Fig. 3. LND-CDN exhibits efficient tumour penetration in vivo.
a, C57Bl/6 mice (n = 3 animals per group) were injected intravenously with Cy5-labelled cGAMP, Cy5-labelled LND-CDN or liposome-CDN (all at 5 nmol CDN) and plasma levels were quantified by fluorescence measurements over time. Dotted lines show two-phase decay curve fits. b, C57Bl/6 mice (n = 4 animals per group) were inoculated in the flank with 5 × 105 MC38 tumour cells, and 10 days later, 5 nmol Cy5-labelled cGAMP, LND-CDN or liposome-CDN were administered intravenously. Shown is the organ-level biodistribution (mean ± s.e.m.) determined from fluorescence measurements on digested tissues 24 h later. n.d., not detectable. c–e, MC38-tumour-bearing mice (n = 4 animals per group) were treated as in b with 5 nmol near-infrared dye-labelled LND-CDN, liposome-CDN or left untreated, and then sacrificed at 4 h. The mice were rapidly frozen and then imaged by cryofluorescence tomography with 50 µm serial sections. c, Representative maximum intensity projections (MIP) of whole mice with tumours identified with a white arrow and outlined with a dotted white line. Lv, liver, Sp, spleen; Bm, bone marrow. d, Enlarged images of a single slice from the middle of representative tumours with the corresponding white-light image shown only for mouse 1. e, Mean fluorescence intensities (±s.e.m.) averaged from three tumour regions of interest per mouse (one at the tumour centre, one 1 mm dorsal and one 1 mm ventral) (n = 4 mice per group). f, MC38-tumour-bearing mice (n = 3 animals per group) were treated with LND or liposomes as in b and tumours were excised 24 h later for histology. High-molecular-weight fluorescein isothiocyanate–dextran (cyan) was injected intravenously 10 min before the mice were sacrificed to label vasculature. Shown are representative whole tumour cross sections and enlarged views of tumour vessels from mice treated with Cy5-labelled LND or liposome (yellow). Scale bars: whole tumour cross sections, 500 µm; enlarged view, 50 µm. g,h, The percentage of the extravascular tumour area with nanoparticle fluorescence (g) and the average fluorescence intensity of the extravascular tumour area (h) was quantified (mean ± s.e.m.). Each point represents one mouse and is the average of two unique tumour cross sections. Statistical comparisons in b, e, g, and h were tested using an ordinary one-way analysis of variance (ANOVA) with Tukey’s multiple-comparisons test.