In order to perform histopathological evaluation, breast and tumor tissues explanted from MMTV-NeuT mice were immersed in a 4% formaldehyde solution for 24 hours in order to obtain chemical fixation. Samples were then dehydrated with ethanol, washed with xylene and included in paraffine; then they were cut into 4 and 10 µm thick sections for hematoxylin/eosin staining and fluorescence imaging, respectively. Slides were then analyzed with a confocal microscope (Nikon A1R laser scanning confocal microscope) to acquire the fluorescent signal released by ICG and compared with the adjacent hematoxylin/eosin sections acquired with a Virtual Slide Microscope (Olympus VS120). Throughout the process of fixing and paraffining, the samples were kept out of the light, in order to avoid the loss of the fluorescent signal. Based on our previous experiments carried out with a similar tumor model and in vivo imaging endpoints 11,12,14 and taken into account the experimental error, we calculated that five mice per experimental group were sufficient to assess the homing capabilities of EV-formulations in mice bearing hyperplastic or transformed mammary gland versus syngeneic healthy controls. Sampling and histology on the resected tumor specimens were determined by professional pathologists (at Pathology Department, Istituto Nazionale Tumori IRCCS Foundation, Milan, Italy). They were not blinded when assessing EV homing with in vivo imaging, while were blinded when assessing EV homing in histopathological samples. Statistical significance was assessed by using one-way ANOVA with Tukey's Multiple Comparison test and nonparametric Mann-Whitney test. All statistical analysis, calculations and tests were performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA).

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