Figure 8

Anti‐miR223 protocell treatment reduces cancer progression in adult zebrafish. A–H) Single‐channel confocal images of immunostained cryosections from 1‐year‐old adult tail tumors at 3 dpt after a single local injection of unlabeled control protocells (A,B,E,F) or unlabeled anti‐miR223 protocells (C,D,G,H); white lines indicate tumor margins; proliferating cells are revealed by anti‐pH3 immunostaining (magenta) in (A)–(D) and apoptotic cells revealed by TUNEL staining (yellow) in (E)–(H). B,D,F,H) High magnification views of (A), (C), (E), and (G). I,J) Graphs showing percentage of pH3‐positive (I) or TUNEL‐positive (J) area over total tumor area from sections after each injection regime quantified from the regions imaged in (A), (C), (E), and (G). K–R) Multi‐channel (K,M,O,Q) or single‐channel (L,N,P,R) images of 1‐year‐old adult tail tumors (red outlines) at 0 and 30 dpt after multiple local injections of unlabeled control protocells (K–N) or unlabeled anti‐miR223 protocells (O–R). S) Graph showing adult tail tumor growth curves after each protocell treatment quantified from the regions imaged in (K)‐(R) and (1). See also Figure S15, Supporting Information. T) Bar chart showing percentage of fish bearing tailfin tumors, and any additional tumor (or not), after each protocell treatment at 0 and 30 dpt quantified from the regions imaged in (2)–(4). Accompanying schematics illustrate fish age, timeline, type of injection (local, single or multiple), and imaged area (black outlined box) used for each experiment. Data are pooled from two independent experiments, and analyzed using Kruskal–Wallis test with Dunn's multiple comparisons test (I,J), two‐way ANOVA test with Bonferroni's multiple comparisons test (S), or Fisher's exact test (T), ns p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Graphs (I), (J), and (S) show mean ± SEM. In graphs (I) and (J), each dot represents one section and blue dots correspond to the representative images shown in the panels. In graph (S), each dot represents the mean of all fish analyzed and curves in lighter color correspond to individual fish. n = number of sections/fish (I,J); n = number of fish (S,T); PCs = protocells. Scale bars = 300 µm (A,C,E,G), 100 µm (B,D,F,H), 2 mm (K,M,O,Q).