title: Tumor-associated macrophages produce interleukin 6 and signal via STAT3 to promote expansion of human hepatocellular carcinoma stem cells.
abstract: OBJECTIVE
  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/Il2Rg(null) 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.
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, Michigan.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Wan
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, Michigan.
authors.forename: Ende
authors.initials: E
authors.lastname: Zhao
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, Michigan.
authors.forename: Ilona
authors.initials: I
authors.lastname: Kryczek
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, Michigan.
authors.forename: Linda
authors.initials: L
authors.lastname: Vatan
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, Michigan.
authors.forename: Anna
authors.initials: A
authors.lastname: Sadovskaya
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, Michigan.
authors.forename: Gregory
authors.initials: G
authors.lastname: Ludema
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, Michigan.
authors.forename: Diane M
authors.initials: DM
authors.lastname: Simeone
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, Michigan.
authors.forename: Weiping
authors.initials: W
authors.lastname: Zou
authors.affiliation: Department of Surgery, University of Michigan, Ann Arbor, Michigan. Electronic address: twelling@med.umich.edu.
authors.forename: Theodore H
authors.initials: TH
authors.lastname: Welling
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D018920:Coculture Techniques; D056945:Hep G2 Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D015850:Interleukin-6 / Q000276:immunology* / Q000378:metabolism; D008113:Liver Neoplasms / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D008264:Macrophages / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014411:Neoplastic Stem Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D014408:Biomarkers, Tumor; C497483:CD44 protein, human; D018960:Hyaluronan Receptors; C508600:IL6 protein, human; D015850:Interleukin-6; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C502936:tocilizumab
keywords: Cancer Biology; IL6 Receptor; Liver Cancer; Mouse Model