Effect of NGR-mTNF on the penetration of doxorubicin in B16F1 and RMA-T tumors. (a) Bright-field (upper panels) and fluorescence (lower panels) microscopy of B16F1 cells incubated in vitro with 100 μg/ml doxorubicin (30 minutes, 37°C). Inset: Merge of bright-field and fluorescence images. (b) Stability of the B16F1 fluorescence signal after in vitro treatment with doxorubicin. B16F1 cells were incubated with various doses of doxorubicin in culture medium (30 minutes, 37°C), washed with 0.9% sodium chloride, and fixed with 4% formaldehyde. The cells were then incubated for 0 hours or 24 hours in culture medium at 4°C, washed again, and analyzed by FACS. (c and f) Representative FACS analysis of cells recovered from B16F1 (c) or RMA-T (f) tumors 2 hours after in vivo administration of doxorubicin alone (320 μg) or in combination with NGR-mTNF (0.1 ng). Dashed lines indicate the fluorescence interval considered positive. (d and g) Mean ± SE fluorescence of B16F1 (d) or RMA-T (g) cells recovered from tumors. (e and h) Mean ± SE of positive cells recovered from B16F1 (e) or RMA-T (h) tumors. *P < 0.05, statistical analysis by two-tailed t test.