Abstract
BackgroundBrain metastasis is becoming increasingly prevalent in breast cancer due to improved extra-cranial disease control. With emerging availability of modern image-guided radiation platforms, mouse models of brain metastases and small animal magnetic resonance imaging (MRI), we examined brain metastases’ responses from radiotherapy in the pre-clinical setting. In this study, we employed half brain irradiation to reduce inter-subject variability in metastases dose-response evaluations.MethodsHalf brain irradiation was performed on a micro-CT/RT system in a human breast cancer (MDA-MB-231-BR) brain metastasis mouse model. Radiation induced DNA double stranded breaks in tumors and normal mouse brain tissue were quantified using γ-H2AX immunohistochemistry at 30 min (acute) and 11 days (longitudinal) after half-brain treatment for doses of 8, 16 and 24 Gy. In addition, tumor responses were assessed volumetrically with in-vivo longitudinal MRI and histologically for tumor cell density and nuclear size.ResultsIn the acute setting, γ-H2AX staining in tumors saturated at higher doses while normal mouse brain tissue continued to increase linearly in the phosphorylation of H2AX. While γ-H2AX fluorescence intensities returned to the background level in the brain 11 days after treatment, the residual γ-H2AX phosphorylation in the radiated tumors remained elevated compared to un-irradiated contralateral tumors. With radiation, MRI-derived relative tumor growth was significantly reduced compared to the un-irradiated side. While there was no difference in MRI tumor volume growth between 16 and 24 Gy, there was a significant reduction in tumor cell density from histology with increasing dose. In the longitudinal study, nuclear size in the residual tumor cells increased significantly as the radiation dose was increased.ConclusionsRadiation damages to the DNAs in the normal brain parenchyma are resolved over time, but remain unrepaired in the treated tumors. Furthermore, there is a radiation dose response in nuclear size of surviving tumor cells. Increase in nuclear size together with unrepaired DNA damage indicated that the surviving tumor cells post radiation had continued to progress in the cell cycle with DNA replication, but failed cytokinesis. Half brain irradiation provides efficient evaluation of dose-response for cancer cell lines, a pre-requisite to perform experiments to understand radio-resistance in brain metastases.
Highlights
Brain metastasis is becoming increasingly prevalent in breast cancer due to improved extra-cranial disease control
The amount of γ-H2A histone family (H2AX) intensity density increased linearly (R2 = 0.78, p < 0.001) with increasing radiation dose. In tumors, this trend stopped at 16 Gy; the level of γ-H2AX intensity density dropped at the dose of 24 Gy compared to 16 Gy
Our results are consistent with this finding as we showed that remaining tumor cells after radiation has a higher sustained level of DNA damage with an elevated γ-H2AX
Summary
Brain metastasis is becoming increasingly prevalent in breast cancer due to improved extra-cranial disease control. We employed half brain irradiation to reduce inter-subject variability in metastases dose-response evaluations. Others, have recently employed whole brain irradiation in mouse models of brain metastasis due to breast cancer to study tumor response after different timing or fractionation regimens of radiotherapy [1,2,3]. Post-sacrifice immunohistochemistry (IHC) slide staining results can vary despite following the same protocol [5] This led us [6] and others [7] to develop and validate platforms for half-brain irradiations [8], allowing us to reduce inter-animal and inter-histological slide variability by using the contralateral brain as the control
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