Tumor-Associated Neutrophils and Macrophages Promote Gender Disparity in Hepatocellular Carcinoma in Zebrafish

Differential responses of neutrophils/macrophages in male and female zebrafish during kras^V12-induced carcinogenesis. Three-month-old kras⁺lyz⁺, lyz⁺, kras⁺mpeg⁺, and mpeg⁺ zebrafish were treated with 30 μg/mL doxycycline for 7 days, and liver-infiltrated neutrophils and macrophages were examined for density and gene expression. Representative liver sections of doxycycline-treated male and female kras⁺lyz⁺ and lyz⁺ (control) zebrafish to show neutrophils are presented in Supplementary Fig. S1A and representative liver sections of doxycycline-treated male and female kras⁺mpeg⁺ and mpeg⁺ (control) zebrafish to show macrophages in Supplementary Fig. S1B. A, Neutrophil density in liver sections of doxycycline-treated male and female kras⁺lyz⁺ and lyz⁺ zebrafish (n = 10 each group). B, Macrophage density in liver sections of doxycycline-treated male and female kras⁺mpeg⁺ and mpeg⁺ (n = 10 each group). C and D, Gene expression change after kras^V12 induction in TANs (C) and TAMs (D). TANs were isolated from livers of doxycycline-treated kras⁺lyz⁺ and lyz⁺ zebrafish, and TAMs from doxycycline-treated kras⁺mpeg⁺ and mpeg⁺ zebrafish by FACS. RNA expression of selected genes was determined by RT-qPCR. Fold changes are shown for TANs (kras⁺lyz⁺) versus naïve neutrophils (lyz⁺) or TAMs (kras⁺mpeg⁺) versus naïve macrophages (mpeg⁺) for both males and females (*, P < 0.05).

Effect of TANs and TAMs on kras^V12-induced carcinogenesis in zebrafish larvae. Zebrafish embryos were injected with various morpholino oligonucleotides at 1-cell stage and treated with 30 μg/mL doxycycline for 48 hours from 4 dpf. Liver sizes and neutrophils/macrophages were examined at 6 dpf. A, Gross morphology of morpholino-injected 6-dpf kras⁺ and WT (control) larvae. Kras^V12-expressing livers (indicated by arrows) decreased in size when neutrophils (MO-gcsfr), macrophages (MO-pu.1), or both (MO-gcsfr + MO-pu.1) were depleted. WT livers are demarcated with dotted lines. Scale bars, 200 μm. B, Quantification of 2D liver sizes under different morpholino conditions as shown in A (n > 20 each group). C, Densities of liver-infiltrated neutrophils (left) and macrophages (right). kras⁺lyz⁺ and lyz⁺ (control) larvae were used for neutrophil counting and kras⁺mpeg⁺ and mpeg⁺ (control) larvae used for macrophage counting. D, Quantification of proliferating (left) and apoptotic cells (right) in the liver sections of morpholino-injected and doxycycline-treated kras⁺ and WT larvae. Proliferation was based on immunofluorescent staining of proliferating cell nuclear antigen (PCNA)⁺ cells and apoptosis based on immunofluorescent staining of caspase-3⁺ cells. E, Gene expression changes after kras^V12 induction in TANs (left) and TAMs (right). TANs were isolated from kras⁺lyz⁺ and lyz⁺ larvae and TAMs from kras⁺mpeg⁺ and mpeg⁺ larvae. RNA expression of selected genes was determined by RT-qPCR. Fold changes are shown for TANs (kras⁺lyz⁺) versus naïve neutrophils (lyz⁺) or TAMs (kras⁺mpeg⁺) versus naïve macrophages (mpeg⁺; *, P < 0.05).

Differential production of cortisol and induction of Tgfβ1 expression by cortisol in kras^V12-expressing livers between genders. Three-month-old zebrafish were treated with 30 μg/mL doxycycline with or without 1 μmol/L of U0126, 10 μg/L of E2, 2 μmol/L mifepristone, or 10 μg/L cortisol for 7 days. The livers were examined for cortisol, Hsd11b, and TgfB1 by immunofluorescent staining and for RNA expression by RT-qPCR. Representative images of immunofluorescent staining of cortisol and Hsd11b on liver sections of kras⁺ male and female zebrafish in the absence or presence of U0126 or E2 are shown in Supplementary Fig. S4A and S4B, respectively. A, Percentages of cortisol positive hepatocytes are shown. B, Gene expression changes in key cortisol synthesis genes (cyp11a1, cyp17a1, and hsd11b1) in FACS-isolated hepatocytes with kras^V12induction in kras⁺ male fish, kras⁺ female fish, Kras⁺ male fish exposed to U0126, and Kras⁺ male fish exposed to E2. C, Percentages of Hsd11b-positive hepatocytes are shown. Expression of tgfb1 mRNA (D) and protein (E) in hepatocytes after alteration of cortisol levels. In addition to doxycycline treatment, male fish were also treated with mifepristone (mife) and female fish with cortisol (cort). RNA expression was determined by RT-qPCR from FACS-isolated hepatocytes. D, Fold changes of tgfb1 mRNA in kras^V12-expressing hepatocytes versus non–kras^V12-expressing controls (fabp10⁺) in both males and females. E, Percentages of Tgfb1 cells based on immunofluorescent staining of Tgfb1 protein (see Supplementary Fig. S4C). n = 10 in each group. *, P < 0.05 in all histograms.

Effects of cortisol on TAN and TAM infiltration in kras^V12-expressing livers. Three-month-old kras⁺, lyz⁺, kras⁺lyz⁺, mpeg⁺, and kras⁺mpeg⁺ zebrafish were treated with 30 μg/mL doxycycline with or without 2 μmol/L mifepristone or 10 mg/L cortisol for 7 days. The livers were examined for neutrophil and macrophage densities as well as for gene expression by RT-qPCR. Liver sections for showing neutrophils and macrophages are shown in Supplementary Fig. S6A and S6B, respectively. Neutrophil (A) and macrophage (B) densities. DsRed- or mCherry-labeled neutrophils and macrophages were counted manually. n = 10 for each group. *, P < 0.05. Gene expression changes after kras^V12 induction in TANs (C) and TAMs (D). TANs were isolated from kras⁺lyz⁺ and lyz⁺ livers and TAMs from kras⁺mpeg⁺ and mpeg⁺ livers. Fold changes are shown for TANs (kras⁺lyz⁺) versus naïve neutrophils (lyz⁺) or TAMs (kras⁺mpeg⁺) versus naïve macrophages (mpeg⁺; *, P < 0.05).

Promotion of kras^V12-induced carcinogenesis by cortisol. Three-month-old kras⁺ male and female zebrafish were treated with 30 μg/mL doxycycline with or without 2 μmol/L mifepristone or 10 mg/L cortisol for 7 days. The livers were examined for histology, proliferation, and apoptosis as described earlier. A, Gross morphology. Scale bars, 3 mm. B, H&E staining of liver sections. Scale bars, 20 μm. C, Quantification of tumor histology based on H&E-stained liver sections. n = 10 in each group. D and E, Immunofluorescent staining of proliferating cell nuclear antigen (PCNA) for cell proliferation (D) and caspase-3 for apoptosis (E) in the livers. Scale bar, 20 μm. Quantification of proliferating and apoptotic cells is shown on the right of each panel. n = 10 in each group. *, P < 0.05.