Adipocytes promote tumor progression and induce PD-L1 expression via TNF-α/IL-6 signaling

Abstract

Background: Obesity confers increased risk for various types of cancer. PD-L1 is a key molecule in tumor immune evasion by inducing T cell exhaustion. The relationship between obesity and PD-L1 is still ambiguous. This study was designed to reveal the development of hepatocellular carcinoma and melanoma in obese mice and to investigate if adipocytes regulate PD-L1 expression and the underlying mechanism.

Methods: Monosodium glutamate-induced obese mice were inoculated with H22 tumor cells and High fat diet (HFD)-induced obese mice were inoculated with B16-F1 mouse melanoma cells. Human hepatoma HepG2 cells and B16-F1 cells were treated with conditional media from 3T3-L1 adipocytes (adi-CM). Neutralized anti-TNF-α and anti-IL-6 antibodies and inhibitor of NF-κB or STAT3 were used to reveal the mechanism of effect of adi-CM.

Results: In obese mice, H22 and B16-F1 tumor tissues grew faster and PD-L1 expression in tumor tissue was increased. Adi-CM up-regulated PD-L1 level in HepG2 and B16-F1 cells in vitro. Differentiated 3T3-L1 adipocytes secreted TNF-α and IL-6, and neutralizing TNF-α and/or IL-6 reduced PD-L1 expression in adi-CM-treated cells. p-NF-κB/NF-κB level was downregulated in HepG2 and B16-F1 cells, and p-STAT3/STAT3 level was also decreased in HepG2 cells. In addition, inhibitor of NF-κB or STAT3 reversed the effect of adi-CM on PD-L1 expression.

Conclusions: TNF-α and IL-6 secreted by adipocytes up-regulates PD-L1 in hepatoma and B16-F1 cells, which may be at least partially involved in the role of obesity in promoting tumor progression.

Keywords: Adipocytes; Hepatocellular carcinoma; IL-6; Melanoma; PD-L1; TNF-α.

Fig. 1

Tumor growth was promoted in MSG-IO and DIO mice. a Representative images of control and MSG-IO mice at 15 weeks of age. b Body weight, waist circumference (c) and Lee’s index (d) measured in MSG-IO model. e Representative images of control and DIO mice at 24 weeks of age. f Body weight, waist circumference (g) and Lee’s index (h) measured in DIO model. i Representative images and weights of tumor tissues in MSG-IO model after 17 days of cell inoculation. j Representative images and weights of tumor tissues in DIO model after 20 days of cell inoculation. Data are expressed as mean ± SEM, n = 12, **P < 0.01 and ***P < 0.001 Vs control

Fig. 2

PD-L1 expression of tumor tissue was increased in obese mice. a PD-L1 protein levels in tumor tissue of mice in MSG-IO model detected by western blot. b PD-L1 protein levels in tumor tissue of mice in DIO model detected by western blot. c Representative immunohistochemistry staining and quantitative analysis of CD8⁺ T cells in H22 tumor tissue. d Representative immunohistochemistry staining and quantitative analysis of CD8⁺ T cells in B16-F1 tumor tissue. Scale bar 50 μM. Data are expressed as mean ± SEM, n = 6 (western blot) and n = 3 (immunohistochemistry), *P < 0.05 and **P < 0.01 Vs control

Fig. 3

Conditional media of 3T3-L1 adipocytes induced PD-L1 expression on HepG2 and B16-F1 cells. a Oil-red O staining and quantification of lipids in 3T3-L1 preadipocytes and adipocytes. Scale bar 10 μm. Data are expressed as mean ± SEM, n = 9, ***P < 0.001 Vs preadipocytes group. b PD-L1 levels of HepG2 cells treated with or without adipocytes conditional media (adi-CM) for 48 h. c PD-L1 levels of B16-F1 cells treated with or without adi-CM for 48 h. Data are expressed as mean ± SEM, n = 6, *P < 0.05 and **P < 0.01 Vs control

Fig. 4

Blockade of TNF-α/IL-6 decreased PD-L1 expression. a Levels of TNF-α and IL-6 in the supernatant of differentiated 3T3-L1 adipocytes detected by ELISA. b Representative western blot images of PD-L1 and phosphorylated-NF-κB protein expression in adi-CM-treated B16-F1 cells. Anti-TNF-α antibody and/or anti-IL-6 antibody were used in neutralizing TNF-α and IL-6. c Quantification of PD-L1 and phosphorylated-NF-κB (d) protein expression on B16-F1 cells treated. e Representative western blot images of PD-L1, phosphorylated-STAT3 and phosphorylated-NF-κB protein expression in adi-CM-treated HepG2 cells with the addition of anti-TNF-α antibody and/or anti-IL-6 antibody. f Quantification of PD-L1, phosphorylated-NF-κB (g) and phosphorylated-STAT3 (h) protein expression on HepG2 cells treated. Data are expressed as mean ± SEM, n = 4 (ELISA) and n = 6 (western blot), ***P < 0.001 Vs adi-CM group. ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 Vs IgG group

Fig. 5

Inhibition of NF-κB or STAT3 downregulated PD-L1 expression in adi-CM model. a PD-L1 protein levels in HepG2 cells treated with different concentration of NF-κB inhibitor, withaferin A (WA). b PD-L1 protein levels in HepG2 cells treated with different concentration of STAT3 inhibitor, BP-1-102. c PD-L1 protein levels in B16-F1 cells incubated with WA. Data are expressed as mean ± SEM, n = 6, **P < 0.01, ***P < 0.001 Vs adi-CM group. ^##P < 0.01 and ^###P < 0.001 Vs DMSO group