The ErbB inhibitors trastuzumab and erlotinib inhibit growth of vestibular schwannoma xenografts in nude mice: a preliminary study

Abstract

Objective: To analyze the ability of ErbB inhibitors to reduce the growth of vestibular schwannoma (VS) xenografts.

Methods: Vestibular schwannoma xenografts were established in the interscapular fat pad in nude mice for 4 weeks. Initially, a small cohort of animals was treated with the ErbB2 inhibitor trastuzumab or saline for 2 weeks. Animals also received bromodeoxyuridine injections to label proliferating cells. In a longer-term experiment, animals were randomized to receive trastuzumab, erlotinib (an ErbB kinase inhibitor), or placebo for 12 weeks. Tumor growth was monitored by magnetic resonance imaging during the treatment period. Cell death was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling of fragmented DNA.

Results: Tumors can be distinguished with T2-weighted magnetic resonance imaging sequences. Trastuzumab significantly reduced the proliferation of VS cells compared with control (p < 0.01) as analyzed by bromodeoxyuridine uptake. Control tumors demonstrated slight growth during the 12-week treatment period. Both trastuzumab and erlotinib significantly reduced the growth of VS xenografts (p < 0.05). Erlotinib, but not trastuzumab, resulted in a significant increase in the percentage of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling of fragmented DNA-positive VS cells (p < 0.01).

Conclusion: In this preliminary study, the ErbB inhibitors trastuzumab and erlotinib decreased growth of VS xenografts in nude mice, raising the possibility of using ErbB inhibitors in the management of patients with schwannomas, particularly those with neurofibromatosis Type 2.

Figure 1

Human vestibular schwannoma xenograft in nude mice. A and B. MRI of xenografts in sagittal (A) and axial (B) planes demonstrating survival of xenografts one month after implantation. Arrows indicate xenografts. C. Immunofluorescent labeling of xenograft frozen section with anti-S100 antibody followed by Alexa 568 (red) secondary antibody. Nuclei are labeled with Hoechst. This section is taken near the capsule, or edge, of the specimen demonstrating the S100 labeling of viable tumor cells and the lack of S100 labeling in the capsule cells.

Figure 2

Trastuzumab inhibits cell proliferation in vestibular schwannoma (VS) xenografts. A and B. Nude mice bearing VS xenografts were treated with saline (A, control) or trastuzumab (B) for 2 weeks. The animals received daily injections of BrdU to allow identification of proliferating cells. Representative images of xenograft frozen sections immunolabeled with anti-BrdU and anti-S100 antibodies with Alexa 568 (red, BrdU-positive) and Alexa 488 (green, S100-positive) conjugated secondary antibodies respectively are shown. Nuclei were labeled with Hoechst (blue). Arrows indicate TUNEL-positive nuclei. Scale bar=50 μm. C. The average number of BrdU-positive, S100-positive nuclei was determined for each condition from 5 random selected fields from 3-4 sections/xenograft from 2 separate animals for each group. Trastuzumab significantly reduced BrdU uptake in these xenografts (p<0.01, Student’s two-tailed t-test). Error bars represent standard error. n=total number of VS cells scored for each condition. N=total number of xenografts analyzed for each condition.

Figure 3

Trastuzumab and Erlotinib reduce the growth of VS xenografts. A. The change in relative tumor volume was determined by subtracting the initial tumor volume (V_i) from the final tumor volume (V_f) and dividing the difference by the initial tumor volume according to the formula: (V_f-V_i)/V_i and is plotted for the 12 week interval for each xenograft. Dashed lines in control group represent animals receiving saline while solid lines represent animals receiving erlotinib vehicle. B. The average relative growth of the xenografts for each condition is plotted.. Differences among the means for the treatment groups were determined by one-way ANOVA followed by a Kruskal-Wallis test. Error bars represent standard error. Both trastuzumab (p=0.025) and erlotinib (p=0.017) reduced the growth of the VS xenografts compared with controls.

Figure 4

Erlotinib but not trastuzumab induces cell death in vestibular schwannoma (VS) xenografts. A and B. Representative images of xenograft frozen sections from control (A) and erlotinib treated (B) animals immunolabeled with anti- S100 antibody and Alexa 488 (green, S100-positive) conjugated secondary antibody. Apoptotic cells were determined by TUNEL staining using biotin labeled dUTP and detected with Alexa 568 labeled streptavidin (red, TUNEL-positive). Nuclei were labeled with Hoechst (blue). Arrows indicate TUNEL-positive nuclei. Scale bar=50 μm. C and D. The average number of TUNEL-positive, S100-positive nuclei was determined for each condition from 5 random selected fields from 3-4 sections/xenograft from 3 separate animals for each group. Erlotinb significantly increased the percent of TUNEL-positive VS cells in these xenografts (p=0.03, Student’s two-tailed t-test) (C) compared with control animals while trastuzumab did not (p=0.61) (D). Error bars in C and D represent standard error. n=total number of VS cells scored for each condition. N=total number of xenografts analyzed for each condition.