In this study, the tumor uptake of ABY-029 was examined as a function of its administered dose and time after injection. The co-localization of the tumor microenvironment and Affibody-fluorescent probe signal was observed through bulk analysis (Figs. 1, 2, and 3). However, the distribution of the fluorescence signal in the tumor was also documented to be highly heterogeneous (Fig. 2). This results from cellular heterogeneity and differentiation level of the glioma cells , and/or the spatial and temporal heterogeneity of the blood supply, leading to high delivery variability from the enhanced permeability and retention effect [40, 41]. The absence of fluorescence usually was observed in the tumor interior and it could be either correlated with necrotic areas (Fig. 2c inset), in which vascular supply is often inadequate or insufficient, or lower interstitial pressure present in peripheral areas of the tumor that allows leakage of macromolecules . Perhaps most importantly, the EGFR overexpression was documented by ex vivo staining with an independent antibody, and the presence of hot spots staining of ABY-029 appeared to be well correlated to the EGFR density (Fig. 2d inset).