Physeal bystander effects in rhabdomyosarcoma radiotherapy: experiments in a new xenograft model

Abstract

Radiotherapy used in the treatment of pediatric musculoskeletal sarcomas may result in crippling defects of skeletal growth. Several radioprotective strategies have shown potential for preserving function of the irradiated epiphysis but have not been evaluated in a tumor-bearing animal model. We developed two bioluminescent human rhabdomyosarcoma cell lines that were used to establish xenograft tumors in skeletally immature mice. Bioluminescence imaging and radiography allowed serial evaluation of tumor growth and tibial elongation following localized radiotherapy. High-dose (10 Gy) radiotherapy significantly reduced tumor growth velocity and prolonged the median survival of tumor-bearing mice but also resulted in a significant 3.3% shortening of the irradiated limb. Exposure to a lower, 2 Gy dose resulted in 4.1% decrease in limb length but did not extend survival. This new model provides a clinically relevant means to test the efficacy and safety of novel radioprotectant and radiorecovery strategies for use in this context.

Figure 1

Luciferin uptake and signal stabilization. Bioluminescence imaging was used to verify tumor placement three hours after (a) subcutaneous or (b) intramuscular inoculation of RD-luc cells. Imaging was initiated one-minute following a single, 150 mg/kg I.P. bolus of d-luciferin mice, and photon flux data was collected in successive one minute intervals. (c) Average log-photon flux ±1 SD of n = 4 mice demonstrating a detectable signal within 1 minute following luciferin injection and was stable for a period ranging from 10 to 24 minutes after-injection. No signal was detected in animals injected with cell-free Matrigel.

Figure 3

Serial bioluminescence imaging demonstrating successful tumor engraftment resulting from subcutaneous and intramuscular inoculation of RD-luc and RC13-luc tumor cells. (a) The growth of RD-luc tumors did not demonstrate substantial tumor growth when implanted either (b) subcutaneously or (c) intramuscularly. (d) Modest growth of RC13-luc cells were implanted (e) subcutaneously, whereas aggressive tumor growth resulted when this cell line was inoculated intramuscularly. Bar graphs show log-photon flux ±1 SD; brackets indicate P ≤ .05 by Student's t-test.

Figure 2

Serial bioluminescence imaging RC13-luc xenografts following radiotherapy with 0 Gy, 2 Gy, or 10 Gy X-rays. All animals in the 0 Gy and 2 Gy groups were euthanized within 63 days of imaging. (a) Data presented as the mean log-transformed photon-flux (photons/second) ±1 SD; brackets indicate P ≤ .05 by ANOVA with Bonferroni-Dunn post hoc test. Representative images demonstrating tumor localization and growth in (b) nonirradiated, (c) 2 Gyirradiated, and (d) 10 Gy irradiated mice.

Figure 4

Representative histology and immunohistochemistry of RC13-luc derived metastatic lesion. (a) H&E, 10x, and (a) 40x; (b) MyoD1; (c) IHC neg. control; (d) Myogenin. (e) Cumulative survival. Kaplan-Meier plot of survival in tumor-bearing mice following localized irradiation with 2 Gy (solid black) or 10 Gy X-rays (dotted blue), or 0 Gy, sham-irradiated mice.