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Molecular and Cellular Pathobiology

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

Chuan Yan, Qiqi Yang and Zhiyuan Gong
DOI: 10.1158/0008-5472.CAN-16-2200 Published March 2017

Abstract

Hepatocellular carcinoma (HCC) occurs more frequently and aggressively in men than women, but the mechanistic basis of this gender disparity is obscure. Chronic inflammation is a major etiologic factor in HCC, so we investigated the role of cortisol in gender discrepancy in a zebrafish model of HCC. Inducible expression of oncogenic KrasV12 in hepatocytes of transgenic zebrafish resulted in accelerated liver tumor progression in males. These tumors were more heavily infiltrated with tumor-associated neutrophils (TAN) and tumor-associated macrophages (TAM) versus females, and they both showed protumor gene expression and promoted tumor progression. Interestingly, the adrenal hormone cortisol was predominantly produced in males to induce Tgfb1 expression, which functioned as an attractant for TAN and TAM. Inhibition of cortisol signaling in males, or increase of cortisol level in females, decreased or increased the numbers of TAN and TAM, respectively, accompanied by corresponding changes in protumor molecular expression. Higher levels of cortisol, TGFB1, and TAN/TAM infiltration in males were also confirmed in human pre-HCC and HCC samples, features that positively correlated in human patients. These results identify increased cortisol production and TAN/TAM infiltration as primary factors in the gender disparity of HCC development in both fish and human. Cancer Res; 77(6); 1395–407. ©2017 AACR.

Introduction

Hepatocellular carcinoma (HCC) is a gender-biased disease, as men are 3 to 5 times more likely to develop HCC than women (1, 2). Men generally have more aggressive HCC than women, and female patients HCC tend to survive about 2 times longer than male patients with HCC (3). Traditionally, this gender bias has been attributed to the fact that men are more predisposed to known risk factors of HCC, such as high alcohol consumption and unhealthy diet (4). However, laboratory mouse experiments under controlled conditions also indicated that males were more likely to develop HCC than females, with 100% of males and 30% of female developing HCC under chronic carcinogen exposure (5). The gender disparity appears to be hormone-dependent. Female hormone, estrogen, has been credited to confer protection against HCC, as postmenopausal women have an increased HCC incidence (6). Similarly, prolactin, dominantly expressed in females, downregulates innate immune activation in an Myc-induced liver cancer model in transgenic mice (7). Notably, the gender disparity in HCC seems to be also mediated by immune cells. For example, estrogen inhibits IL6 production in Kupffer cells (liver resident macrophages) and reduces liver cancer risks in female mice (8). Interestingly, numerous HCC studies have demonstrated a more robust immune cell infiltration in males than in females. For example, infiltrating TAM density has been found to be higher in males than in females in a mouse HCC model (9). In human patients with HCC, men have considerably high numbers of intratumoral infiltrated CD66b+ neutrophils and CD8+ T cells, both of which are indicators of poor disease prognosis (10).

However, despite these studies, several previous clinical trials targeting sex hormone pathways showed no significant improvement in HCC patient survival (11). Clearly, the mechanisms regarding male biasness in HCC remain to be elucidated for clinical benefits. Recently, we have generated several inducible HCC models by transgenic expression of an oncogene in hepatocytes in zebrafish (12–16). With the inducible system, the oncogene can be activated at a given and controlled timing in both genders, providing an excellent platform to study gender disparity in HCC initiation and progression in a controlled environment. In this study, we found in our krasV12-expressing tumor model that male transgenic zebrafish showed accelerated carcinogenesis compared with female counterparts. Interestingly, cortisol was highly produced in males and it contributed to enhanced infiltration of tumor-associated neutrophils (TAN) and tumor-associated macrophages (TAM) in male oncogenic liver by stimulation of Tgfb1. Consequently, the protumor TANs and TAMs accelerated HCC in a male-biased manner. The increased cortisol level, TGFB1 expression, and TAN/TAM infiltration in male HCC were also confirmed from human patient samples; thus, cortisol is likely also involved in gender disparity of human HCC.

Materials and Methods

Zebrafish husbandry

Zebrafish were maintained in compliance with Institutional Animal Care and Use Committee guidelines, National University of Singapore (Singapore, Singapore). Four transgenic lines [Tg(fabp10:rtTA2s-M2; TRE2:EGFP-krasG12V) (gz32Tg) in a Tet-On system for inducible hepatocyte-specific expression of oncogenic krasG12V(12), Tg(fabp10a:DsRed; ela3l:GFP) (gz15Tg) with DsRed-labeled liver and GFP-labeled exocrine pancreas (17), Tg(lyz:DsRed2) (nz50Tg) with DsRed-labeled neutrophils under the lysozyme C (lyz) promoter (18), Tg(mpeg1:mCherry) (gl22Tg), with mCherry-labeled macrophages under the macrophage expressed gene promoter (19)] were used in this study and referred to as kras, fabp, lyz, and mpeg, respectively, in the present report.

Chemical treatments

All chemical/reagent treatments were conducted in larvae from 3 to 6 days postfertilization (dpf) and in 3-month-old adult fish for 7 days unless otherwise specified. The chemicals/reagents used included doxycycline (Sigma), U0126 (Sigma), 17β-estradiol (Sigma), mifepristone (Sigma), and hydrocortisone (Sigma). Doxycycline (30 μg/mL) was used for induction of krasV12 expression as previously reported (20). U0126, 17β-estradiol, mifepristone, and hydrocortisone were used for adult exposure at 1 μmol/L, 10 μg/mL, 2 μmol/L, and 10 mg/L, respectively. The dosages were selected on the basis of the highest all-survival concentrations in preliminary experiments.

Morpholino knockdown

For knockdown of Gcsfr and Pu.1, previously validated gene-specific morpholinos were used, including MO-gcsfr (5′-GAAGCACAAGCGAGACGGATGCCAT-3′; ref. 21) and MO-pu.1 (5′-GATATACTGATACTCCATTGGTGGT-3′; ref. 22). A standard control morpholino, MO-control (5′-CCTCTTACCTCAGTTACAATTTATA-3′) targeting a human β-globin intron (GeneTools), was used as a negative control. Aliquots of morpholino (1 mmol/L) and 1% (wt/vol) phenol red in Danieau solution were injected into embryos at the 1-cell stage. Doxycycline was added to all larvae from 3 to 6 dpf.

Photography and image analysis

At each time point of chemical treatment and morpholino knockdown experiments, 10 adult fish or 15 to 20 larvae of each group were randomly collected for imaging analyses. Adult zebrafish were anesthetized in 0.08% tricaine (Sigma) and larvae immobilized in 3% methylcellulose (Sigma) before imaging. Adult zebrafish were photographed with Olympus microscope, and larvae were photographed using a confocal microscope (Carl Zeiss LSM510). Measurement of 2-dimensional (2D) liver sizes was performed using ImageJ as previously described (14, 23) and neutrophils and macrophages counted manually.

Isolation of hepatocytes, neutrophils, and macrophages by FACS

Five to 8 adult zebrafish livers were sampled and pooled. In larvae, to enrich the TANs, TAMs, and hepatocytes, central part of 6-dpf larvae (after removal of the head and tail regions) was used for FACS using a cell sorter (BD Aria). Adult livers or enriched larval livers were dissociated into single cells using a 40-μm mesh (BD Biosciences) and enzymatically digested with 0.05% trypsin (Sigma), as previously described (24). GFP+ oncogenic hepatocytes (OH), Ds-Red+ TANs, and mCherry+ TAMs were isolated from doxycycline-induced kras+, kras+/lyz+, kras+/mpeg+ transgenic larvae, whereas nononcogenic control hepatocytes, naïve neutrophils, and naïve macrophages were isolated, respectively, from doxycycline-treated fabp10+, lyz+, and mpeg+ control larvae on the basis of Ds-Red or mCherry expression.

RNA extraction, cDNA amplification, and RT-qPCR

Total RNA was extracted using RNeasy Mini Kit (Qiagen). A total of 5 ng RNA was used as a template to synthesize and amplify cDNA using QuantiTect Whole Transcriptome Kit (Qiagen). Amplified cDNA was used for real-time quantitative PCR with LightCycler 480 SYBR Green I Master (Roche Diagnostics). Interested genes were amplified for 40 cycles (95°C, 20 seconds; 65°C, 15 seconds; 72°C, 30 seconds). The sequences of primers used are presented in Supplementary Table S1.

Histologic and cytologic analyses

Ten adult livers or twenty 6-dpf larvae were fixed in 4% paraformaldehyde in PBS (Sigma) and paraffin-sectioned at 5-μm thickness using a microtome, followed by hematoxylin and eosin (H&E) staining or IHC. For immunofluorescent staining, the primary antibodies used included: rabbit anti-PCNA (Santa Cruz Technology), anti-caspase-3 (BD Biosciences), anti-cortisol (Sigma), anti-HSD11b (Novus Bio), anti-Tgfb1 (Millipore), anti-neutrophil elastase (Abcam), and anti-cd68 (Abcam).

Human patient samples

Paraffin-embedded human liver disease progression tissue microarray slides were purchased from Biomax Inc. (LV8011a). Patients were classified into 3 groups: normal (n = 5), pre-HCC (n = 23), and HCC (n = 30). These patient samples were subjected to H&E staining and IHC staining for HSD11B, cortisol, and TGFB1, respectively.

Statistical analysis

Statistical significance between 2 groups was evaluated by 2-tailed unpaired Student t test using inStat version 5.0 for Windows. Statistical data are presented as mean values ± SEM.

Results

More aggressive HCC promotion in male krasV12-expressing livers

In human, male patients develop more aggressive HCC, with larger tumor and lower survival rate, than female patients. To examine whether krasV12-induced zebrafish HCC is similar in a male biased manner, 3-month-old male and female kras+ zebrafish were exposed to doxycycline for 7 days, and their state of tumor progression was examined. Morphologically, male krasV12-expressing livers cover a larger area in the abdomen than female krasV12-expressing livers (Fig. 1A), accompanied with more severe histopathologic features. In male krasV12-expressing livers, oncogenic hepatocytes were densely arranged with prominent nucleolus; the typical 2-cell plate of hepatocytes observed in wild-type (WT) siblings were completely abrogated, denoting an early carcinoma status. In comparison, although female krasV12-expressing livers had more prominent nucleoli and vacuolated cytoplasm, the integrity of the 2-cell plate was largely maintained, with the majority having only hyperplasic histology (Fig. 1B). As summarized in Fig. 1D, 80% of the krasV12-expressing males had reached either adenoma or carcinoma stage, whereas only 30% of krasV12-expressing females showed adenoma phenotypes. To further investigate the mechanism of the gender disparity in liver morphology and histology, cell proliferation analysis was performed. As expected, krasV12-expressing hepatocytes had a higher rate of cell proliferation than their WT siblings (Fig. 1C and E). When comparing between genders, male krasV12-expressing hepatocytes had significantly higher numbers of proliferating cells than female krasV12-expressing hepatocytes, thus accounting for the difference in liver size between krasV12-expressing genders.

Figure 1.
Figure 1.

Gender disparity in krasV12-induced carcinogenesis. Three-month-old adult zebrafish were treated with 30 μg/mL doxycycline for 7 days and examined by various assays. A, Gross liver morphology of kras+ and fabp+ (control) male and female zebrafish after doxycycline exposure. Male krasV12-expressing liver (green) was significantly enlarged as compared with female krasV12-expressing liver and also to fabp+ male and female livers (red). B, H&E staining of liver sections of doxycycline-treated kras+ and WT male and female zebrafish. Examples of prominent nucleolus and vacuolated cytoplasm are indicated by white and red arrows, respectively. C, Immunofluorescent staining of liver sections of doxycycline-treated kras+ and WT male and female zebrafish with antibody against proliferating cell nuclear antigen (PCNA; red). D, Quantification of liver tumor histology based on H&E-stained liver sections of doxycycline-treated kras+ male and female zebrafish (n = 10 each group). E, Quantification of proliferating cells based on PCNA staining (n = 10 each group). F, Comparison of gene expression change after krasV12 induction in hepatocytes isolated from doxycycline-treated kras+ and fabp+ males and females. RNA expression of selected genes was determined by RT-qPCR. Fold changes are shown for FACS-isolated krasV12-expressing hepatocytes versus fabp10+ control hepatocytes in both males and females (*, P < 0.05). Scale bars, 3 mm in A and 20 μm in B and C.

To further elucidate the molecular mechanism of the gender disparity, expression of selected biomarker genes in FACS isolated krasV12-expressing oncogenic hepatocytes was compared with control hepatocytes from WT siblings of the same gender. Liver fibrosis–related genes (col1a1b and lamα5), angiogenesis genes (vegfab and vegfc), epithelial-to-mesenchymal transition genes (snail and slug), and neutrophil and macrophage chemoattractant genes (il8 and csf1a) all showed higher levels of increased expression in male oncogenic hepatocytes than female oncogenic hepatocytes as compared with their WT siblings of the same gender (Fig. 1F). In contrast, known antitumor cytokines, il12 and tnfa, were more upregulated in female oncogenic hepatocytes than in male OHs. Expression of egfp-krasV12 was consistent and similar between male and female kras+ zebrafish, suggesting that the difference in expression of these biomarker genes was due to a gender-mediated mechanism rather than a difference in induced krasV12expression (Fig. 1F).

Higher TAN and TAM infiltration in male krasV12-expressing livers

Gender disparity in HCC has been linked to differential immune cell responses between genders (8). To observe for a similar gender-discrepant response of neutrophils and macrophages in the kras+ zebrafish, kras+/lyz+, and kras+/mpeg+ double transgenic zebrafish were generated to observe for liver infiltration of neutrophils and macrophages. As shown in Fig. 2A and B and Supplementary Fig. S1, krasV12-expressing livers attracted neutrophils and macrophages within 7 days of oncogene activation with higher infiltration of neutrophils (defined as TANs) and macrophages (defined as TAMs) in male krasV12-expressing liver than in female krasV12-expressing liver.

Figure 2.
Figure 2.

Differential responses of neutrophils/macrophages in male and female zebrafish during krasV12-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 krasV12 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).

Because TAN and TAM infiltration showed a male biased manner, a more protumor behavior of these innate immune cells would explain the advanced disease status in male krasV12-expressing zebrafish. Gene expression profiles of FACS-isolated TANs and TAMs from livers of male and female adult krasV12-expressing zebrafish were compared with naïve neutrophils and naïve macrophages of their own gender. Both male TANs and TAMs showed higher increased expression of protumor (il10) and chemoattractant genes (il8 and csf1a) than female TANs/TAMs (Fig. 2C and D). In contrast, phagocytic enzyme genes (mpx, lyz, and mpeg) and an antitumor gene (il12) were downregulated in male TANs/TAMs as compared with female TANs/TAMs. Naïve neutrophils and naïve macrophages of wild-type males and females were quite similar in expression of these genes, with fold changes less than 1.5 (Supplementary Fig. S2). Thus, both TANs and TAMs in male krasV12-expressing livers showed higher protumor activity than those in female counterparts and they may contribute to the more severe HCC phenotype in males.

Acceleration of hepatocarcinogenesis by TANs and TAMs

To elucidate the roles of TANs and TAMs during krasV12-induced carcinogenesis, splice-blocking morpholinos, MO-gcsfr and MO-pu.1, were used to inhibit differentiation of neutrophils or macrophages in krasV12-expressing larvae, respectively. The effects of these morpholinos were validated by injection into lyz+ or mpeg+ embryos (Supplementary Fig. S3). As we previously reported (20), MO-gcsfr greatly decreased both counts and density of neutrophils and caused dramatic decrease of liver size in kras+ larvae as compared with kras+ larvae injected with MO-control (Fig. 3A–C). When MO-pu.1 was used to inhibit macrophage differentiation in kras+ larvae, macrophage density in both the liver and liver size was significantly decreased as compared with kras+ controls injected with MO-control (Fig. 3B and C). Hence, both TANs and TAMs appear to play an accelerating role during liver carcinogenesis. Finally, on co-injecting both MO-gcsfr and MO-pu.1 to deplete both TANs and TAMs in kras+ larvae, a more profound decrease in liver size was observed (Fig. 3B and C). To investigate whether the changes of liver size were due to cell-cycle aberration, cell proliferation and apoptosis analyses were performed. As summarized in Fig. 3D, decreased infiltration of TANs and TAMs into krasV12-expressing livers was corresponded to a decreased cell proliferation and increased apoptosis (Fig. 3D). Molecularly, TANs and TAMs showed a consistent change of gene expression profiles: chemoattractant genes (il8 and csf1a), protumor and inflammatory genes (nfκb2, cxcl1, and il1b) were significantly upregulated, whereas expression of antitumor and phagocytic genes (il12, tnfa, mpx, lyz, and mpeg) was less increased or downregulated (Fig. 3E).

Figure 3.
Figure 3.

Effect of TANs and TAMs on krasV12-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. KrasV12-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 krasV12 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 cortisol activities in genders induced differential activities of TANs and TAMs

To further understand the roles of TANs and TAMs in gender disparity in krasV12-expressing livers, possible molecular mechanisms were interrogated. Inconclusive clinical trial results with sex hormone therapy on patients with HCC suggested potential involvement of non–sex hormone during HCC. One potential candidate is cortisol, a well-known adrenal gland hormone involved in stress response (25, 26). Cortisol signaling is known to be linked to fatty liver and can influence both neutrophil and macrophage gene expression profile (27–29). Cortisol is secreted in a male biased manner (30), and estrogen can suppress the production of cortisol synthesis via downregulation of important cortisol synthesis genes (31). Hence, to investigate whether cortisol was involved in gender disparity in liver tumors, immunostaining with cortisol antibody confirmed a significantly higher cortisol level in the liver section of krasV12-expressing males than in krasV12-expressing females (Fig. 4A; Supplementary Fig. S4A). RT-qPCR analyses of 3 key genes in cortisol synthesis, cyp11a1, cyp17a1, and hsd11b1, showed significantly higher upregulation in OHs in males than in females as compared with control hepatocytes from WT siblings (Fig. 4B). This was also confirmed by immunostaining with Hsd11b antibody in the liver section of krasV12-expressing males than in females (Fig. 4C; Supplementary Fig. S4B).

Figure 4.
Figure 4.

Differential production of cortisol and induction of Tgfβ1 expression by cortisol in krasV12-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 krasV12induction 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 krasV12-expressing hepatocytes versus non–krasV12-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.

To further explain for the male dominant cortisol expression observed in krasV12-expressing zebrafish, possible molecular mechanisms were interrogated. As MAPK/ERK signaling is a major pathway of Kras activation, to investigate whether MAPK/ERK is involved in cortisol production, U0126, an Erk phosphorylation inhibitor, was used to treat kras+ male zebrafish, significant decreases of both 11Hsdb1 and cortisol were observed (Fig. 4A–C, Supplementary Fig. S4), suggesting that MAPK/ERK signaling promotes cortisol synthesis in krasV12-expressing hepatocytes as well. To further explain for the lack of cortisol expression in female krasV12-expressing zebrafish, male kras+ fish were exposed to 17β-estradiol (E2). Indeed, when E2 was exposure to male krasV12-expressing zebrafish, both cortisol and 11Hsdb1 were downregulated (Fig. 4A and C). Similarly, the 3 cortisol synthesis genes, cyp11a1, cyp17a1, and hsd11b1, were also down regulated compared with their respective controls (Fig. 4B),

Tgfb1 is a well-known inducer of protumor phenotype in neutrophils and macrophages (32) and has been demonstrated to be required for recruitment of TANs into krasV12-expressing zebrafish livers in our previous study (20). Interestingly, cortisol, a well-known adrenal gland hormone involved in stress response (25, 26), has been reported to upregulate the expression of tgfb1 in a variety of cell types such as T-cell, fibroblast, and osteoblasts (33, 34). To investigate whether the role of cortisol was through Tgfb, male kras+ fish were co-treated with doxycycline and mifepristone (a glucocorticoid receptor inhibitor) and female kras+ fish were co-treated with doxycycline and hydrocortisone (a cortisol analog). Both tgfb gene and protein expression were consistently inhibited by mifepristone in kras+ males; in contrast, additional cortisol in kras+ females caused increase of tgfb1 mRNA and protein (Fig. 4D and E).

To demonstrate whether cortisol could cause an increase of neutrophils and macrophages, 4-dpf larvae were treated with 10 mg/L cortisol for 4 days. Indeed, there were general increases of total neutrophils and macrophages after cortisol treatment accompanied with increases of liver size (Supplementary Fig. S5). To further demonstrate whether changes of cortisol activity could affect infiltration of TANs and TAMs in krasV12-expressing livers, kras+mpeg+ and kras+lyz+ males were co-treated with doxycycline and mifepristone whereas kras+mpeg+ and kras+lyz+ females were co-treated with doxycycline and hydrocortisone. As shown in Fig. 5A and B and Supplementary Fig. S6A and S6B, inhibition of cortisol signaling in kras+mpeg+ and kras+lyz+ males decreased TAM and TAN infiltration significantly, whereas increase of cortisol in kras+mpeg+ and kras+lyz+ females increased TAM and TAN infiltration significantly. Molecularly, TANs and TAMs in both co-treated krasV12-expressing males and females showed reversals of gene expression in phagocytic (lyz, mpx, and mpeg), protumor (il10), antitumor (il12), and chemoattractant (csf1a and il8) genes (Fig. 5C and D). These experiments implied a role of cortisol in induction of Tgfb expression, which in turn induced liver tumor infiltration of TANs and TAMs, thus promoting hepatocarcinogenesis.

Figure 5.
Figure 5.

Effects of cortisol on TAN and TAM infiltration in krasV12-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 krasV12 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).

High cortisol in adult male krasV12-expressing liver accelerates carcinogenesis

To further demonstrate whether cortisol-induced TAN and TAM infiltration could enhance carcinogenesis, liver tumor progression was investigated in male and female kras+ zebrafish treated with mifepristone or hydrocortisone together with doxycycline. Morphologically, doxycycline and mifepristone double-treated kras+ males showed slightly smaller livers than the kras+ males treated with doxycycline only. In contrast, kras+ female fish treated with hydrocortisone showed significantly enlarged livers even when compared with the 2 kras+ male groups, suggesting accelerated carcinogenesis (Fig. 6A).

Figure 6.
Figure 6.

Promotion of krasV12-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.

Histologically, doxycycline/mifepristone double-treated kras+ male fish had a lower cell density and were more hypervacuolated than kras+ males treated with doxycycline alone, with fewer fish reaching adenoma or carcinoma (Fig. 6B and C). In contrast, all kras+ female fish treated with hydrocortisone and doxycycline showed either adenoma or carcinoma (Fig. 6B and C). From cell proliferation and apoptosis analyses, inhibition of cortisol in krasV12-expressing males effectively reduced liver cell proliferation and apoptosis to levels even below those in krasV12-expressing female. Cortisol/doxycycline double-treated krasV12-expressing females showed much higher cell proliferation and apoptosis, which were even comparable with those in krasV12-expressing males (Fig. 6D and E). Thus, it is evident that the cortisol-induced increase in TANs and TAMs could, at least in part, contribute to the significant acceleration of carcinogenesis observed in male kras+ fish.

Gender difference in cortisol and TGFB1 level in human liver disease patients

In human liver diseases, increased cortisol levels have been observed in morbidly obese patients and, although less frequently, in patients with HCC (29, 35). To observe whether the gender disparate cortisol levels and the Tgfb1 induction observed in our krasV12 HCC zebrafish model would also be reflected in human patients, a panel of liver disease samples, including normal, pre-HCC (patients with liver inflammation and cirrhosis), and HCC, was analyzed for levels of HSD11B1, cortisol, and TGFB1 as well as infiltration of TANs (demarcated by neutrophil elastase) and TAMs (demarcated by CD68). Each sample was stained by H&E for confirmation of their histology (Fig. 7A) and then stained for HSD11B, cortisol, and TGFB1 (Fig. 7B–D). HSD11B1 expression was significantly higher in both pre-HCC and HCC males than in female patients (Fig. 7G). In contrast, cortisol level in liver disease patients appeared to be either highly detected or not detected at all. About 24% pre-HCC males (4 of 17) and 33% HCC male (7 of 21) patients had increased cortisol whereas only a single female patient with HCC (of 8) had a high cortisol level in the liver (Fig. 7G). Interestingly, both expression of HSD11B1 and accumulation of cortisol were restricted to liver sinusoids (Fig. 7B and C), but not within hepatic plates, suggesting the origin of cortisol from nonhepatocytes. Male-dominant TGFB1 expression was observed to be significantly higher in both pre-HCC and HCC samples (Fig. 7D and G). Similarly, both neutrophil and macrophage infiltrations were significantly higher in male patients with HCC than in female patients with HCC (Fig. 7E–G). Of the 8 patients with HCC (7 males and 1 female) with high levels of cortisol, correlation study between cortisol/TGFB1 (R = 0.76, P = 0.02), TGFB1/neutrophil elastase (R = 0.68, P = 0.08), and TGB1/CD68 (R = 0.88, P = 0.004) showed strong and positive linear relationship (Fig. 7H), suggesting that the molecular mechanism of cortisol-induced TGFB1 expression is potentially translated in this cohort of patients with liver disease.

Figure 7.
Figure 7.

Gender difference in levels of HSD11B, cortisol, and TGFB1 in human liver disease samples. A panel of liver disease samples from human patients was examined for histology by H&E staining and for levels of HSD11B, cortisol, and TGFB1 by antibody staining. These samples were categorized into normal, pre-HCC, and HCC for both males and females. A, H&E staining of human liver disease samples. B–F, IHC staining of antibody against HSD11B (B), cortisol (C), TGFB1 (D), neutrophil elastase (E), and CD68 (F). For cortisol staining in male pre-HCC and HCC groups, both positive and negative samples are shown in C. G, Quantification of the percentages of HSD11B, cortisol, TGFB1, neutrophil elastase (for neutrophil density), and CD68 (for macrophage density)-positive liver cells in pre-HCC and HCC samples. H, Correlations of cortisol to TGFB1, neutrophil elastase, and CD68 in cortisol-positive patients with HCC. *, P < 0.05. Scale bar, 20 μm.

Discussion

Previous studies on HCC gender disparity have been mainly focused on the effects of sex hormones because of 2 main reasons: (i) these hormones are produced in obvious gender-biased manners; (ii) expression of estrogen and androgen receptors in patients with HCC, suggesting susceptibility of the diseased cells to these hormones (36, 37). However, failure of estrogen- and androgen-related HCC clinical trials as well as inconclusive results from these clinical trials (2, 38, 39) has suggested the important roles of other potential factors. For example, prolactin, a female luteotropic hormone, has been recently shown to confer protection in females in an Myc-induced HCC mouse model, indicating the potential roles of other hormones in HCC gender disparity (7).

In this study, we demonstrated a novel role of cortisol in promoting gender disparity in HCC carcinogenesis by using a combination of human liver disease clinical samples and a krasV12-induced zebrafish HCC model (12). The gender differential production of cortisol and infiltration of immune cells were also similarly observed in another oncogene (xmrk) induced zebrafish HCC model (our unpublished data); thus, the involvement of cortisol in gender disparity of HCC is not oncogene-specific. Zebrafish is an ideal model for study of cortisol-elicited effects, as both human and zebrafish utilize cortisol as their main stress hormone whereas mouse and rat make use of corticosterone instead (40). We found a male dominant increase of cortisol synthesis in the krasV12-expressing livers in our zebrafish HCC model, which is in line with our human clinical studies. Several other independent studies have also demonstrated that cortisol is at a higher level in males than in females (26, 30, 41), and a high level of cortisol has been associated with a more aggressive breast, ovarian, cervical, and lymphoma cancer phenotype, mediated by a cancer-associated inflammation response (42). Notably, cortisol promotes liver disease progression by promoting hepatic dyslipidemia, and increased cortisol synthesis has been observed in livers of morbidly obese patients (29).

In our current study, we demonstrated a male-dominant cortisol expression in both male kras+ zebrafish and patients with HCC. The increased cortisol level in males could be attributed to an upregulated MAPK/ERK signaling, which can be inhibited by estrogen in females (Supplementary Fig. S7).

Furthermore, we showed a significant and positive correlation between cortisol, Tgfb1, and TAN/TAM infiltration in both human and zebrafish HCC. As zebrafish do not have Kupffer cells (liver resident macrophages), the increased TAM infiltration would be contributed by circulating macrophages (43). Previous studies conducted in both fish and humans have indicated that corticosteroids are capable of increasing circulating white blood cell count (44, 45). In addition, glucocorticoid can increase tgfb1 expression in a variety of human cell lines including T cells, fibroblasts, osteoclasts, etc. (33, 46). Mechanistically, glucocorticoids antagonize Tgfb-specific RNases, thus indirectly stabilizing TGFB1 mRNA (34). Tgfb1 has been shown to be a macrophage and neutrophil chemoattractant in human (47, 48), and it is also the best known inducer of protumorigenicity of TANs and TAMs (32). Hence, cortisol-induced Tgfb1 overexpression would possibly explain for the increased infiltration of TANs and TAMs as well as their more vivid behavior observed in male kras+ zebrafish. Our morpholino knockdown experiments to suppress differentiation of neutrophils and/or macrophages also indicated that TANs and TAMs are protumor during the initiation of hepatocarcinogenesis in zebrafish. This is in line with the prevailing knowledge in human and mouse studies that TANs and TAMs can promote tumor proliferation, angiogenesis, and epithelial–mesenchymal transition (20, 49). Furthermore, in human HCCs, both TAN and TAM infiltrations are indicators of poor disease prognosis (10, 50).

In conclusion, using both a zebrafish HCC model and human liver disease samples, cortisol was shown to contribute to gender disparity in HCC carcinogenesis via Tgfb1 to initiate the protumor responses of TANs and TAMs. This study demonstrated that hormones other than estrogen and androgen are not simply bystanders but may also actively contribute to gender disparity of HCC.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: C. Yan, Z. Gong

Development of methodology: C. Yan, Q. Yang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Yan, Q. Yang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Yan, Q. Yang, Z. Gong

Writing, review, and/or revision of the manuscript: C. Yan, Z. Gong

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Yan, Z. Gong

Study supervision: Z. Gong

Grant Support

The study was supported by National Medical Research Council of Singapore (grant numbers: NMRC/CBRG/0016/2002 and NMRC/CIRG/1373/2013) and Ministry of Education of Singapore (grant number: R154000667112).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

  • Received August 10, 2016.
  • Revision received December 12, 2016.
  • Accepted December 26, 2016.

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Tumor-Associated Neutrophils and Macrophages Promote Gender Disparity in Hepatocellular Carcinoma in Zebrafish
Chuan Yan, Qiqi Yang and Zhiyuan Gong
Cancer Res March 15 2017 (77) (6) 1395-1407; DOI: 10.1158/0008-5472.CAN-16-2200
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