Figure 9.

Evaluating drug uptake and vasculature changes in NGP tumors 24 h after sonopermeation. (A) Ex vivo evaluation of NGP tumors using histology and immunostaining to evaluate levels of doxorubicin and liposomal DiD uptake, cell death (TUNEL), lectin, αSMA, and Endomucin (lumen). Untreated tumors (left column) were compared with tumors given L-DOX and DiD liposomes only (no sonopermeation, middle column), and sonopermeated tumors (right column). Low magnification of the apoptosis marker TUNEL reveals that sonopermeation with L-DOX led to large clusters of apoptotic tumor cells at 24 h (second row, white dots, yellow arrows) although no observable changes in blood vessel (lectin) or pericyte coverage (αSMA) were observed (third row). Sonopermeation caused enlargement of vascular lumens (marked L) in the presence of L-DOX: lumens were measured using Pannoramic Viewer, the longest distance separating Endomucin immunostained cells was measured, and over 100 measurements of randomized locations per tissue were analyzed (see Supplementary Figure 5). (B) Quantification of doxorubicin in NGP tumors calculated by average fluorescent intensity within the tumors. Image intensities are ~2 and 5-fold higher for L-DOX/DiD only and sonopermeated tumors with L-DOX/DiD respectively. Untreated controls show a small level of background signal due to tissue autofluorescence. (C) Comparison of liposome uptake in NGP tumors using average DiD fluorescent intensity. Infusion of L-DOX without sonopermeation did not alter DiD uptake in tumors compared to the untreated controls. Sonopermeation increased DiD presence in the tumors approximately 5-fold. (D) Quantification from low magnification (10x) images reveals no significant changes in endothelial marker lectin or pericyte marker αSMA. N = 3 mice per group. * Indicates p<0.05 and ** indicates p<0.01.