Figure 1

Induction of krasG12V overexpression in zebrafish liver cancer by Tet-On system. (A) Components of the Tet-On system and doxycycline (DOX) treatment strategy for induction of liver cancer. (B) Confocal images showed zebrafish liver after DOX activation of krasG12V from 6 days postfertilization (dpf) to 10 dpf. (C,D) antibody staining results of proliferating cell nuclear antigen (PCNA) in the control group (n = 11) and krasG12V+ group (n = 13). (E) Confocal images showed the presence of a green, fluorescent signal in the intestine outside the liver after DOX activation of krasG12V. (F) H&E staining to confirm the morphology of the liver in the control and krasG12V+ groups. (G) Statistics of the percentage fibrosis/necrosis area in the liver in the control (n = 5) and krasG12V+ (n = 4) groups. (H) Sirius Red staining of the liver in the control and krasG12V+ groups. (I) Statistics of the percentage fibrosis area in the liver in the control (n = 5) and krasG12V+ (n = 5) groups. Numbers indicate the percentage of larvae exhibiting this expression. Asterisks show significance: *—p < 0.05; ***—p < 0.001; ****—p < 0.0001. Scale bars—100 μm; error bars—S.D.
Figure 2

Zebrafish hepatocytes undergo dedifferentiation and biliary duct activation after krasG12V induction. (A) Whole-mount in situ hybridization (WISH) results showed the expression pattern of sox9b, hhex, and foxa3 at 6 dpf, 8 dpf, and 10 dpf, respectively, after DOX activation of krasG12V. (B,C) Antibody staining of biliary duct marker Anxa4 in zebrafish liver expression at 6 dpf, 8 dpf, and 10 dpf after DOX induction, and statistics of Anxa4+ area in the liver in the control (n = 10) and krasG12V+ (n = 11) groups. Numbers indicate the percentage of larvae exhibiting this expression. Asterisks show significance: *—p < 0.05; ***—p < 0.001; ****—p < 0.0001. Scale bars—100 μm; error bars—S.D.
Figure 3

The Notch signaling pathway was upregulated after hepatocellular carcinoma induction, and the inhibition of Notch signaling suppressed hepatocyte dedifferentiation. (A) WISH results showed the expression pattern of notch1a, notch1b, notch2, notch3, and her15 at 10 dpf after DOX activation of krasG12V. (B) Tg(fabp10a:Tet3G;TRE3G:krasG12V-ZsGreen1) with Tg(Hsp70l:dnMAML-GFP) double transgenic fish line treatment strategy. (C) Monolayer images of Anxa4 antibody staining showed expression changes in the DOX-induced krasG12V+ and krasG12V+ and dnMAML+ groups at 8 dpf and 10 dpf. (D) Statistics of Anxa4 + and ZsGreen1 + /ZsGreen1+ ratio in the DOX-induced krasG12V+ (n = 11) and krasG12V+ and dnMAML+ (n = 13) groups at 8 dpf and 10 dpf. (E) The results of fluorescent in situ hybridization (FISH) showed sox9b expression in the DOX-induced control and krasG12V+ and krasG12V+ and dnMAML+ groups at 10 dpf. (F) The results of FISH showed cp expression in the DOX-induced control, krasG12V+ and krasG12V+ and dnMAML+ groups at 10 dpf. Numbers indicate the proportion of larvae exhibiting that expression. Asterisks show significance: ****—p < 0.0001. Scale bars—100 μm; error bars—S.D.
Figure 4

Hepatic Sox9 expression is upregulated after hepatocellular carcinoma induction, and the sox9bfh313 mutant suppressed hepatocyte dedifferentiation. (A) Tg(fabp10a:Tet3G;TRE3G:krasG12V-ZsGreen1) with Tg(Hsp70l:dnMAML-GFP) double transgenic fish line treatment strategy. (B,C) Sox9 antibody staining indicated the number of Sox9+ cells in the liver at 10 dpf in the DOX-induced control (n = 5), krasG12V+ (n = 5) and krasG12V+ and dnMAML+ (n = 5) groups with statistical results. (D) Tg(fabp10a:Tet3G;TRE3G:krasG12V-ZsGreen1) treatment strategy in sox9bfh313 mutant. (E) Three-dimensional images of Anxa4 antibody staining showed biliary duct changes in the DOX-induced krasG12V+ and krasG12V+ sox9bfh313 mutants at 8 dpf and 10 dpf. (F) Monolayer images of Anxa4 antibody staining biliary duct changes in the DOX-induced krasG12V+ and krasG12V+ sox9bfh313 mutant at 8 dpf and 10 dpf. Asterisks show significance: *—p < 0.05; **—p < 0.01. Scale bars—100 μm; error bars—S.D.
Figure 5

Inhibition of the Notch signaling pathway after liver cancer induction reduced cancer cell migration and improved survival. (A) Tg(fabp10a:Tet3G;TRE3G:krasG12V -ZsGreen1) with Tg(Hsp70l:dnMAML-GFP) double transgenic fish line treatment strategy to observe the migration of cancer cells. (B) Zebrafish larvae were classified into three classes, I, II, and III, by the number of cancer cells outside the liver and the number of locations of metastases. (C) The proportion of the total number of larvae in different metastasis classes in the krasG12V+ (n = 46) and krasG12V+ and dnMAML+ (n = 130) groups at 9 dpf. (D) Kaplan–Meier survival curves of the DOX-induced krasG12V+ (n = 191) and krasG12V+ and dnMAML+ (n = 163) groups. p values for survival curves were calculated by log-rank test. Scale bars, 100 μm.
Acknowledgments
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