Alcohol reshapes a liver premetastatic niche for cancer by extra- and intrahepatic crosstalk-mediated immune evasion

Abstract

Cancer metastatic organotropism is still a mystery. The liver is known to be susceptible to cancer metastasis and alcoholic injury. However, it is unclear whether and how alcohol facilitates liver metastasis and how to intervene. Here, we show that alcohol preferentially promotes liver metastasis in colon-cancer-bearing mice and post-surgery pancreatic cancer patients. The mechanism is that alcohol triggers an extra- and intrahepatic crosstalk to reshape an immunosuppressive liver microenvironment. In detail, alcohol upregulates extrahepatic IL-6 and hepatocellular IL-6 receptor expression, resulting in hepatocyte STAT3 signaling activation and downstream lipocalin-2 (Lcn2) upregulation. Furthermore, LCN2 promotes T cell-exhaustion neutrophil recruitment and cancer cell epithelial plasticity. In contrast, knocking out hepatocellular Stat3 or systemic Il6 in alcohol-treated mice preserves the liver microenvironment and suppresses liver metastasis. This mechanism is reflected in hepatocellular carcinoma patients, in that alcohol-associated signaling elevation in noncancerous liver tissue indicates adverse prognosis. Accordingly, we discover a novel application for BBI608, a small molecular STAT3 inhibitor that can prevent liver metastasis. BBI608 pretreatment protects the liver and suppresses alcohol-triggered premetastatic niche formation. In conclusion, under extra- and intrahepatic crosstalk, the alcoholic injured liver forms a favorable niche for cancer cell metastasis, while BBI608 is a promising anti-metastatic agent targeting such microenvironments.

Keywords: BBI608; STAT3; alcohol; lipocalin-2; liver; premetastatic niche; tumor targeting strategy.

Graphical abstract

Figure 1

Alcohol facilitates liver metastasis (A) Alcohol pretreatment promoted colon cancer cell metastasis to the liver in BALB/c (Ctrl, n = 8; Alco, n = 7) and C57BL/6 (n = 5) mice, but did not influence tumor growth in the spleen or the correlation between tumor burdens in liver and spleen. The metastases on liver were quantified by the gross liver weight, the tumor net weight, and (B) the relative metastatic area on the liver histopathological sections (n = 5). (C) Alcohol pretreatment had no effect on subcutaneous MC38 tumor growth in C57BL/6 mice (n = 7). (D) Analysis workflow for post-surgery patients with pancreatic cancer. (E) Alcohol exposure resulted in higher rates of liver metastasis. (F) Patients with liver metastasis had a higher proportion of alcohol exposure than those with other metastatic sites and those free of metastasis. (G) Alcohol exposure and liver metastasis influenced patient pancreatic cancer progression-free time. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 2

Alcohol promotes liver metastasis by activating hepatocyte STAT3 signaling (A) RNA sequencing revealed the differentially expressed genes in the alcohol-treated mouse liver (Ctrl, n = 3; Alco, n = 5). (B) Upregulated genes were highly enriched in IL-6-JAK-STAT3 signaling. (C) GSEA of the upregulated genes (Ctrl, n = 3; Alco, n = 5). (D) Analysis of mouse liver early micrometastases 48 h after splenic injection of MC38 cells. p-STAT3-positive hepatocytes around the tumor clones increased following alcohol pretreatment (n = 5). (E) Schematic gene organization of the Stat3^liverΔ mouse. (F) By Stat3 conditional knockout, RNA sequencing revealed differentially expressed genes and negatively enriched pathways from alcohol-treated mouse liver tissue (n = 4). (G) The qRT-PCR validation of the most dramatically changed genes modulated by alcohol and STAT3. (H) Suppressing STAT3 phosphorylation in hepatocyte Stat3 knockout restricted alcohol-induced liver metastasis, which is quantified by (I) the gross liver weight and the tumor net weight (n = 5) and (J) the relative liver metastatic area (n = 5). Red arrowheads indicate metastases. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 3

Extrahepatic IL-6 elevation is necessary for alcohol-induced liver metastasis (A) Liver Il6 mRNA (n = 5) and serum IL-6 protein (n = 8) levels in the alcohol-treated mouse. (B) Schematic gene organization of the Il6^{em1(Luc-eGFP)Smoc} mouse. (C) Luciferase and (D) GFP signals in different organs. (E) Alcohol-induced STAT3 phosphorylation and (F and G) liver metastasis were restricted by systemic Il6 knockout (n = 5). Red arrowheads indicate metastases. ∗p < 0.05, ∗∗p < 0.01.

Figure 4

Alcohol enhances hepatocyte Lcn2 expression and intrahepatic neutrophil infiltration via the IL-6/STAT3 axis (A) Lcn2 mRNA expression in alcohol-treated C57BL/6 mouse liver tissue (n = 5). (B) Lcn2 mRNA levels in primary C57BL/6 mouse hepatocytes treated with alcohol (50 mM), rmIL-6 (50 ng/mL), and BBI608 (0.1 mM) (n = 4). (C) Lcn2 mRNA levels in primary Stat3^flox/flox and Stat3^liverΔ mouse hepatocytes treated with alcohol and rmIL-6 (n = 4). (D) Neutrophil Lcn2 mRNA expression by alcohol treatment in vitro (n = 6). (E) Immunofluorescence of C57BL/6 mouse liver tissue sections under low-power field. (F) Alcohol-promoted liver LCN2 expression and neutrophil (labeled by Ly6G) infiltration (n = 5), which was reversed by knocking out (G) hepatocellular Stat3 (n = 5) and (H) systemic Il6 (n = 5), respectively. LCN2 expression and neutrophil infiltration were positively correlated. ‘L’ and ‘M’ denote liver and metastases, respectively. IOD, integral optical density. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 5

Alcohol induces liver CD8 T cell exhaustion in the liver (A) Neutrophil and exhausted T cell signatures were enhanced by alcohol treatment. (B) Correlation of Lcn2 FPKM value, neutrophil signature, and exhausted T cell signatures. In athymic mice, alcohol enhanced hepatocyte (C) p-STAT3 and (D) LCN2 expression and LCN2-positive neutrophil liver infiltration (n = 5), but (E and F) did not promote liver metastasis (n = 5). Red arrowheads indicate metastases. In immune-competent C57BL/6 mice, (G) CD8 T cell proportion was decreased by alcohol treatment in the liver, but (H) not influenced in the spleen (n = 5). (I) In vivo (n = 5), alcohol increased the hepatic PD-1⁺CTLA-4⁺CD8 cell proportion but decreased the PD-1⁻CTLA-4⁻ proportion. PD-1⁺CTLA-4⁺ cells showed low (J) Ki-67 and (K) IFN-γ expression, and alcohol further decreased it. (G), (H), (J), and (K) correspond to Figures S6A–S6D, respectively. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 6

LCN2 promotes T cell-exhausting neutrophil recruitment and cancer cell epithelial plasticity (A) Lymphocytes cocultured with neutrophils showed elevated PD-1 and CTLA-4 levels, characterized by IFN-γ downregulation. (B) Neutrophil morphology and (C) polarization (n = 4) were not altered by rmLCN2. (D) Neutrophil chemotaxis was enhanced by rmLCN2 and (E) alcohol-treated liver tissue homogenate, which was ameliorated by anti-LCN2 neutralizing antibodies. (F) MC38 cell proliferation was not altered by rmLCN2 treatment, but (G) adhesiveness and (H) epithelial plasticity were enhanced. ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 7

Alcohol-characteristic signaling activation in liver tissue promotes cancer progression (A–D) Survival analysis of hepatocellular carcinoma patients by alcohol-characteristic signaling activation in noncancerous liver tissue. (A) Schematic analysis workflow. (B) The human-homologous genes that were upregulated by alcohol in the mouse liver showed a higher average overall survival hazard ratio (HR) than the downregulated homologous genes. (C) HR distribution of the upregulated and downregulated genes. (D) The patients were categorized using the AlcoMetas score. High AlcoMetas score indicated adverse overall survival. (E) Correlation analysis of the IL-6-JAK-STAT3 signature and the LCN2 and LY6GC count values each with the AlcoMetas Score. (F) Kaplan-Meier overall survival analysis by the IL-6-JAK-STAT3 signature and LCN2 and LY6GC count values.

Figure 8

BBI608 prevents liver metastasis by targeting the alcohol-induced premetastatic liver niche (A) The expression of mouse liver genes sharing the backgrounds of both alcoholic injury and STAT3 modulation was assessed under the conditions of alcohol and/or BBI608. Analyses of (B) p-STAT3 expression by IHC, (C) LCN2 and Ly6G expression by immunofluorescence, and (D) infiltrated neutrophil and exhausted T lymphocyte proportions by flow cytometry (B, n = 5; C, n = 9; D, n = 4) in mouse liver treated with BBI608 and alcohol. (E and F) BBI608 administration against alcohol pretreatment prevented liver metastasis quantified by (E) the liver and tumor weight (n = 5) and (F) the liver metastatic area. Red arrowheads indicate metastases. (G) A schematic summarizing the current study. Alcohol induces hepatocyte STAT3 phosphorylation by upregulating hepatocellular IL-6R and extrahepatic IL-6. Consequently, hepatocytes upregulate and secrete LCN2, which induces neutrophil recruitment and cancer cell mesenchymal-to-epithelial transition. Neutrophils direct a T cell-exhausted immunosuppressive niche for cancer cell settlement. BBI608 is demonstrated as a promising drug against the alcohol-induced liver premetastatic niche. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.