Activated hepatic stellate cells promote tumorigenicity of hepatocellular carcinoma

Abstract

Liver cirrhosis is the main risk factor for the development of hepatocellular carcinoma (HCC). Activated hepatic stellate cells (HSC) are the effector cells of hepatic fibrosis and also infiltrate the HCC stroma where they might play a critical role in HCC progression. Here we aimed to analyze the effects of activated HSC on the proliferation and growth of HCC cell lines in vitro and in vivo. Conditioned media (CM) collected from HSC significantly induced proliferation and migration of HCC cells cultured in monolayers. In a 3-dimensional spheroid coculture system, HSC promoted HCC growth and diminished the extent of central necrosis. In accordance, in vivo simultaneous implantation of HSC and HCC cells into nude mice promoted tumor growth and invasiveness, and inhibited necrosis formation. As potential mechanism of the tumorigenic effects of HSC we identified activation of NFkappaB and extracellular-regulated kinase (ERK) in HCC cells, two signaling cascades that play a crucial role in HCC progression. In summary, our data indicate that stromal HSC promotes HCC progression and suggest the HSC-HCC interaction as an interesting tumor differentiation-independent target for therapy of this highly aggressive cancer.

Figure 1

Effect of conditioned medium collected from activated hepatic stellate cells (HSC) on hepatocellular carcinoma (HCC) cells in vitro. HCC cell lines (HepG2, Hep3B and PLC) were stimulated with conditioned medium (CM) collected from in vitro activated HSC. (A) Proliferation was analyzed applying XTT‐assays. Migratory potential analyzed in (B) Boyden chamber and (C) monolayer scratch assays. (D) Representative pictures of the areas between scratch fronts after 2 days (I: Hep3B; II: Hep3B plus CM). (*P < 0.05 compared to control).

Figure 2

Effect of activated hepatic stellate cells (HSC) on hepatocellular carcinoma (HCC) cells in a three dimensional in vitro coculture system. (A) Volume of spheroids formed by in vitro activated HSC, Hep3B cells, and a 1:1 mixture of activated HSC and Hep3B cells after 10 days. The dashed bar indicates the virtual sum of the mean volumes of pure HSC and Hep3B spheroids. (*P < 0.05). (B) Histochemical staining (hematoxylin and eosin) of spheroids formed by pure Hep3B cells or a 1:1 mixture of activated HSC and Hep3B cells after 10 days. The diameter of central necrosis (white arrow) and the outer diameter of the spheroid (black arrow), respectively, are indicated. (I: HCC; II: HCC plus HSC).

Figure 3

Effect of activated hepatic stellate cells (HSC) on hepatocellular carcinoma (HCC) cells in vivo. (A) Growth kinetic of tumors formed in nude mice after implantation of pure HepG2 cells or HepG2 cells together with activated HSC. (*P < 0.05). (B–E) (Immuno‐)histochemical staining pictures of tumors formed in nude mice after implantation of pure HepG2 cells (I) or HepG2 cells together with activated HSC (II) (day 16): (B) CD31 (C) hematoxylin and eosin (D) alpha‐smooth muscle actin, and (E) sirius‐red fast green staining. (100×; representative staining results are depicted).

Figure 4

Effect of activated hepatic stellate cells (HSC) on activation of extracellular‐regulated kinase (ERK) and NFkappaB in hepatocellular carcinoma (HCC) cells. HCC cells were stimulated with conditioned medium (CM) collected from in vitro activated HSC. Non‐stimulated cells served as control. (A) Western blot analysis using an antibody against phosphorylated ERK, p44 and p42, designed ERK1 and ERK2. Analysis of activated nuclear NFkappaB (B) and (C) interleukin (IL)‐8 mRNA expression. (*P < 0.05 compared to control). (D–F) Prior to stimulation with CM Hep3B cells were incubated with MG132 (2 µM), SU 5402 (15 µM), or aminoguanidin (AG; 500 µM) for 30 min. Subsequently, proliferation was analyzed applying XTT‐assays (D), and the migratory potential was analyzed in Boyden chamber assays (E, F). (*P < 0.05).

Figure 5

Effect of anti‐hepatocyte growth factor (HGF) antibodies on the proliferation and migration of hepatocellular carcinoma (HCC) cells in the presence of conditioned medium from activated hepatic stellate cells (HSC). Conditioned medium (CM) from activated HSC was preincubated with anti‐HGF antibodies or isotype matched control antibodies (contr. IgG) before addition to HCC cells. Subsequently (A) proliferation was analyzed applying XTT‐assays, and (B) migratory potential was analyzed in Boyden chamber assays. (*P < 0.05).