Gastroenterology

Gastroenterology

Volume 147, Issue 6, December 2014, Pages 1393-1404
Gastroenterology

Original Research
Full Report: Basic and Translational—Liver
Tumor-Associated Macrophages Produce Interleukin 6 and Signal via STAT3 to Promote Expansion of Human Hepatocellular Carcinoma Stem Cells

https://doi.org/10.1053/j.gastro.2014.08.039Get rights and content

Background & Aims

Cancer stem cells (CSCs) can contribute to hepatocellular carcinoma (HCC) progression and recurrence after therapy. The presence of tumor-associated macrophages (TAMs) in patients with HCC is associated with poor outcomes. It is not clear whether TAMs interact with CSCs during HCC development. We investigated whether TAMs affect the activities of CSCs in the microenvironment of human HCCs.

Methods

HCCs were collected from 17 patients during surgical resection and single-cell suspensions were analyzed by flow cytometry. CD14+ TAMs were isolated from the HCC cell suspensions and placed into co-culture with HepG2 or Hep3B cells, and CSC functions were measured. The interleukin 6 (IL6) receptor was blocked with a monoclonal antibody (tocilizumab), and signal transducer and activator of transcription 3 was knocked down with small hairpin RNAs in HepG2 cells. Xenograft tumors were grown in NOD-SCID/Il2Rgnull mice from human primary HCC cells or HepG2 cells.

Results

CD44+ cells from human HCCs and cell lines formed more spheres in culture and more xenograft tumors in mice than CD44- cells, indicating that CD44+ cells are CSCs. Incubation of the CD44+ cells with TAMs promoted expansion of CD44+ cells, and increased their sphere formation in culture and formation of xenograft tumors in mice. In human HCC samples, the numbers of TAMs correlated with the numbers of CD44+ cells. Of all cytokines expressed by TAMs, IL6 was increased at the highest level in human HCC co-cultures, compared with TAMs not undergoing co-culture. IL6 was detected in the microenvironment of HCC samples and induced expansion of CD44+ cells in culture. Levels of IL6 correlated with stages of human HCCs and detection of CSC markers. Incubation of HCC cell lines with tocilizumab or knockdown of signal transducer and activator of transcription 3 in HCC cells reduced the ability of TAMs to promote sphere formation by CD44+ cells in culture and growth of xenograft tumors in mice.

Conclusions

CD44+ cells isolated from human HCC tissues and cell lines have CSC activities in vitro and form a larger number of xenograft tumors in mice than CD44- cells. TAMs produce IL6, which promotes expansion of these CSCs and tumorigenesis. Levels of IL6 in human HCC samples correlate with tumor stage and markers of CSCs. Blockade of IL6 signaling with tocilizumab, a drug approved by the Food and Drug Administration for treatment of rheumatoid arthritis, inhibits TAM-stimulated activity of CD44+ cells. This drug might be used to treat patients with HCC.

Section snippets

Human Subjects

HCC tissues were obtained from patients undergoing surgical resection as described.13 All patients provided written informed consent. The study was approved by and followed the University of Michigan Institutional Review Board guidelines.

Additional Methods can be found online in the Supplementary information.

HCC Cells Express Multiple Potential CSC Markers

Many cell surface markers have been described to define human HCC CSCs, including CD24, CD44, CD90, CD117, CD133, and epithelial cell adhesion molecule (EpCAM).15, 16, 17 To test whether these markers identify CSCs in a Western HCC patient cohort, surgically resected HCCs (n = 17 patients) (Supplementary Table 1) underwent single-cell suspension and flow cytometry by gating on 7-AAD-CD45- (excluding nonviable and immune cells), and were stained for putative CSC markers (Figure 1A and B). HCC

Discussion

In this study, we examined the CSC profile of a Western HCC patient cohort, patient xenografts, and cell lines and defined CD44 as an important marker of HCC CSCs. We show that HCC CSCs are expanded by patient TAMs. A significant correlation between TAMs and CD44+ HCC cells in patient tumors along with stemness-related gene expression was shown. This was supported by CSC subset expansion after HCC cell exposure to TAMs in vitro. IL6 secretion by TAMs accounts for this effect because IL6 was the

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by National Institutes of Health grants CA151414 (T.H.W.), and 5P30CA46592-24 and CA152470 (W.Z.), and the GI SPORE Career Development Award (T.H.W.).

    Author names in bold designate shared co-first authors.

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