXR at a dose of 5 mg/kg on the fifth day of gefitinib treatment. Arrows and arrow heads indicate the days of drug treatment. The tumor size represents the sum of the left and right adrenal gland volumes calculated from MR images. The tumor size values are given as the mean ± SD (n = 4–6). *P < 0.05 versus control at 22 weeks of age.

Figure 6

Tumor vascular change after the oral gefitinib treatment (100 mg/[kg·day] for five consecutive days), as determined by DCE-MRI (A) and Hoechst 33342 staining (B and C). (A) Tumor Gd concentration was assessed by DCE-MRI using Gd-DTPA. Each data point represents the dot plots of an individual mouse. (B and C) Tumor vessels stained by intravenous injection of Hoechst 33342 as a perfusion marker in the saline- (B) and gefitinib-treated mice (C). Scale bar, 200 μm.