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Activated hepatic stellate cells promote angiogenesis via interleukin-8 in hepatocellular carcinoma

  • †1,
  • †1,
  • †2, 5,
  • 3,
  • 4,
  • 1,
  • 1,
  • 1 and
  • 1Email author
†Contributed equally
Journal of Translational Medicine201513:365

https://doi.org/10.1186/s12967-015-0730-7

©  Zhu et al. 2015

  • Received: 30 May 2015
  • Accepted: 13 November 2015
  • Published:

Abstract

Background

Chemokines have been recognized as important modulators of angiogenesis, and they play critical roles in the development and metastasis of hepatocellular carcinoma (HCC), although their origins and latent molecular mechanisms remain elusive. The aim of this study was to investigate how activated hepatic stellate cells (a-HSCs) promote angiogenesis in HCC.

Methods

A total of 22 HCC patients were enrolled randomly. We used immunohistochemistry, western blotting, and enzyme-linked immunosorbent assay (ELISA) to analyse the production of interleukin-8 (IL-8) in a-HSCs derived from HCC tissues. The angiogenic effects of IL-8 in vitro and in vivo were assessed by ELISA, real-time quantitative polymerase chain reaction, capillary tube formation assay, and chick embryo chorioallantoic membrane assay.

Results

The present study showed that IL-8 was enriched predominantly in the tumour stroma of HCC tissues and was mainly derived from a-HSCs, rather than from hepatoma cells, in vivo and in vitro. Angiogenesis was most active at the invading edge, which was close to the a-HSCs. The angiogenic effect was dramatically attenuated by an IL-8 neutralizing antibody both in vitro and in vivo. Moreover, the IL-8 neutralizing antibody down-regulated Ser727-phosphorylated STAT3 levels in hepatoma cells treated with a-HSCs conditioned medium.

Conclusions

These findings reveal that a-HSCs within the stroma of HCC contribute to tumour angiogenesis via IL-8.

Keywords

  • Hepatocellular carcinoma
  • Activated hepatic stellate cells
  • Interleukin-8
  • Angiogenesis
  • Phospho-signal transducer and activator of transcription 3

Background

Hepatocellular carcinoma (HCC) is one of the most common solid malignant neoplasms and a leading cause of cancer-related death worldwide [1, 2]. Tumour progression relies on interactions between tumour cells and their surrounding microenvironment [3, 4]. The tumour microenvironment consists of stromal components, growth factors, proteolytic enzymes, extracellular matrix (ECM) proteins, and inflammatory cytokines [5, 6]. These elements play critical roles in tumour cell proliferation and survival as well as in angiogenesis and metastasis.

Angiogenesis plays a pivotal role in tumour growth and metastasis [7–9]. Dense tumour vasculature is associated with poor prognosis in certain types of cancer [10, 11]. Angiogenesis is regulated by transcription factors such as hypoxia inducible factor-1 (HIF-1), nuclear factor κB (NF-κB), and signal transducer and activator of transcription 3 (STAT3) [12]. In HCC, a net excess of angiogenic factors, including vascular endothelial growth factor (VEGF)-A, angiopoietins (Angs), and platelet-derived growth factor (PDGF)-B, is produced by tumour cells, vascular endothelial cells, immune cells, and pericytes [8, 13, 14].

The tumour microenvironment is composed of various cell types that influence the angiogenic response. Hepatic stellate cells (HSCs), the main type of stromal cell in the liver, are activated by tumour cells, inflammatory cytokines, and tumour-derived factors that are present in the surrounding milieu [15, 16]. Studies have suggested that activated HSCs (a-HSCs) play a crucial role in tumour angiogenesis and tumourigenesis [14, 17]. A-HSCs can promote the invasion of HCC cells by secreting matrix metalloproteinases (MMPs), urokinase-type plasminogen activator (uPA), interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), and other factors [18–21].

IL-8, alternatively known as CXC motif ligand 8 (CXCL-8), is a proinflammatory CXC chemokine [22–24]. Its biological effects are mediated through binding to two cell surface G protein-coupled receptors, CXC motif receptor 1 (CXCR1) and CXCR2 [12, 25, 26]. Accumulating evidence has demonstrated that IL-8 is associated with tumour angiogenesis, metastasis, and poor prognosis in many types of cancer [27–30]. However, the precise mechanism by which IL-8 promotes tumour angiogenesis in HCC is not fully understood.

In this paper, we demonstrated that a-HSCs played an important role in angiogenesis associated with HCC. A-HSCs secreted IL-8, which in turn stimulated hepatoma cells (Hep3B and Huh-7) to express angiogenic factors, such as VEGF-A, to promote angiogenesis. Furthermore, we showed that a-HSCs promoted angiogenesis via IL-8 by up-regulating Ser727-phosphorylated STAT3 levels in hepatoma cells. These findings characterized certain interactions within the tumour microenvironment, and the data may help researchers develop more effective therapeutic strategies against HCC.

Methods

Patients and specimens

Pathologically confirmed HCC samples were obtained from 22 patients undergoing hepatectomy between 2013 and 2014 at the Department of Hepatobiliary Surgery of the Third Affiliated Hospital of Sun Yat-sen University in Guangzhou. These patients provided signed informed consent, and they had received no previous local or systemic treatment before the operation. Individuals with autoimmune disease, HIV or syphilis were excluded. The clinical stage was classified according to the guidelines of the International Union against Cancer. The clinical characteristics of all the patients are summarized in Additional file 1: Table S1. All the samples were anonymously coded in accordance with the local ethical guidelines (as stipulated by the Declaration of Helsinki). The experiments were conducted in strict accordance with a study design approved by the Clinical Research Ethics Committee at the Third Affiliated Hospital of Sun Yat-sen University in Guangzhou.

Cell lines

Human hepatoma cell lines (Hep3B and Huh-7) were purchased from the Chinese Academy of Sciences. Primary HSCs were separated from the fresh tissue as described previously [31]. To minimize culture stress and clonal selection, HSCs passaged for up to 4-10 doublings were used for the experiments. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical cords with collagenase I (1 mg/mL; Sigma, USA) [32]. Only primary HUVECs at passages 3-6 were used in this study to avoid age-dependent cellular modifications [33].

Capillary tube formation assay

Diluted Matrigel (Matrigel: Medium 199 = 1:2) (BD, Bioscience, USA) was added to a 96-well plate (50 mL/well) and allowed to polymerize for 2 h at 37 °C. HUVECs were then added at a density of 3 × 104 cells per well in the 96-well plate with serum-free conditioned media from hepatoma cells, which were treated or not with HSC serum-free culture supernatant with or without the IL-8 neutralizing antibody (R&D Systems, USA) for 8 h in a 5 % CO2 incubator at 37 °C. Bloodvessel branch points were visualized by phase microscopy and quantitated in 3 random fields per well (10×) [34].

Immunohistochemical staining

Paraffin-embedded and formalin-fixed samples were cut into 4-µm-thick sections, which were then processed for immunohistochemical staining as previously described [35].

Immunofluorescence assay

A-HSCs growing in the 24-well flat-bottom plates were fixed, and stained with rabbit-anti-human monoclonal antibodies against alpha smooth muscle actin (α-SMA), vimentin and immunoglobulin (IgG, control) (Abcam, Cambridge, MA, USA) followed by treatment of AF488-conjugated donkey-anti-rabbit IgG (Molecular Probes, Carlsbad, CA, USA). The cell nuclei were counterstained with 4′-6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO, USA) as previously described [36, 37]. These images were assessed using a fluorescence microscope (Leica DMI 4000B, Germany) at a wavelength of 488 nm and analysed with Leica Application suite software (version 4.0).

Enzyme-linked immunosorbent assay (ELISA)

Concentrations of IL-8 and VEGF-A were detected by ELISA (R&D Systems, USA) as previously described [38]

Chick embryo chorioallantoic membrane (CAM) assay

White leghorn chicken eggs on the fifth day after fertilization were purchased from the Animal Husbandry Institute of South China Agricultural University. The air chambers of these eggs were windowed after disinfection with 75 % alcohol. The shell membranes were then removed carefully, and a 50 μL droplet was pipetted onto the CAM. These windows were sealed with sterile ventilate surgical tapes, and the eggs were incubated without rotation at 80 % relative humidity and at 37.8 °C in the incubator. After 48 h, the results were observed under a ZEISS Lumar.V12 stereomicroscope.

Multiplex bead–based enzyme–linked immunosorbent assay

According to the manufacturer’s instructions and a previously described method [39], conditioned supernatants were analysed using the multiplex bead–based Enzyme–Linked Immunosorbent Assay system (Millipore, Billerica, MA, USA). The results were analysed using a Luminex plate reader and the Milliplex analyst software (Luminex 200 System).

Western blotting

For western blotting, proteins were separated by 10 or 15 % SDS-acrylamide gels and then transferred to 0.20 μM nitrocellulose membranes. After immunoblotting with an antibody against glyceraldehyde-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, USA), the protein expression of IL-8 and other markers was detected with an ECL kit. The antibodies included the following: NF-κB p65 (C22B4; #4764) and STAT3 (124H6; #9139), as well as their phosphorylated forms (Ser536-phosphorylated NF-κB p65: 93H1, #3033; Ser727-phosphorylated STAT3: #9134) (Cell Signaling, Beverly, MA, USA); HIF-1α (Cell Signaling, Beverly, MA, USA); and GAPDH and β-actin (Santa Cruz Biotechnology, USA).

Real-time quantitative polymerase chain reaction (qPCR) analysis

Total RNA was extracted from cultured hepatoma cells using the TriPure Isolation Reagent (Roche Applied Science, Germany). The qPCR analysis of mRNA was executed according to the manufacturer’s instructions. The qPCR primer sequences corresponding to angiogenic factors are described in Table 1. All the reactions were performed in triplicate.
Table 1

Primer sequences for angiogenic factors

Factor

Primer sequence

VEGF-A

 Forward

5′-ATTCCCCACTTGAATCGGGC-3′

 Reverse

5′-TCACTCACTTTGCCCCTGTC-3′

VEGF-B

 

 Forward

5′-AGGCTATATCCCAGTGGGGG-3′

 Reverse

5′-ACAAGGGATGGCAGAAGAGC-3′

PDGF-A

 

 Forward

5′-CACTAAGCATGTGCCCGAGA-3′

 Reverse

5′-AGATCAGGAAGTTGGCGGAC-3′

PDGF-B

 Forward

5′-ACTGATGGGGTCGCTCTTTG-3′

 Reverse

5′-CAGGGATCAGGCAGGCTATG-3′

PDGF-C

 Forward

5′-GAGTCGCTGCTTCCAAAGTG-3′

 Reverse

5′-TCTTGTACTCCGTTCTGTTCCTT-3′

Ang-1

 Forward

5′-AGCAACTGGAGCTGATGGAC-3′

 Reverse

5′-TCCTCCCTTTAGTAAAACACCTTCT-3′

VEGF vascular endothelial growth factor, PDGF platelet-derived growth factor, Ang-1 angiopoietin-1

Statistical analysis

The data are presented as the mean ± standard error of the mean (SEM). The differences between groups were analysed using Student’s t test if only two groups were compared or using one-way analysis of variance (ANOVA) if more than two groups were compared. All the statistical tests were two-sided. All the experiments were performed at least three independent times. P values < 0.05 were considered statistically significant.

Results

A-HSCs secreted high levels of the inflammatory chemokine IL-8

To confirm that a-HSCs facilitate tumour angiogenesis, we first isolated a-HSCs from HCC tissues. A-HSCs were identified based on the high expression of the fibroblast-specific markers α-SMA and vimentin using fluorescence microscopy (Fig. 1A). Using the Multiplex bead-based Enzyme–Linked Immunosorbent Assay system, the levels of various inflammatory chemokines that are closely associated with angiogenesis, including GRO (α, β, and γ), CXCL-5, CXCL-6, CXCL-7 and IL-8, were detected in the 50 % a-HSC conditioned medium (HSC-CM) (CM:HSC-CM = 1:1) [23]. A-HSCs secreted significantly higher levels of IL-8 than of any other inflammatory chemokines (Fig. 1B).
Fig. 1
Fig. 1

A-HSCs expressed high levels of IL-8. A Immunofluorescent staining of primary human a-HSCs isolated from a representative sample of HCC with an anti-α-SMA antibody,anti-vimentin antibody, and IgG. Scale bar, 50 μM. B The levels of various angiogenic chemokines in the cell-free culture supernatants of the a-HSCs were measured by Multiplex bead–based enzyme–linked immunosorbent assay at day 2. The data are expressed as mean ± SEM of triplicates. (C) The concentrations of IL-8 (pg/mL) in the supernatants of a-HSCs and hepatoma cells were determined by ELISA. The concentration of IL-8 in the a-HSCs culture medium was markedly higher compared with hepatoma cells. (D) The expression of IL-8 in the abovementioned cells was assessed by Western blotting. A-HSCs spontaneously released large amounts of IL-8, whereas hepatoma cells produced low levels of IL-8. (E) Hepatocarcinoma samples were stained with various antibodies, and different levels of antibodies expression can be seen on the same section. E (a) Immunohistochemical staining for α-SMA (1:1000) to identify a-HSCs in the tumour stromal region. E (b) The IL-8 distribution in hepatocarcinoma samples was visualized by immunohistochemical staining using the IL-8 neutralizing antibody (1:400). E (c) Neovascularization in hepatocarcinoma samples was visualized by immunohistochemical staining for CD31 (1:400). Scale bar 200 μM. One of the 22 representative micrographs is shown

It was reported that tumour cells also secret the angiogenic factor IL-8 [12]. Therefore, we compared the levels of IL-8 secreted by a-HSCs with those secreted by hepatoma cells. The ELISA assay revealed that the concentration of IL-8 in the a-HSC culture medium was markedly higher than that in the culture medium of hepatoma cells (Fig. 1C). Consistently, IL-8 production by a-HSCs and hepatoma cells was further confirmed by Western blotting (Fig. 1D).

IL-8 was mainly enriched in the HCC stroma in vivo

To further study the role of IL-8 in tumour angiogenesis, we detected the distribution of IL-8 in tumour tissues from patients with HCC by immunohistochemistry. As shown in Fig. 1E (a and b), IL-8 was mainly enriched in the stroma surrounding the tumour, where numerous a-HSCs, as detected based on the fibroblast-specific marker α-SMA, were also present. This finding further confirmed that a-HSCs were the main source of IL-8 in HCC tissues. Furthermore, immunohistochemical staining for CD31 (Abcam, Cambridge, MA, USA), a microvessel marker, revealed that neovascularization occurred largely at the invading tumour edge, and close to the tumour stroma (Fig. 1E (c)).

IL-8 neutralizing antibody suppresses tumour angiogenesis in vitro and in vivo

To study the effect of IL-8 secreted by a-HSCs on angiogenesis, we collected the supernatants of a-HSCs and hepatoma cells cultured in the 50 % serum-free medium. Supernatants from untreated hepatoma cells afterculture for 24 h had only a slight effect on HUVEC tube formation. Supernatants from hepatoma cells that had been exposed to HSC-CM for 24 h significantly promoted angiogenic tube formation (Fig. 2a, b). Furthermore, the number of branch points within the capillary-like structures was dramatically reduced by the IL-8 neutralizing antibody (Fig. 2d, e).
Fig. 2
Fig. 2

IL-8 neutralizing antibody repressed tumour angiogenesis in vitro and in vivo. a, b Soluble factors derived from HSC-CM-treated hepatoma cells induced angiogenic tube formation in vitro. The tube formation assay was done using HUVECs in the presence of serum-free conditioned medium from hepatoma cells, HSC-CM-treated hepatoma cells alone or supplemented with an IL-8 neutralizing antibody, and the IL-8 neutralizing antibody dramatically inhibited HUVEC tube formation. The illustrated results represent six separate experiments. c In the CAM assay, more capillary-like structures developed in the presence of serum-free supernatants from Hep3B cells exposed to the 50 % serum-free HSC-CM compared with supernatants from untreated Hep3B cells. The number of these capillary-like structures was significantly reduced by the IL-8 neutralizing antibody. d, e Representative images of the number of branch points generated by HUVECs in vitro. The data are presented as mean ± SEM of four independent experiments. *p < 0.05; **p < 0.01

A similar effect of a-HSCs and IL-8 on tumour angiogenesis was also observed in the CAM animal model (Fig. 2c). The group of eggs that was treated with HSC-CM showed more marked signs of angiogenesis compared to the other two groups in the CAM assay. Consistently, treatment with the IL-8 neutralizing antibody markedly inhibited angiogenesis.

IL-8 neutralizing antibody down-regulated the expression of angiogenic factors in HSC-CM-treated hepatoma cells

We next investigated the secretion of angiogenic factors in hepatoma cells under various conditions. VEGF-A levels were higher in the medium from HSC-CM-treated hepatoma cells (Hep3B and Huh-7) than from normal medium-treated hepatoma cells (Hep3B: p = 0.0193; Huh-7: p = 0.0155). Notably, the concentration of VEGF-A was dramatically decreased when the HSC-CM-treated hepatoma cells were treated with the IL-8 neutralizing antibody (Hep3B: p = 0.0152; Huh-7: p = 0.0065) (Fig. 3). Consistent with the above findings, HSC-CM up-regulated the levels of VEGF-A mRNA (p = 0.010) and VEGF-B mRNA (p = 0.034) in Hep3B cells compared to the normal medium (Fig. 4). Additionally, the IL-8 neutralizing antibody effectively down-regulated VEGF-A mRNA (p = 0.034) and VEGF-B mRNA (p = 0.048) levels. However, the mRNA levels of other angiogenic factors (PDGF-A, PDGF-B, PDGF-C and Angiopoietin-1 (Ang-1)) did not change significantly. In Huh-7 cells, the mRNA expression of angiogenic factors (VEGF-A (p = 0.008), VEGF-B (p = 0.031), PDGF-B (p = 0.007), and PDGF-C (p = 0.047)) was up-regulated in response to stimulation with HSC-CM. The mRNA levels of these angiogenic factors (VEGF-A (p = 0.005), VEGF-B (p = 0.0244), PDGF-B (p = 0.001), and PDGF-C (p = 0.022)) were significantly reduced by the IL-8 neutralizing antibody. Nevertheless, PDGF-A mRNA and Ang-1 mRNA levels remained unchanged in response to various conditioned media.
Fig. 3
Fig. 3

VEGF-A levels in the supernatant of hepatoma cells subjected to various treatments. The concentrations of VEGF-A (pg/mL) were assessed in the presence of serum-free conditioned medium from hepatoma cells, HSC-CM-treated hepatoma cells alone or supplemented with an IL-8 neutralizing antibody by ELISA. The IL-8 neutralizing antibody significantly down-regulated VEGF-A levels in the supernatant of hepatoma cells that were treated with 50 % HSC-CM for 24 h. The data are expressed as mean ± SEM of triplicates. *p < 0.05; **p < 0.01

Fig. 4
Fig. 4

IL-8 neutralizing antibody down-regulated the mRNA expression of angiogenic factors in HSC-CM-treated hepatoma cells. a–f mRNA expression of angiogenic factors (VEGF-A, VEGF-B, PDGF-A, PDGF-B, PDGF-C, and Ang-1) in hepatoma cells after various treatments. In Hep3B cells, the mRNA levels of VEGF-A and VEGF-B were up-regulated in response to stimulation with HSC-CM and were down-regulated by IL-8 neutralizing antibody. The mRNA levels of PDGF-A, PDGF-B, PDGF-C, and Ang-1 remained unchanged in response to various conditioned media. In Huh-7 cells, the mRNA levels of VEGF-A, VEGF-B, PDGF-B, and PDGF-C were up-regulated in response to stimulation with HSC-CM and were down-regulated by IL-8 neutralizing antibody. The mRNA levels of PDGF-A and Ang-1 remained unchanged in response to various conditioned media. The data are presented as mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001

IL-8 neutralizing antibody down-regulated Ser727-phosphorylated STAT3 levels in HSC-CM-treated hepatoma cells

Evidence suggests that IL-8 modulates tumour angiogenesis by up-regulating the expression of the HIF-1, NF-κB, and STAT3 transcription factors [12]. We used western blotting to analyse these signalling pathways in hepatoma cells exposed to HSC-CM (Fig. 5a). Amongst these pathways, only Ser727-phosphorylated STAT3 was activated in HSC-CM-treated hepatoma cells (Fig. 5b, c). Moreover, Ser727-phosphorylated STAT3 was effectively inhibited in HSC-CM-treated hepatoma cells by the IL-8 neutralizing antibody, whereas HIF-1α and NF-κB p65 activation was not affected (Fig. 5d–f). Taken together, these results demonstrated that a-HSCs partially exerted their angiogenic function via IL-8, which up-regulated Ser727-phosphorylated STAT3 levels in hepatoma cells.
Fig. 5
Fig. 5

IL-8 neutralizing antibody exerted its function by suppressing Ser727-phosphorylated STAT3 signalling. a Hepatoma cells were cultured in CM alone (lanes 1 and 4), 50 % HSC-CM (lanes 2 and 5), or 50 % HSC-CM and the IL-8 neutralizing antibody (lanes 3 and 6). Twenty-four hours after treatment, HIF-1α, Ser727-phosphorylated STAT3, and Ser536-phosphorylated NF-κB p65 levels were determined by Western blotting. b–f The results of Western blot analyses were quantified by densitometry (relative ratio to GAPDH). Western blotting showed differences in Ser727-phosphorylated STAT3 expression among the hepatoma cells after various treatments. Data are presented as mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01. GAPDH was used as the internal control

Discussion

Although a series of studies has indicated that a-HSCs play a key role in tumour angiogenesis, recurrence, and metastasis [31, 40, 41], the underlying mechanism remains incompletely understood. Our study demonstrated that a-HSCs produce a large amount of IL-8 in vitro; IL-8 was enriched predominantly in the tumour stroma rather than in the cancer nests of HCC samples. Furthermore, the IL-8 neutralizing antibody partially attenuated the ability of HSC-CM to induce angiogenesis and down-regulated Ser727-phosphorylated STAT3 levels in hepatoma cells. These findings revealed a distinct interaction between a-HSCs and hepatoma cells within the tumour microenvironment, which promoted tumour angiogenesis.

It is generally believed that the tumour stroma plays a key role in angiogenesis associated with HCC, and that a-HSCs are enriched predominantly in the tumour stroma [5, 18, 42]. However, the specific contributions of these a-HSCs to HCC angiogenesis have not been extensively studied. The present study provides evidence that a-HSC-derived IL-8 promoted angiogenesis in HCC. Several of our observations support this notion. First, a-HSCs were present primarily in the tumour stroma. Second, neovascularization was more active at the invading tumour edge, which is closer to the tumour stroma. Third, IL-8 was secreted in vitro mainly by a-HSCs, rather than by hepatoma cells. Finally, IL-8 was enriched predominantly in the tumour stroma, and the main distribution area of IL-8 corresponded with the location of a-HSCs in vivo.

IL-8 is a potent pro-inflammatory cytokine that plays a critical role in tumour angiogenesis, a crucial step in tumour progression [30, 43]. When CD1 nude mice bearing DLD-1HIF−kd xenografts were treated with the IL-8 neutralizing antibody, the vessels were dramatically narrowed and fragmented, and the vascular density decreased [44]. In the current study, we observed that culture supernatants from HSC-CM-treated hepatoma cells significantly promoted angiogenic tube formation, and the angiogenic effects were significantly reduced by the IL-8 neutralizing antibody in vitro and in vivo. These results indicated that a-HSC-derived IL-8 is essential for HCC angiogenesis.

Previous studies have confirmed that IL-8 receptors (CXCR1 and CXCR2) are expressed on the surface of hepatoma cells [30]. IL-8 functions via its high-affinity receptors, CXCR1 and CXCR2 [26]. Evidence has indicated that IL-8 is closely associated with angiogenic factors during tumourigenesis [12, 45]. As a key mediator, IL-8 stimulates VEGF expression by activating the NF-κB pathway in endothelial cells [46]. Tumour angiogenesis is modulated by IL-8 via the up-regulated expression of the HIF-1, NF-κB, and STAT3 transcription factors [12]. However, in HIF-1-deficient tumours, IL-8 neutralization evokes a marked inhibition of tumour growth and angiogenesis [44]. In the current study, we found that IL-8 up-regulated angiogenic factors in hepatoma cells. Surprisingly, this process did not involve the transcription factor HIF-1α, NF-κB p65, Ser536-phosphorylated NF-κB p65, or STAT3, but instead required the activation of Ser727-phosphorylated STAT3. Our results provide direct evidence supporting the critical role of a-HSCs in HCC progression and reveal a fine-tuned collaboration between stromal cells and cancer cells in the tumour milieu.

The current understanding of the effect of a-HSCs on tumour angiogenesis strongly supports our findings [19, 40, 47]. Our research has some limitations. First, the 22 tumour samples evaluated in our study were not representative of all types of HCC, and the cohort was small considering the different conditions of these HCC patients. Second, further studies are needed to elucidate the molecular mechanism by which a-HSCs promote angiogenesis. Despite these shortcomings, our analysis shows that a-HSC-derived IL-8 plays a pivotal role in HCC-associated angiogenesis and reveals a fine-tuned collaboration between stromal cells and cancer cells in the tumour milieu. Larger studies are necessary to fully address this important question.

Conclusions

Our results indicate that a-HSCs promote HCC angiogenesis via IL-8, which provides important new insights into the roles of a-HSCs in HCC progression. Understanding the mechanisms of a-HSC-mediated angiogenesis in HCC will aid in the rational design of promising targets for anti-tumour therapy.

Notes

Abbreviations

HCC: 

hepatocellular carcinoma

a-HSCs: 

activated hepatic stellate cells

CXCL-8: 

C-X-C motif ligand 8

CXCR 1: 

C-X-C motif receptor 1

HUVECs: 

human umbilical vein endothelial cells

α-SMA: 

α-smooth muscle actin

IgG: 

immunoglobulin

ELISA: 

enzyme-linked immunosorbent assay

CM: 

conditioned medium

VEGF-A: 

vascular endothelial growth factor-A

CAM: 

chick embryo chorioallantoic membrane assay

GAPDH: 

glyceraldehyde-phosphate dehydrogenase

NF-κB: 

nuclear factor κB

STAT3: 

signal transducer and activator of transcription 3

HIF-1: 

hypoxia-inducible factor-1

qPCR: 

quantitative polymerase chain reaction

SEM: 

standard error of the mean

ANOVA: 

one-way analysis of variance

Declarations

Authors’ contributions

BZ, NL, and MZ (the principle investigators) performed the cell isolation, cell co-culture study, capillary tube formation assay, chick embryo chorioallantoic membrane assay, multiplex bead-based enzyme-linked immunosorbent assay, and immunofluorescence, generated the figures, and contributed to the design of the study and the writing of the manuscript. YZ, HC, and SC performed the immunohistochemistry, cell co-culture study, Western blotting, ELISA, and qPCR, and participated in the manuscript preparation. YL and DP acquired specimens and contributed to the design of the study. BZ and RX designed and coordinated the study, contributed to the experimental setup, data analysis and interpretation, and drafted and edited the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (NSFC-81272642) and the Natural Science Foundation of Guangdong Province, China (2014A030313144 and 2014A030313067).

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Hepatobiliary Surgery, The 3rd Affiliated Hospital of Sun Yat-sen University, No. 600 Tianhe Road, Guangzhou, 510630, Guangdong, China
(2)
Department of Infectious Diseases, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
(3)
Department of Gastrointestinal Surgery, The 4th Affiliated Hospital of Anhui Medical University, Hefei, China
(4)
Department of Ophthalmology, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
(5)
Guangdong Provincial Key Laboratory of Liver Disease Research, Guangzhou, China

References

  1. Fernandez M, Semela D, Bruix J, Colle I, Pinzani M, Bosch J. Angiogenesis in liver disease. J Hepatol. 2009;50:604–20.View ArticlePubMedGoogle Scholar
  2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61:69–90.View ArticlePubMedGoogle Scholar
  3. Quail DF, Joyce JA. Microenvironmental regulation of tumor progression and metastasis. Nat Med. 2013;19:1423–37.PubMed CentralView ArticlePubMedGoogle Scholar
  4. Berns A, Pandolfi PP. Tumor microenvironment revisited. EMBO Rep. 2014;15:458–9.PubMed CentralView ArticlePubMedGoogle Scholar
  5. Li T, Yang Y, Hua X, Wang G, Liu W, Jia C, Tai Y, Zhang Q, Chen G. Hepatocellular carcinoma-associated fibroblasts trigger NK cell dysfunction via PGE2 and IDO. Cancer Lett. 2012;318:154–61.View ArticlePubMedGoogle Scholar
  6. Leonardi GC, Candido S, Cervello M, Nicolosi D, Raiti F, Travali S, Spandidos DA, Libra M. The tumor microenvironment in hepatocellular carcinoma (review). Int J Oncol. 2012;40:1733–47.PubMedGoogle Scholar
  7. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285:1182–6.View ArticlePubMedGoogle Scholar
  8. Weis SM, Cheresh DA. Tumor angiogenesis: molecular pathways and therapeutic targets. Nat Med. 2011;17:1359–70.View ArticlePubMedGoogle Scholar
  9. Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol. 2002;29:15–8.View ArticlePubMedGoogle Scholar
  10. Uzzan B, Nicolas P, Cucherat M, Perret GY. Microvessel density as a prognostic factor in women with breast cancer: a systematic review of the literature and meta-analysis. Cancer Res. 2004;64:2941–55.View ArticlePubMedGoogle Scholar
  11. Ushijima C, Tsukamoto S, Yamazaki K, Yoshino I, Sugio K, Sugimachi K. High vascularity in the peripheral region of non-small cell lung cancer tissue is associated with tumor progression. Lung Cancer. 2001;34:233–41.View ArticlePubMedGoogle Scholar
  12. Waugh DJ, Wilson C. The interleukin-8 pathway in cancer. Clin Cancer Res. 2008;14:6735–41.View ArticlePubMedGoogle Scholar
  13. Gerald D, Chintharlapalli S, Augustin HG, Benjamin LE. Angiopoietin-2: an attractive target for improved antiangiogenic tumor therapy. Cancer Res. 2013;73:1649–57.View ArticlePubMedGoogle Scholar
  14. Zhu AX, Duda DG, Sahani DV, Jain RK. HCC and angiogenesis: possible targets and future directions. Nat Rev Clin Oncol. 2011;8:292–301.PubMed CentralView ArticlePubMedGoogle Scholar
  15. Casazza A, Di Conza G, Wenes M, Finisguerra V, Deschoemaeker S, Mazzone M. Tumor stroma: a complexity dictated by the hypoxic tumor microenvironment. Oncogene. 2014;33:1743–54.View ArticlePubMedGoogle Scholar
  16. Shi C, Washington MK, Chaturvedi R, Drosos Y, Revetta FL, Weaver CJ, Buzhardt E, Yull FE, Blackwell TS, Sosa-Pineda B, et al. Fibrogenesis in pancreatic cancer is a dynamic process regulated by macrophage-stellate cell interaction. Lab Invest. 2014;94:409–21.PubMed CentralView ArticlePubMedGoogle Scholar
  17. Erkan M, Hausmann S, Michalski CW, Fingerle AA, Dobritz M, Kleeff J, Friess H. The role of stroma in pancreatic cancer: diagnostic and therapeutic implications. Nat Rev Gastroenterol Hepatol. 2012;9:454–67.View ArticlePubMedGoogle Scholar
  18. Coulouarn C, Corlu A, Glaise D, Guenon I, Thorgeirsson SS, Clement B. Hepatocyte-stellate cell cross-talk in the liver engenders a permissive inflammatory microenvironment that drives progression in hepatocellular carcinoma. Cancer Res. 2012;72:2533–42.PubMed CentralView ArticlePubMedGoogle Scholar
  19. Thompson AI, Conroy KP, Henderson NC. Hepatic stellate cells: central modulators of hepatic carcinogenesis. BMC Gastroenterol. 2015;15:63.PubMed CentralView ArticlePubMedGoogle Scholar
  20. Amann T, Bataille F, Spruss T, Muhlbauer M, Gabele E, Scholmerich J, Kiefer P, Bosserhoff AK, Hellerbrand C. Activated hepatic stellate cells promote tumorigenicity of hepatocellular carcinoma. Cancer Sci. 2009;100:646–53.View ArticlePubMedGoogle Scholar
  21. Wu SD, Ma YS, Fang Y, Liu LL, Fu D, Shen XZ. Role of the microenvironment in hepatocellular carcinoma development and progression. Cancer Treat Rev. 2012;38:218–25.View ArticlePubMedGoogle Scholar
  22. Rossi D, Zlotnik A. The biology of chemokines and their receptors. Annu Rev Immunol. 2000;18:217–42.View ArticlePubMedGoogle Scholar
  23. Zlotnik A, Yoshie O. The chemokine superfamily revisited. Immunity. 2012;36:705–16.PubMed CentralView ArticlePubMedGoogle Scholar
  24. Waugh DJ, Wilson C, Seaton A, Maxwell PJ. Multi-faceted roles for CXC-chemokines in prostate cancer progression. Front Biosci. 2008;13:4595–604.View ArticlePubMedGoogle Scholar
  25. Holmes WE, Lee J, Kuang WJ, Rice GC, Wood WI. Structure and functional expression of a human interleukin-8 receptor. Science. 1991;253:1278–80.View ArticlePubMedGoogle Scholar
  26. Campbell LM, Maxwell PJ, Waugh DJ. Rationale and means to target pro-inflammatory interleukin-8 (CXCL8) signaling in cancer. Pharmaceuticals (Basel). 2013;6:929–59.View ArticleGoogle Scholar
  27. Bendrik C, Dabrosin C. Estradiol increases IL-8 secretion of normal human breast tissue and breast cancer in vivo. J Immunol. 2009;182:371–8.View ArticlePubMedGoogle Scholar
  28. Brat DJ, Bellail AC, Van Meir EG. The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis. Neuro Oncol. 2005;7:122–33.PubMed CentralView ArticlePubMedGoogle Scholar
  29. Maxwell PJ, Coulter J, Walker SM, McKechnie M, Neisen J, McCabe N, Kennedy RD, Salto-Tellez M, Albanese C, Waugh DJ. Potentiation of inflammatory CXCL8 signalling sustains cell survival in PTEN-deficient prostate carcinoma. Eur Urol. 2013;64:177–88.PubMed CentralView ArticlePubMedGoogle Scholar
  30. Tang KH, Ma S, Lee TK, Chan YP, Kwan PS, Tong CM, Ng IO, Man K, To KF, Lai PB, et al. CD133(+) liver tumor-initiating cells promote tumor angiogenesis, growth, and self-renewal through neurotensin/interleukin-8/CXCL1 signaling. Hepatology. 2012;55:807–20.View ArticlePubMedGoogle Scholar
  31. Santamato A, Fransvea E, Dituri F, Caligiuri A, Quaranta M, Niimi T, Pinzani M, Antonaci S, Giannelli G. Hepatic stellate cells stimulate HCC cell migration via laminin-5 production. Clin Sci (Lond). 2011;121:159–68.View ArticleGoogle Scholar
  32. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973;52:2745–56.PubMed CentralView ArticlePubMedGoogle Scholar
  33. Hu L, Roth JM, Brooks P, Luty J, Karpatkin S. Thrombin up-regulates cathepsin D which enhances angiogenesis, growth, and metastasis. Cancer Res. 2008;68:4666–73.View ArticlePubMedGoogle Scholar
  34. Huang YQ, Li JJ, Hu L, Lee M, Karpatkin S. Thrombin induces increased expression and secretion of angiopoietin-2 from human umbilical vein endothelial cells. Blood. 2002;99:1646–50.View ArticlePubMedGoogle Scholar
  35. Unitt E, Rushbrook SM, Marshall A, Davies S, Gibbs P, Morris LS, Coleman N, Alexander GJ. Compromised lymphocytes infiltrate hepatocellular carcinoma: the role of T-regulatory cells. Hepatology. 2005;41:722–30.View ArticlePubMedGoogle Scholar
  36. Zhang M, Wang F, Chong Y, Tai Q, Zhao Q, Zheng Y, Peng L, Lin S, Gao Z. Liver myofibroblasts from hepatitis B related liver failure patients may regulate natural killer cell function via PGE2. J Transl Med. 2014;12:308.PubMed CentralView ArticlePubMedGoogle Scholar
  37. Li T, Yi S, Liu W, Jia C, Wang G, Hua X, Tai Y, Zhang Q, Chen G. Colorectal carcinoma-derived fibroblasts modulate natural killer cell phenotype and antitumor cytotoxicity. Med Oncol. 2013;30:663.View ArticlePubMedGoogle Scholar
  38. Zhang M, Ye Y, Wang F, Zhu J, Zhao Q, Zheng Y, Gu Y, Xie C, Huang Z, Tai Q, et al. Liver myofibroblasts up-regulate monocyte CD163 expression via PGE2 during hepatitis B induced liver failure. J Transl Med. 2014;12:60.PubMed CentralView ArticlePubMedGoogle Scholar
  39. Zhang M, Wang FL, Zhu JY, Zheng YB, Zhao QY, Gu YR, Zhang Q, Chong YT, Gao ZL. Liver myofibroblasts regulate the phenotype and function of monocytes through soluble factors in cirrhosis. Exp Ther Med. 2013;5:143–9.PubMed CentralPubMedGoogle Scholar
  40. Coulouarn C, Clement B. Stellate cells and the development of liver cancer: therapeutic potential of targeting the stroma. J Hepatol. 2014;60:1306–9.View ArticlePubMedGoogle Scholar
  41. Yin C, Evason KJ, Asahina K, Stainier DY. Hepatic stellate cells in liver development, regeneration, and cancer. J Clin Invest. 2013;123:1902–10.PubMed CentralView ArticlePubMedGoogle Scholar
  42. Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer. 2006;6:392–401.View ArticlePubMedGoogle Scholar
  43. Sparmann A, Bar-Sagi D. Ras-induced interleukin-8 expression plays a critical role in tumor growth and angiogenesis. Cancer Cell. 2004;6:447–58.View ArticlePubMedGoogle Scholar
  44. Mizukami Y, Jo WS, Duerr EM, Gala M, Li J, Zhang X, Zimmer MA, Iliopoulos O, Zukerberg LR, Kohgo Y, et al. Induction of interleukin-8 preserves the angiogenic response in HIF-1alpha-deficient colon cancer cells. Nat Med. 2005;11:992–7.PubMedGoogle Scholar
  45. Singh JK, Simoes BM, Howell SJ, Farnie G, Clarke RB. Recent advances reveal IL-8 signaling as a potential key to targeting breast cancer stem cells. Breast Cancer Res. 2013; 15:210.PubMed CentralView ArticlePubMedGoogle Scholar
  46. Martin D, Galisteo R, Gutkind JS. CXCL8/IL8 stimulates vascular endothelial growth factor (VEGF) expression and the autocrine activation of VEGFR2 in endothelial cells by activating NFkappaB through the CBM (Carma3/Bcl10/Malt1) complex. J Biol Chem. 2009; 284:6038–42.PubMed CentralView ArticlePubMedGoogle Scholar
  47. Jung JO, Gwak GY, Lim YS, Kim CY, Lee HS. Role of hepatic stellate cells in the angiogenesis of hepatoma. Korean J Gastroenterol. 2003;42:142–8.PubMedGoogle Scholar

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