Figure 6.

Combined targeting of endothelial and perivascular tumor cells enhances anti-tumor efficacy of liposomal doxorubicin (DXR) in neuroblastoma. (A,B) Accumulation of APN- and APA-targeted, DXR-loaded, nanocarriers in nude mice orthotopically implanted with GI-LI-N neuroblastoma cells. (A) ³H-labeled, endothelial- (via NGR peptide) and perivascular- (via CPRECES peptide) targeted, DXR-loaded nanocarriers were injected intravenously in a single bolus. Treatment groups consisted of NGR-³H-SL(DXR), CPRECES-³H-SL(DXR), and combination of targeted liposomes (COMBO). At selected time points post-injection, blood was measured for ³H in a beta-counter. Points, average of three mice; bars, ±95% C.I. (B) Tumor accumulation of DXR visualized by fluorescence microscopy of NB tissue sections. Magnitude, 40×. (C) Effects of the combination therapy on endothelial, perivascular, and tumor cells in vivo. Immunohistochemistry was performed on established NB tumors removed from untreated mice (CTR) or from mice treated with DXR-loaded, NGR-targeted or CPRECES-targeted nanocarriers, or with a combination of the two liposomal formulations (COMBO). Tumors were removed on day 36 and tissue sections were immunostained for CD31 and SMA to detect tumor vasculature (scale bar, 250 μm). TUNEL was performed to detect tumor apoptosis (scale bar, 100 μm). Cell nuclei were stained with DAPI. Columns, mean of CD31, SMA, and TUNEL staining intensities; error bars represent 95% C.I. **P < 0.01; ***P < 0.001, COMBO vs. single treatments.