Expression of TREM-1 in hepatic stellate cells and prognostic value in hepatitis B-related hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a typical inflammation-related malignancy characterized by high postoperative recurrence and metastasis. Although several inflammatory cells and inflammatory signatures have been linked to poor prognosis, the inflammation-associated molecular mechanisms of HCC development and progression are largely unknown. Here we show that triggering receptor expressed in myeloid cells (TREM)-1, a transmembrane receptor expressing in myeloid cells, was also expressed in tumor-activated hepatic stellate cells (HSCs) and associated with the aggressive behavior of HCC cells. Enzyme-linked immunosorbent assay was used to measure the expression levels of soluble TREM-1 (sTREM-1) in activated hepatic stellate cells supernatant and 92 preoperative and postoperative plasmas of patients with malignancy and/or benign liver tumor/disease, respectively. Expression levels of TREM-1 were assessed by immunohistochemistry in tissue microarray from 240 patients with HCC. As a result, increased secretion of sTREM-1 from activated HSCs was observed after co-culture with HCC cell lines (P < 0.001), and conditioned medium collected from activated HSCs/cancer associated myofibroblasts (CAMFs) with or without agonist/inhibitor of TREM-1 significantly changed the migratory ability of HCC cells. The levels of sTREM-1 were significantly higher in patients with HCC than those with benign liver tumors (P < 0.005). Peritumoral density of TREM-1 was shown to be an independent prognosis predictor according to univariate (P < 0.001 for both overall survival and time to recurrence) and multivariate analysis (P = 0.008 for overall survival; P = 0.005 for time to recurrence). Thus, these observations suggest that TREM-1 is related to the aggressive tumor behavior and has potential value as a prognostic factor for HCC.

Figure 1

Expression levels of triggering receptor expressed on myeloid cells (TREM)‐1 by immunohistochemistry and immunofluorescent analysis. High (a,c) and low (b,d) density of TREM‐1 staining cells in intratumoral (a,b) and peritumoral areas (c,d). (e) Results of immunofluorescent analysis of primary hepatic stellate cells (HSCs) and cancer associated myofibroblasts (CAMFs) at 2, 7, and 14 days after isolation from human peritumoral and cancer tissues using fluorescence‐labeled TREM‐1 (red), Desmin (green), α‐SMA (green), and DAPI (blue) stained cell nuclei.

Figure 2

Release of triggering receptor expressed on myeloid cells (TREM)‐1 by lipopolysaccharide (LPS)‐induced inflammation. (a–c) Effects of various doses of LPS stimulation (10 μg–1 ng/mL). Data shown are the mean ± SD (n = 3). aHSC, activated hepatic stellate cell; CAMFs, cancer associated myofibroblasts; qHSC, quiescent phenotype hepatic stellate cell.

Figure 3

Enzyme‐linked immunosorbent assay for triggering receptor expressed on myeloid cells (TREM)‐1, interleukin (IL)‐6, and tumor necrosis factor (TNF)‐α. (a) Activated hepatic stellate cells (HSCs) and cancer associated myofibroblasts (CAMFs) released high levels of IL‐6 and TNF‐α, and increased the release of TREM‐1, IL‐6, and TNF‐α after co‐culture with cancer cells or normal hepatocytes (P < 0.05). Cancer cells and normal hepatocytes had no significant effect on secretion of TREM‐1, IL‐6, or TNF‐α by non‐activated HSCs. (b) Expression levels of soluble TREM‐1 (sTREM‐1), IL‐6, and TNF‐α were upregulated in plasmas of hepatocellular carcinoma (HCC) patients compared with patients with benign disease or postoperative HCC at 5 days. *P < 0.005, vs other groups; #P < 0.005, vs other groups; ▴P < 0.005, vs other groups; **P < 0.05, vs other groups; ##P < 0.05, vs other groups; ▴▴P < 0.05, vs other groups. aHSC, activated HSC; qHSC, quiescent phenotype HSC.

Figure 4

Migration assay of cancer cells. (A) Representative pictures of Hep3B cells' in vitro migration assay using conditioned medium (CM) of activated hepatic stellate cells (aHSCs) and/or inhibitor (peptide) of triggering receptor expressed on myeloid cells (TREM)‐1 (LP17), or control peptide (LP17C), or anti‐TREM‐1 agonist mAb. (b,c) Number of migrated Hep3B (B) and HepG2 (C) cells (×200) both showed that CM of activated HSCs/CAMFs increase migration of cancer cells compared with control (P < 0.05). Furthermore, migration was greatly increased after incubation with CM and mAb (CM + mAb, P < 0.05). Migration was decreased after adding inhibitor of TREM‐1 LP17 into CM (CM+LP17) compared with those cultured in CM of activated HSCs/cancer associated myofibroblasts (CAMFs) (P < 0.01). Non‐activated HSCs had no effect on migration of HCC cell lines. *P < 0.05, vs CM or CM + LP17 or control; **P < 0.05, vs CM or control; ***P < 0.05, compared with control.

Figure 5

Prognostic roles of triggering receptor expressed on myeloid cells (TREM)‐1 by Kaplan–Meier analysis. (a,b) Kaplan–Meier estimates of overall survival according to high or low expression levels of TREM‐1 positive cells in testing (a) and validation cohorts (b). (c,d) Kaplan–Meier estimates of time to recurrence of peritumoral TREM‐1 positive staining cells in testing (c) and validation cohorts (d).