Orthotopic Glioblastoma Models for Evaluation of the Clinical Target Volume Concept

SIMPLE SUMMARY: The accurate and precise definition of the target volume is of enormous importance for the treatment success of radiotherapy. In glioblastoma, the microscopic tumor extension is unclear, which limits the specificity of irradiation leading to either increased risk of local failure or enhanced toxicity rates. In this study, we investigated the microscopic tumor extensions of two different untreated and irradiated orthotopic brain tumor models and correlated this with histologically stained cancer stem cell markers as well as invasion markers and analyses using Matrix-Assisted Laser Desorption/Ionization (MALDI). We found specific MALDI peaks as potential markers for normal brain tissue but also others for demarcation of tumor areas. Furthermore, MMP14 staining revealed mainly positive cells in the tumor border, which could reflect the invasive front in both models. Altogether, the results of this study indicate that an individualized target volume definition for radiotherapy based on biological tumor characteristics in glioblastoma models seems possible. ABSTRACT: In times of high-precision radiotherapy, the accurate and precise definition of the primary tumor localization and its microscopic spread is of enormous importance. In glioblastoma, the microscopic tumor extension is uncertain and, therefore, population-based margins for Clinical Target Volume (CTV) definition are clinically used, which could either be too small—leading to increased risk of loco-regional recurrences—or too large, thus, enhancing the probability of normal tissue toxicity. Therefore, the aim of this project is to investigate an individualized definition of the CTV in preclinical glioblastoma models based on specific biological tumor characteristics. The microscopic tumor extensions of two different orthotopic brain tumor models (U87MG_mCherry; G7_mCherry) were evaluated before and during fractionated radiotherapy and correlated with corresponding histological data. Representative tumor slices were analyzed using Matrix-Assisted Laser Desorption/Ionization (MALDI) and stained for putative stem-like cell markers as well as invasion markers. The edges of the tumor are clearly shown by the MALDI segmentation via unsupervised clustering of mass spectra and are consistent with the histologically defined border in H&E staining in both models. MALDI component analysis identified specific peaks as potential markers for normal brain tissue (e.g., 1339 m/z), whereas other peaks demarcated the tumors very well (e.g., 1562 m/z for U87MG_mCherry) irrespective of treatment. MMP14 staining revealed only a few positive cells, mainly in the tumor border, which could reflect the invasive front in both models. The results of this study indicate that MALDI information correlates with microscopic tumor spread in glioblastoma models. Therefore, an individualized CTV definition based on biological tumor characteristics seems possible, whereby the visualization of tumor volume and protein heterogeneity can be potentially used to define radiotherapy-sensitive and resistant areas.