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. 2013 Nov 20;8(11):e80212.
doi: 10.1371/journal.pone.0080212. eCollection 2013.

Intratumoral hepatic stellate cells as a poor prognostic marker and a new treatment target for hepatocellular carcinoma

Bin Sun  1 , Xiaofeng Zhang, Xianshuo Cheng, Yu Zhang, Lei Chen, Lehua Shi, Zhenyu Liu, Haihua Qian, Mengchao Wu, Zhengfeng Yin
Affiliations

Intratumoral hepatic stellate cells as a poor prognostic marker and a new treatment target for hepatocellular carcinoma

Bin Sun et al. PLoS One. .

Abstract

Hepatic stellate cells (HSCs), a specialized stromal cytotype in the liver, have been demonstrated to actively contribute to hepatocellular carcinoma (HCC) development. However, the previous studies were performed using HSC cell lines, and the prognostic value of intratumoral HSCs (tHSCs) was unclear. Here we isolated tHSCs from fresh human HCC tissues, and analyzed the abilities of tHSCs to promote HCC progression by using in vitro assays for cell viability, migration and invasion as well as epithelial-mesenchymal transition (EMT) phenotype. 252 HCC patients who underwent hepatectomy were enrolled for analysis of tHSCs and E-cadherin expression in tumor tissues, and 55 HCC patients for analysis of tHSCs in tumor tissues and circulating tumor cells (CTCs) in blood. Prognostic factors were then identified. The results showed that coculture of tHSCs with HCC cells had a stronger effect on HCC cell viability, migration and invasion, accompanied with the acquisition of epithelial-mesenchymal transition (EMT) phenotype. In vivo cotransplantation of HCC cells with tHSCs into nude mice more efficiently promoted tumor formation and growth. Icaritin, a known apoptosis inducer of HSCs, was demonstrated to effectively inhibit tHSC proliferation in vitro and tHSC-induced HCC-promoting effects in vivo. Clinical evidence indicated that tHSCs were rich in 45% of the HCC specimens, tHSC-rich subtypes were negatively correlated either with E-cadherin expression in tumor tissues (r = -0.256, p < 0.001) or with preoperative CTCs in blood (r = -0.287, p = 0.033), and were significantly correlated with tumor size (p = 0.027), TNM staging (p = 0.018), and vascular invasion (p = 0.008). Overall and recurrence-free survival rates of tHSC-rich patients were significantly worse than those for tHSC-poor patients. Multivariate analysis revealed tHSC-rich as an independent factor for overall and recurrence-free survival. In conclusion, tHSCs provide a promising prognostic biomarker and a new treatment target for HCC.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. tHSC-CM promoted proliferation, migration and invasion of PLC/PRF/5 cells.
(A and B) tHSC-CM significantly promoted HCC cell growth with time- and concentration-dependent manner. (C) Representative images of wound migration assays (left panel). Results are expressed as the percentage of the wounded area (right panel). (D) Representative images of invasion assays performed by transwell chamber (left panel). Results are expressed as the number of cells per field (right panel). *p < 0.05; **p < 0.01.
Figure 2
Figure 2. tHSC-CM induces EMT-like phenotype in HCC cells.
(A) The morphology changes in PLC/PRF/5 cells (magnification, ×200). (B) Western blot analysis of E-cadherin and vimentin expressions in PLC/PRF/5 cells (left panel). Results are expressed as the fold value of protein levels compared with GAPDH (right panel). *p < 0.05. (C and D) Representative images of E-cadherin and vimentin expressions in PLC/PRF/5 cells by immunoflurescence staining (magnification, ×400).
Figure 3
Figure 3. tHSCs promote HCC growth, and icaritin effectively inhibits tHSC-induced HCC-promoting effects in vivo.
(A) The tumor volumes were monitored every 4 days up to 23 days after cell implantation. (B) The tumor weights at the end of experiments (*p < 0.05). (C) Representative immunohistochemical staining of α-SMA, E-cadherin, Ki-67, and CD34 expressions in different xenografts. (D) Representative images of TUNEL assay in different xenografts (magnification, ×200). PLC: PLC/PRF/5.
Figure 4
Figure 4. tHSCs are associated with E-cadherin expression, HCC cell invasion in human HCC specimens, and poor survival outcome.
(A) Representative immunostaining of HSCs in normal liver tissues and HCC tissues with α-SMA and desmin antibodies. (B) Representative samples of the tHSC density determined by α-SMA immunostaining (magnification, ×200). Typical grades 0, 1, 2, and 3 are shown in a, b, c, and d, respectively. (C) Representative pictures of α-SMA and E-cadherin expressions in human HCC tissues detected by immunostaining (magnification, ×400). tHSC-rich is accompanied by deccreased E-cadherin expression in case 1 (upper panel), and tHSC-poor is accompanied by increase E-cadherin expression in case 2 (lower panel). (D) Immunofluorescence staining separately with Hep Par 1 and E-cadherin antibody on serial sections of one HCC sample (magnification, ×400). The white arrows indicated the tumor cells with both Hep Par 1-positive and E-cadherin-negative staining that have infiltrated from their nests into the surrounding stroma. (E) The impact of tHSCs on the survival of HCC patients. (a) tHSC-associated overall survival rate. (b) tHSC-associated recurrence-free survival rate.
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Grants and funding

This research was supported by the Grant from China National Key Projects for Infectious Disease (No. 2012ZX10002012-010) and National Nature Science Foundation of China (No. 81172207, 81272669, 81272668). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.